Anti-inflammatory mechanisms of dietary restriction in slowing aging processes

Mechanisms of Dietary Restriction in Aging and Disease

Interdisciplinary Topics inGerontology

Vol. 35

Series Editors Patrick R. Hof, New York, N.Y.

Charles V. Mobbs, New York, N.Y.

Editorial Board Constantin Bouras, Geneva

Caleb E. Finch, Los Angeles, Calif.

Kevin Flurkey, Bar Harbor, Me.

Terry Fulmer, New York, N.Y.

Jack Guralnik, Bethesda, Md.

Jeffrey H. Kordower, Chicago, Ill.

Bruce S. McEwen, New York, N.Y.

Diane Meier, New York, N.Y.

Jean-Pierre Michel, Geneva

John H. Morrison, New York, N.Y.

Mark Moss, Boston, Mass.

Nancy Nichols, Melbourne

S. Jay Olshansky, Chicago, Ill.

James L. Roberts, San Antonio, Tex.

Albert Siu, New York, N.Y.

John Q. Trojanowski, Philadelphia, Pa.

Mechanisms of Dietary Restriction inAging and Disease

Basel · Freiburg · Paris · London · New York ·

Bangalore · Bangkok · Singapore · Tokyo · Sydney

Volume Editors Charles V. Mobbs, New York, N.Y.

Kelvin Yen, New York, N.Y.

Patrick R. Hof, New York, N.Y.

23 figures and 5 tables, 2007

Charles V. Mobbs, PhD Patrick R. Hof, MDDepartment of Neuroscience Department of Neuroscience

Mount Sinai School of Medicine Mount Sinai School of Medicine

New York, N.Y., USA New York, N.Y., USA

Kelvin Yen, BADepartment of Neuroscience

Mount Sinai School of Medicine

New York, N.Y., USA

Bibliographic Indices. This publication is listed in bibliographic services, including Current Contents® andMEDLINE/Index Medicus.

Disclaimer. The statements, options and data contained in this publication are solely those of the individ-ual authors and contributors and not of the publisher and the editor(s). The appearance of advertisements in thebook is not a warranty, endorsement, or approval of the products or services advertised or of their effectiveness,quality or safety. The publisher and the editor(s) disclaim responsibility for any injury to persons or propertyresulting from any ideas, methods, instructions or products referred to in the content or advertisements.

Drug Dosage. The authors and the publisher have exerted every effort to ensure that drug selection anddosage set forth in this text are in accord with current recommendations and practice at the time of publication.However, in view of ongoing research, changes in government regulations, and the constant flow of informationrelating to drug therapy and drug reactions, the reader is urged to check the package insert for each drug for any change in indications and dosage and for added warnings and precautions. This is particularly important whenthe recommended agent is a new and/or infrequently employed drug.

All rights reserved. No part of this publication may be translated into other languages, reproduced orutilized in any form or by any means electronic or mechanical, including photocopying, recording, microcopying,or by any information storage and retrieval system, without permission in writing from the publisher.

© Copyright 2007 by S. Karger AG, P.O. Box, CH–4009 Basel (Switzerland) www.karger.comPrinted in Switzerland on acid-free paper by Reinhardt Druck, BaselISSN 0074–1132ISBN–10: 3–8055–8170–XISBN–13: 978–3–8055–8170–7

Library of Congress Cataloging-in-Publication DataMechanism of dietary restriction in aging and disease / volume editors, Charles V. Mobbs,Kelvin Yen, Patrick R. Hof.

p. ; cm – (Interdisciplinary topics in gerontology, ISSN 0074-1132 ;v. 35)Includes bibliographical references and index.ISBN-13: 978-3-8055-8170-7 (hardcover : alk. paper)ISBN-10: 3-8055-8170-X (hardcover : alk. paper)

1. Nutrition disorders in old age. 2. Older people–Nutrition.I. Mobbs, Charles V. II. Yen, Kelvin. III. Hof, Patrick R. IV. Series.[DNLM: 1. Caloric Restriction. 2. Longevity–physiology.

3. Aging–physiology. W1 IN679 v.35 2007 / WT.116 M486 2007]RC620.6.M43 2007618.97–dc22 2006022533

V

Contents

VII Preface

1 The Role of Hormesis in Life Extension by Dietary Restriction

Masoro, E.J. (San Antonio, Tex.)

18 Metabolic Reprogramming in Dietary Restriction

Anderson, R.M.; Weindruch, R. (Madison, Wisc.)

39 Secrets of the lac Operon

Glucose Hysteresis as a Mechanism in Dietary Restriction,

Aging and Disease

Mobbs, C.V.; Mastaitis, J.W.; Zhang, M.; Isoda, F.; Cheng, H.;

Yen, K. (New York, N.Y.)

69 Effects of Dietary Restriction on the Expression of Insulin-

Signaling-Related Genes in Long-Lived Mutant Mice

Bartke, A.; Masternak, M.M.; Al-Regaiey, K.A.; Bonkowski, M.S. (Springfield, Ill.)

83 Anti-Inflammatory Mechanisms of Dietary Restriction in

Slowing Aging Processes

Morgan, T.E.; Wong, A.M.; Finch, C.E. (Los Angeles, Calif.)

98 Dietary Restriction in the Nematode Caenorhabditis elegans

Houthoofd, K. (Ghent/London); Gems, D. (London);

Johnson, T.E. (Boulder, Colo.); Vanfleteren, J.R. (Ghent)

115 Diet Restriction in Drosophila melanogaster

Design and Analysis

Tatar, M. (Providence, R.I.)

137 Dietary Restriction in Aging Nonhuman Primates

Mattison, J.A.; Roth, G.S.; Lane, M.A.; Ingram, D.K. (Baltimore, Md.)

159 Caloric Intake and Alzheimer’s Disease

Experimental Approaches and Therapeutic Implications

Pasinetti, G.M.; Zhao, Z.; Qin, W.; Ho, L.; Shrishailam, Y.; MacGrogan, D.;

Ressmann, W.; Humala, N.; Liu, X.; Romero, C.; Stetka, B.; Chen, L.;

Ksiezak-Reding, H.; Wang, J. (New York, N.Y./Bronx, N.Y.)

176 Can Short-Term Dietary Restriction and Fasting Have a

Long-Term Anticarcinogenic Effect?

Klebanov, S. (New York, N.Y.)

193 Author Index

194 Subject Index

Contents VI

Preface

This volume arose from a program announcement made by the National

Institutes on Aging (NIA) requesting applications to study the basic mecha-

nisms by which dietary restriction decreases disease burden and increases life

span (RFA: AG–01–002, ‘Molecular and neural mechanisms underlying the

effects of caloric restriction on health and longevity’). Most of the applicants

who were funded had long been fascinated with the significance of this phe-

nomenon, potentially the most far-reaching in biomedical science. A particu-

larly valuable aspect of this program announcement was the requirement that

funded applicants, along with a few other selected invitees, were to meet near

the NIH on an annual basis to present and discuss their most recent results, pub-

lished and unpublished. As these meetings progressed, it became clear that the

field had experienced profound progress since the classic volume presenting a

comprehensive view of the field, The Retardation of Aging and Disease by

Dietary Restriction, by Richard Weindruch and Roy Walford, published in

1988, now sadly out of print. While it would be impossible for a multi-author

volume to capture the coherence, tone and focus that made that classic so valu-

able to so many of us early in our careers, nevertheless it seemed as if some

kind of review along those lines would be of some value. Therefore we con-

tacted the participants in the NIA workshops, as well as many of the other lead-

ing authorities in the field, and many were gracious enough to accept our

invitations to contribute reviews. We are particularly grateful to Dr. Masoro and

Dr. Weindruch, who did so much to create this field, and Dr. Finch, who has

VII

contributed so much to so many areas of gerontology, for agreeing to contribute

papers representing their current thoughts on the subject. Indeed, we very much

appreciate the time and effort that all our contributors made, and hope that our

readers benefit as much from reading the articles as we did editing them.

Charles V. Mobbs, Kelvin Yen, Patrick R. Hof

New York, N.Y.

Preface VIII

Mobbs CV, Yen K, Hof PR (eds): Mechanisms of Dietary Restriction in Aging and Disease.

Interdiscipl Top Gerontol. Basel, Karger, 2007, vol 35, pp 1–17

The Role of Hormesis in Life Extensionby Dietary Restriction

Edward J. Masoro

Barshop Institute for Longevity and Aging Studies, University of Texas Health

Science Center, San Antonio, Tex., USA

AbstractThe level of food restriction that results in life extension and retarded aging in rodents

also enhances their ability to cope with intense stressors. Moreover, this level of dietary

restriction (DR) leads to a modest increase in the daily peak concentration of plasma free

corticosterone, which strongly points to DR as a low-intensity stressor. These findings sug-

gest that hormesis plays a role in the life-extending and anti-aging actions of DR. The evi-

dence for and against this possibility is considered, and it is concluded that hormesis does

have an important role.

Copyright © 2007 S. Karger AG, Basel

It is some 70 years since McCay et al. [1] showed that long-term restriction

of food intake markedly extends the life of rats. This finding has been con-

firmed many times in studies involving a variety of strains of rats and mice as

well as a spectrum of other species, including hamsters, dogs, invertebrate ani-

mals and yeast [2]. The dietary factor responsible for the life extension of rats is

a reduction of caloric intake rather than a specific nutrient [2]. The relevant

studies have not been done to determine if this is also true of mice. One study

indicates that in the case of Drosophila melanogaster, a decreased intake of

protein or fat or both may underlie the life-prolonging action of food restriction

[3]. The term dietary restriction (DR) will be used in this article when referring

to life extension due to food restriction, since in some species, dietary factors

other than reduction in caloric intake could be responsible for life extension.

The mechanisms underlying the anti-aging and life-extending actions of

DR remain to be defined, although many hypotheses have been proposed over

the past 70 years. These include, but are not limited to, the effects of the

following: retardation of growth; reduction of body fat content; reduction of

Masoro 2

metabolic rate; decreased body temperature; increased physical activity;

enhancement of apoptosis; increased protein turnover; attenuation of oxidative

stress; attenuation of glycation and glycoxidation, and attenuation of insulin-

IGF-1 signaling [2]. In 1998, Masoro [4] and Turturro et al. [5] independently

proposed the hormesis hypothesis. This hypothesis, which may embrace many

of the specific hypotheses just mentioned, will be discussed in detail in this

article.

Concept of Hormesis

Hormesis refers to the phenomenon in which the response of an organism

to a chemical or physical agent is qualitatively different when the agent is of

high intensity than when it is of low intensity. An example would be a carcino-

gen that promotes the occurrence of cancer when administered at medium or

high levels but protects the organism from cancer when used at low levels (fig. 1).

Indeed, a variety of toxic chemical agents involving a spectrum of endpoints

(growth, metabolic effects, reproduction, disease processes and longevity) have

been shown to have hormetic actions at low concentrations in a wide range

of taxonomic groups [6]. Ionizing radiation has also been claimed to exhibit

hormesis, e.g. high doses of X-rays and gamma rays have been found to

decrease and low doses to increase the life spans of mice, rats, houseflies, flour

0

5

10

15

20

25

30

35

40

45

50

0 10 20 30 40 50 60 70

Can

cer

incid

en

ce p

er

1,0

00

Concentration of carcinogen

Fig. 1. Effects of a hypothetical carcinogen on cancer incidence. Low concentrations

of the hypothetical carcinogen decrease and high doses increase cancer incidence, a phenom-

enon termed hormesis. The horizontal dashed line refers to no effect on cancer incidence.

Hormesis and Dietary Restriction 3

beetles, codling moths and house crickets [7, 8]. However, a recent National

Research Council report claims that such is not the case for the human response

to ionizing radiation [9].

Within the realm of toxicology, hormesis has come to be viewed as a bene-

ficial phenomenon, i.e. there are many examples of low doses of toxic com-

pounds resulting in protective or other favorable actions. Indeed, hormesis is

now generally used to refer to the low-dose beneficial effects of otherwise

harmful agents. Rattan [10] has proposed the following definition of hormesis

as it relates to aging: hormesis in aging is characterized by the beneficial effects

resulting from the cellular responses to mild repeated stress. That definition

serves as the basis for this discourse.

Dietary Restriction – A Low-Intensity Stressor

In considering the possibility that hormesis plays an important role in DR’s

anti-aging and life-extending actions, the first question that must be addressed

is whether DR is a low-intensity stressor. It has long been known that stressors

markedly elevate the plasma level of glucocorticoids in mammalian species,

and the elevation of the plasma level of this class of steroid hormones is now

generally considered to be a signature response to a stressor [11]. Sabatino et al.

[12] compared the daily circadian rhythms of plasma glucocorticoid levels of ad

libitum fed male rats with those of male rats on a DR regimen; they found that

the daily afternoon peak plasma concentration of free corticosterone of the DR

rats is moderately but significantly greater throughout life than that of ad libi-

tum fed rats. Armario et al. [13] reported that the concentration of plasma total

corticosterone is elevated in young male rats on a DR regimen for 34 days.

Stewart et al. [14] found that DR elevated the plasma total corticosterone con-

centration in 5-month-old rats but not in 24-month-old rats. In the study of

Sabatino et al. [12], plasma total corticosterone was also found to be elevated

only in young rats, while plasma free corticosterone exhibits a modest daily ele-

vation throughout life. The reason for the apparent discrepancy is the decrease

in the level of plasma corticosteroid-binding globulin with increasing age in the

rats on a DR regimen, resulting in an elevation of free corticosterone in the

absence of an increase in total corticosterone. That the plasma free cortico-

sterone remains elevated is important because it is not the total concentration

but rather the free corticosterone concentration that is believed responsible for

the physiological actions of this hormone [15]. Indeed, Han et al. [16] reported

that as the rat ages, there are changes in the mechanism underlying the increase

in the level of plasma free corticosterone induced by DR. The fact that two or

more mechanisms are employed to sustain the life-long DR-induced daily

Masoro 4

elevation of the peak level of plasma free corticosterone is indicative of its

importance.

In summary, the evidence is convincing that long-term DR in rats causes

the animal to experience daily periods of mild stress throughout life. Although

not as thoroughly studied, it appears that such is also the case for mice [17].

Thus, the answer is yes, long-term DR is a sustained low-intensity stressor.

Dietary Restriction and Coping with Acute Intense Stressors

Rattan [18] proposed that the concept of hormesis as an aging retardant is

based on the principle that repeated exposure to mild stress stimulates mainte-

nance and repair processes. In their consideration of aging, from the viewpoint

of far from equilibrium thermodynamics, Toussaint et al. [19] have come to a

similar conclusion; they state that conditions of moderate stress that enhance

the efficiency of the stress response are known as hormesis. Assuming that DR

has a hormetic action, it should enhance the ability of the organism to cope with

intense stressors. There is much evidence that DR, indeed, has this action. In

both young and old rats, DR attenuates the acute loss of body weight due to

surgical stress [4]. It also reduces the inflammatory response in young mice fol-

lowing the injection of an inflammatory substance into their footpad [17], and it

increases the ability of rats to survive a sudden marked increase in environmen-

tal temperature [20]. DR protects rodents from the damaging action of a spec-

trum of toxic chemicals [21]. Testing the effects of four potentially toxic drug

candidates on Sprague-Dawley rats, Keenan et al. [22] found that DR increased

the maximum tolerated dose of each. Berg et al. [23] reported that following the

administration of ganciclovir sodium, B6C3F1 mice on a DR regimen had a

markedly reduced mortality, compared to those fed ad libitum.

Thus, DR meets the classic criteria of hormesis. A marked reduction in

food intake is clearly harmful to the point of being lethal, while a long-term

moderate reduction in food intake enables the organisms to more successfully

cope with damaging environments and toxic agents.

Dietary Restriction, Hormesis and Aging Processes

So DR does have a hormetic action. How is that relevant to aging? Aging,

defined in terms of senescence, is characterized by a progressive deterioration

of function throughout most of the adult life of an organism. Most biological

gerontologists believe that this functional deterioration is caused by the

accumulation of molecular damage from both endogenous (e.g. the metabolic


generation of reactive oxygen molecules) and exogenous (e.g. damage due to

toxic chemicals) sources, which is not successfully prevented by the organism’s

protective and repair processes. Kirkwood’s Disposable Soma of Aging Theory

[24] poses that less energy is used for somatic maintenance than is needed for

indefinite survival. Indeed, it is likely that the rate of aging is determined by the

extent of imbalance in favor of damaging processes over repair and protective

processes. Thus DR, acting as a moderate stressor, protects the organisms from

acute, intense stressors, which suggests that it may also protect against the long-

term damage that underlies aging. Moreover, it is likely that DR does so, in

part, by augmenting protective and repair processes, i.e. Rattan’s view [18] of

the role of hormesis in the retardation of aging.

Indeed, there is evidence that moderate stressors, other than DR, extend

life. In 1958, Maynard Smith [25] reported that a transient heat stressor

increased the life span of female fruit flies; Khazaeli et al. [26] confirmed that

finding and, in addition, showed that it occurs in males as well. Shama et al.

[27] observed that a transient mild heat stressor extends the replicative life span

of Saccharomyces cerevisiae. Lithgow et al. [28] and Yashin et al. [29] reported

a similar effect of transient, mild heat stressors on the longevity of

Caenorhabditis elegans; this, they propose, is because of an increased adapta-

tion of the organism resulting from the mild heat stress stimulus. Indeed,

Cypser and Johnson [30] found that several different low-intensity stressors

extend the life of C. elegans. Moreover, upon a review of the literature, Johnson

et al. [31] concluded that it is not uncommon for longevity to be increased in

organisms exposed to moderately stressful conditions. The study of Kapahi

et al. [32] also links longevity to the ability to cope with a damaging environ-

ment; in their study on culture of fibroblasts derived from eight mammalian

species, resistance to damaging agents correlated positively with the life span

of the species.

There is concern that unlike the modest effects of other low-intensity stres-

sors, DR markedly extends life. It has been suggested that this quantitative dif-

ference tends to negate a role for hormesis in the life-extending action of DR. In

my opinion, this view is illusory. For example, although DR from 6 weeks of

age on increased the median length of life of male rats by 51%, those on this

dietary regime from 6 weeks to 6 months of age exhibited only a 15% increase

[33]. In the report of Le Bourg et al. [34], exposing young D. melanogaster

males for 14–24 days to a moderate hypergravity stressor frequently resulted in

a 10–15% increase in mean length of life. Clearly, the quantitative difference

between these two stressors is not great when each is applied for only a fraction

of the life span.

There is genetic evidence of a link between longevity and the ability to

cope with intense stressors. Organisms with genetic manipulations that result in

Masoro 6

increased longevity usually, though not always, have an increased ability to

cope with intense stressors [2, 35]. For example, single-gene mutations experi-

mentally generated in C. elegans [36, 37], yeast [38] and D. melanogaster [39]

have yielded organisms with this characteristic.

Thus, DR extends life and enhances the ability to cope with intense stres-

sors. Other moderate stressors extend life and increase the ability to cope with

intense stressors. These findings suggest that hormesis plays a role in the life-

extending action of DR. What is missing is an understanding of the molecular,

cellular and organismic processes involved in the hormesis-based life-extending

actions of DR. There are, however, promising leads.

The study of Anderson et al. [40] has provided a partial description of the

DR-induced hormetic pathway responsible for the increase in the replicative life

span of a PSY strain of S. cerevisiae. They found that both a functional PNC1

gene and Sir2 gene are required for DR to induce replicative life extension in this

yeast strain. PNC1 encodes the PNC1 protein, a nicotinamidase, and DR was

found to increase the amount of this enzyme. Sir2 encodes the SIR2 protein,

which has deacetylase activity; a product of the deacetylase reaction is nicotin-

amide, an inhibitor of the SIR2 deacetylase activity. Thus, by increasing the level

of the nicotinamidase protein, DR maintains a low level of nicotinamide in the

cells of this yeast strain, thereby increasing SIR2 deacetylase activity. It is this

increased deacetylase activity that plays a key role in the DR-induced replicative

life extension. Moreover, Anderson et al. [40] found that the same pathway is

involved in replicative life extension induced by two other low-intensity stres-

sors, heat stress and osmotic stress. Thus, Anderson et al. have begun to delineate

the hormetic pathway in this yeast strain that connects the stressor to an increase

in SIR2 deacetylase activity. The pathway linking the increased deacetylase

activity to an increase in replicative life span remains to be defined.

However, the finding that DR can extend the replicative life span of yeast

strains lacking the SIR2 protein has cast doubt on the general relevancy of the

work of Anderson et al. Recently, Lamming et al. [41] reported that one yeast

strain lacking SIR2 contains HST2, a sirtuin protein homologue of SIR2, and

that this homologue is also a deacetylase and functions in the actions of DR in a

fashion similar to SIR2. The question that arises is whether sirtuin proteins play

a similar role in the action of DR in other species. Indeed, there is such evidence

[42]. Rogina and Helfand [43] reported that SIR2 is directly involved in the

DR-induced life extension of Drosophila.Also, Tissenbaum and Guarente [44]

reported that increasing the dosage of the Sir2.1 gene, which codes for a sirtuin

protein, extends the life of C. elegans and that this effect requires an active

DAF-16 gene. Nevertheless, the role of sirtuin proteins in DR-induced life span

extension in C. elegans is in doubt since Lakowski and Hekimi [45] found that

mutation of the eat-2 gene extends the life of this species, presumably by


decreasing food intake, and that the life extension does not require an active

DAF-16 gene.

Furthermore, several very recent findings have to be reconciled with the

view that sirtuin proteins play a key role in the pathway linking low-intensity

stressors to life extension. In contrast to its role in the replicative life span,

Fabrizio et al. [46] reported that Sir2 decreases the chronological life of yeast

rather than increasing it. Kaeberlein et al. [47] have found that mutations of

Tor1 or Sch9, as well as other genes in their nutrient-sensing pathways, extend

the replicative life of yeast independent of Sir2. However, it is not clear whether

HST2 or some other sirtuin homologue of SIR2 is required. Tor and Sch9 muta-

tions have also been found to extend the life of D. melanogaster [48] and

C. elegans [49].

Stress response genes, which protect against cellular damage [50], are also

likely to be part of the DR-induced hormetic pathway. For example, DR

enhances the expression of heat shock proteins in rats exposed to damaging

agents [19, 51–55]. Moreover, several studies, which do not involve DR,

strongly implicate stress response gene expression in life extension. Tatar et al.

[56] studied a transgenic strain of D. melanogaster with an extended life span

and found that this strain exhibits an enhanced expression of stress response

genes. Garigan et al. [57] presented evidence that heat shock proteins slow the

rate of aging of C. elegans. Indeed, several studies show that stress response

proteins and the transcription factor promoting their expression have an anti-

aging and life-extending action in this species of worm. Hsu et al. [58] reported

a regulatory relationship between heat shock transcription factor and the aging

of C. elegans, and Yokoyama et al. [59] found that the constitutive overexpres-

sion of hsp70F extended the life of this organism. Morley and Morimoto [60]

expanded our understanding by showing that HSF-1, a transcriptional factor

that regulates stress-inducible gene expression, enhances the ability of C. elegans

to cope with intense stressors and, in addition, extends the life of these worms.

Significantly, Walker and Lithgow [61] found that insulin-like signaling plays a

regulatory role in the life-extending action of stress response genes in C. elegans.

Thus, there is strong evidence to support the view that an enhancement of the

expression of stress response genes is a component of the hormetic pathway by

which DR retards aging processes and extends life.

An enhancement of repair processes is also likely to be a component of

DR-induced hormesis. Indeed, DR has been found to retard the age-associated

decrease in the ability of rats to repair the transcribed strands of DNA [62, 63].

Also, DR increases the rate of whole-body protein turnover in adult and old rats

[64, 65]. These actions should slow the accumulation with increasing age of

damaged DNA and protein molecules; indeed, that has been found to be the

case for both rats and mice [66–72].

Masoro 8

In addition to its effects at a cellular level, DR also has potential hormetic

actions at the organismic level. As mentioned above, DR causes a lifetime ele-

vation of the daily peak concentration of plasma free corticosterone in rats [12],

the major glucocorticoid in this species. The hypothalamic-hypophyseal-

adrenal cortical glucocorticoid system plays a key role in enabling mammals to

cope with damage [11]. Thus, the hormetic pathway for the DR-induced anti-

aging activity may well involve the daily elevation in the level of plasma free

glucocorticoid, which, in turn, retards aging by its effects on target cells.

Indeed, Leakey et al. [73] proposed that increased levels of glucocorticoids con-

tribute to the anti-aging actions of DR. The work of Pashko and Schwartz [74]

on carcinogenesis provides experimental support for this proposal. They found

that DR’s ability to protect mice against chemically induced tumors is lost if the

animals have been adrenalectomized. Schwartz and Pashko [75] hypothesized

that elevated levels of adrenal steroids underlie the ability of DR to inhibit both

carcinogen-induced tumors and those that occur spontaneously with advancing

age. Birt et al. [76] have also found that the ability of DR to inhibit skin car-

cinogenesis in mice requires elevated levels of glucocorticoids.

Analyses of Findings Opposing the Hormesis Hypothesis

Up to this point, the focus has been primarily on findings that support the

hormesis hypothesis. In this section, an assessment is made of findings that

question its validity.

Neafsey [77] compared the lifetime age-specific mortality pattern in the

hormesis-based increase in longevity induced by DR with that induced by low

levels of methylene chloride or gamma radiation. Because the pattern in the

DR studies that she assessed differed from that of the other two stressors,

Neafsey concluded that hormesis does not underlie the life-extending actions

of DR. However, it has recently become clear that the lifetime age-specific

mortality pattern in response to DR exhibits interspecies as well as

intraspecies variation [69, 78–80]. Moreover, the classic interpretation of age-

specific mortality analyses has recently been questioned [81] and needs to be

reevaluated [82]. Thus, the basis of Neafsey’s challenge [77] is currently open

to question.

Although DR enhances the ability of organisms to cope with several

intense stressors, it does not do so for all of them. Indeed, animals on a DR reg-

imen cope less effectively with some intense stressors. Of course, such occur-

rences do not lessen the fact that DR has beneficial actions in response to many

stressors. Even in the case of those stressors where organisms fare less well,

other effects of DR may mask the beneficial hormetic action.


The healing of skin wounds appears to be a case in point. It is known that

DR adversely affects the healing of such wounds in mice and rats [83, 84]. Reed

et al. [85] investigated the effect of age on the healing of such wounds in ad libi-

tum fed mice and found, as have others, that this ability deteriorates with

increasing age. However, if mice that had been on DR until advanced ages were

fed ad libitum starting 4 weeks prior to wounding, their wounds healed as

rapidly as those of young mice. Wound healing requires the expenditure energy

for the biosynthesis of collagen and other matrix molecules and for augmented

cell proliferation; it appears that the reduced energy intake by the mouse on DR

masks its beneficial effect on wound healing.

The effect of DR on the response of rats to cold stress may be another

example. Campbell and Richardson [86] reported that rats on DR are less able

to cope with cold stress. It is well known that an increase in metabolic heat pro-

duction is the major way that small mammals like rats cope with cold stress. It

is likely that this is another case where the reduced energy intake and storage

mask a beneficial hormetic action of DR. A study, such as that done by Reed

et al. [85] on wound healing, has yet to be done in regard to cold stress.

The findings are mixed regarding the effect of DR on coping with the chal-

lenge of infectious agents. In 1975, Gerbase-Delima et al. [87] reported that in

young C57BL/6 mice, DR decreases the in vitro proliferative response of their

splenic lymphocytes to mitogens; in contrast, it enhances the in vitro prolifera-

tive responses of splenic lymphocytes from middle-aged and old mice. It was

concluded that DR initiated in young mice slows the maturation of the immune

system, but enhances its function once maturation is achieved. Subsequent

studies using a variety of mouse and rat strains have yielded similar results as

well as other indicators of immune function enhancement. See Pahlavani [88]

for a review of these many findings. However, other studies have yielded con-

flicting results. Roecker et al. [89] found that adult rhesus monkeys on DR for

2–4 years exhibit a reduction in the mitogen-induced proliferative response of

peripheral blood mononuclear cells. Weindruch et al. [90] studied the immune

function of rhesus monkeys that had been on DR for 7 years starting at 1 year of

age or 3–5 years of age; they found that DR decreased the peripheral blood

mononuclear cell proliferative response in the younger but not the older group,

and that both groups exhibited lymphopenia. Sun et al. [91] tested 6-month-old

C57BL/6 mice for their response to polymicrobial sepsis induced by cecal liga-

tion and puncture; they found that mice on DR for 5 months died earlier than

those fed ad libitum. This may have been due to DR’s effect on the maturation

of the immune system since DR was started at 1 month of age in these mice.

The findings on the effect of DR on the response of animal models to influenza

are also conflicting. Effros et al. [92] reported that long-term DR enhances the

immune response of mice to influenza vaccination, as evidenced by increased

Masoro 10

antigen-specific lymphoproliferation, antigen presentation, antibody produc-

tion and T-cell function. In contrast, Roecker et al. [89] found that adult rhesus

monkeys on DR for 2–4 years exhibit a reduced plasma antibody response to

influenza vaccine. Recently, Gardner [93] has reported that long-term DR

decreased the survival of mice after primary influenza infection. Further

research is clearly needed to understand the basis of these conflicting findings.

The impressive studies of Sapolsky [94], showing that glucocorticoids can

adversely affect the aging of the nervous system, have led to some doubt that

the increased daily elevation in the level of plasma free glucocorticoid in DR

rodents plays a role in the life extension. However, this concern is countered by

the fact that adrenalectomy results in a loss in the ability of DR to retard car-

cinogenesis in mice [74]. Indeed, it seems likely that a level of glucocorticoid,

either too low or too high, adversely affects longevity, and that the level in DR

rodents is nearly optimal. Further detailed studies are needed to fully define the

relationship between long-term glucocorticoid levels and longevity.

Conclusions

Although the currently available database strongly supports the concept

that hormesis plays an important role in the life-extending and anti-aging

actions of DR, it is not a view held by most biological gerontologists. There are

several possible reasons for this disconnection between the database and the

skepticism of its critics.

Calebrese [95] points out that biologists in general have long discounted the

importance or even the existence of hormesis. Thus, part of the negative attitude

of biological gerontologists may stem from biologists’ disregard for hormesis.

Calabrese feels that this attitude relates to several factors. First, in many cases,

the hormetic effect of chemical agents occurs at extremely low doses, and it is

thus often missed in dose-response studies. Second, the hormetic effect is often

small, leading one to doubt that it is real. Third, hormesis presents difficulties for

those in regulatory agencies charged with determining safe limits for toxic

agents, which they prefer to avoid. Fourth, hormesis is often confused with

homeopathy, an approach to medicine that has been in disrepute for some time.

Biological gerontologists have encountered similar problems when they

attempt to explore hormesis. For example, Michalski et al. [96] found that if

3-day-old C. elegans undergo heat stress for less than 2 h, their longevity is

increased; heating for one half-hour had the greatest effect. However, the magni-

tude of the effect decreased with longer heating periods and disappeared when the

heating period exceeded 2 h. Another example is the D. melanogaster heat stress

study of Le Bourg et al. [97]. They found that heating young flies for a 5-min


period for 5 consecutive days increased longevity, but heating them for 10, 20,

30 min or longer had either no effect or decreased longevity. It is clear that in such

studies, hormesis can easily be missed or interpreted as noise rather than real.

However, in my opinion, the major reason that hormesis is discounted

relates to the fact that stressors often cause damage and accelerate aging.

Indeed, McEwen [98] has promoted the concept of allostatic load, which he

defines as the cumulative physiological toll over time by the organism’s efforts

to adapt to stressors. Certainly allostatic load is an important gerontological

concept, but it should not overshadow the fact that stressors can also have ben-

eficial actions. Clearly, whether chronic stressors are detrimental or beneficial

depends on the nature of the stressor and its intensity.

Returning to DR, it is clear that a significant reduction in food intake is a

beneficial stressor. Also, the hormetic component of this chronic stressor is not

likely to be missed, because it has beneficial actions over such a wide range of

restrictions, with benefits documented for levels of food restriction ranging

from 10 to 50% of the ad libitum intake [2]. Moreover, by focusing on the retar-

dation of the accumulation of molecular and cellular damage, the hormesis

hypothesis unifies many of the other hypotheses proposed to explain the anti-

aging and life-extending actions of DR. For example, in the attenuation of

oxidative stress hypothesis, the retardation of oxidative damage to cellular

structures is viewed as the basis of the anti-aging actions of DR; the hormesis

hypothesis encompasses such protection. In the attenuation of the glycation and

glycoxidation hypothesis, the focus is on the damage caused by non-enzymatic

glycation and glycoxidation; again, the hormesis hypothesis encompasses pro-

tection against such damage.

However, it should be pointed out that although hormesis is an important

component of the anti-aging actions of DR, it is not likely to be the only one.

Hormesis can explain all the protective components of DR, but in addition, it is

likely that DR has a different general action, namely that of decreasing the gen-

eration of damaging agents. For example, in regard to the attenuation of oxida-

tive stress hypothesis, there is evidence that DR decreases the generation of

harmful reactive oxygen molecular species; this action should decrease the for-

mation of oxidatively damaged cellular macromolecules [99]. However, Merry

[100] points out that a caveat is in order since the effect of DR on the generation

of reactive molecules has been found to occur in in vitro preparations such as

isolated mitochondria, but it has yet to be shown in the intact organism. Another

example relates to the attenuation of glycation and glycoxidation hypothesis; it

has been shown that DR causes the blood glucose level to be significantly less

throughout the lifetime of rats on DR compared to those fed ad libitum [101].

And this reduction in glycemia, which should result in a decreased glycation

and glycoxidation of macromolecules, is also not likely to have a hormetic link.

Masoro 12

Ideally the hormesis hypothesis of DR action should be tested by studies

that can falsify it. Given the current state of knowledge, such experiments are

difficult, if not impossible, to design for the following reasons. First, hormesis is

only one of the two general mechanisms proposed for DR action, which makes

interpretation of findings equivocal. Second, more than one hormetic pathway is

likely to be involved in DR’s actions, and the number and nature of such path-

ways have yet to be defined. The study of Pashko and Schwartz [74] illustrates

both of these problems. As mentioned above, these investigators tested the role

of glucocorticoids in DR’s protection against cancer in mice (a major disease

affecting longevity of this species), and they found that adrenalectomy abolishes

this protective effect. This finding is in accordance with a role of elevated

plasma glucocorticoid levels in DR’s anticancer action, which provides some

support for the hormesis hypothesis. However, if adrenalectomy had not abol-

ished the anticancer action of DR, that finding would not have falsified the

hormesis hypothesis for two reasons: hormesis is not likely the only process

underlying the life-extending action of DR and the pathway involving glucocor-

ticoids is probably not the only hormetic pathway involved. Both of these prob-

lems require further research before meaningful studies can be designed that can

falsify the hormesis hypothesis. It may be possible to address the first problem

through analyses of the effect of DR on the lifetime characteristics of age-

specific mortality (see Masoro [82] for a discussion of this possibility). The sec-

ond problem – i.e. the hormesis pathway(s) involved – should initially be

explored in young animals, e.g. the pathway(s) by which hormesis modulates the

inflammatory response can be readily investigated in young animals. Armed

with this information, the lengthy process of determining the pathway(s) under-

lying life extension can then be more efficiently explored.

In summary, current evidence suggests that DR retards aging and extends

life by two general processes. The first is the reduction in the generation of

damaging agents. The second is the enhancement of protective and repair

processes, and hormesis is the basis of this enhancement. The relative impor-

tance of DR’s two general processes probably depends on both genetic and

environmental factors and their interaction. Indeed, there are interspecies and

intraspecies differences in age-specific mortality characteristics underlying the

life-extending action of DR [82] and this strongly indicates the involvement of

more than one general process.

References

1 McCay CM, Crowell MF, Maynard LA: The effect of retarded growth upon the length of life and

upon the ultimate body size. J Nutr 1935;10:63–79.

2 Masoro EJ: Caloric Restriction: A Key to Understanding and Modulating Aging. Amsterdam,

Elsevier, 2002.


3 Mair W, Piper MDW, Partridge L: Calories do not explain extension of life by dietary restriction in

Drosophila. Plos Biol 2005;3:e223.

4 Masoro EJ: Hormesis and the antiaging action of dietary restriction. Exp Gerontol 1998;33:61–66.

5 Turturro A, Hass B, Hart RW: Hormesis – Implications for risk assessment caloric intake (body

weight) as an example. Hum Exp Toxicol 1998;17:454–459.

6 Calabrese EJ, Baldwin LA: Hormesis as a biological hypothesis. Environ Health Perspect

1998;106(suppl 1):357–362.

7 Kauffman JL: Radiation hormesis: demonstrated, deconstructed, denied, dismissed and some

implications for public policy. J Sci Explor 2003;17:389–407.

8 Caratero A, Courtade M, Bonnet L, Planel H, Caratero C: Effect of continuous gamma irradiation

at very low dose on the life span of mice. Gerontology 1998;44:272–276.

9 Kaiser J: Radiation dangerous even at low doses. Science 2005;309:233.

10 Rattan SIS: Applying hormesis in aging research and therapy. Hum Exp Toxicol 2001;20:

281–285.

11 Munck CV, Guyre PM, Holbrook NJ: Physiologic functions of glucocorticoids in stress and their

relation to pharmacological actions. Endocr Rev 1984;5:25–44.

12 Sabatino F, Masoro EJ, McMahan CA, Kuhn RW: An assessment of the role of the glucocorticoid

system in aging processes and in the action of food restriction. J Gerontol Biol Sci 1991;46:

B171–B179.

13 Armario A, Montero JL, Jolin T: Chronic food restriction and the circadian rhythms of pituitary-

adrenal hormones. Ann Nutr Metab 1990;31:81–87.

14 Stewart J, Meaney MJ, Aitken D, Jensen L, Kalant N: The effects of acute and life-long food

restriction on basal and stress-induced serum corticosterone levels in young and aged rats.

Endocrinology 1988;123:1934–1941.

15 Mendel CM: The free hormone hypothesis: a physiologically based mathematical model. Endocr

Rev 1989;10:232–274.

16 Han ES, Evans TR, Shu JH, Lee S, Nelson JF: Food restriction enhances endogeneous and

corticotropin-induced plasma elevations of free but not total corticosterone throughout life in rats.

J Gerontol Biol Sci 2001;56A:B391–B397.

17 Klebanov S, Shehab D, Stavinoha WB, Yongman S, Nelson JF: Hyperadrenocorticism attenuated

inflammation, and the life-prolonging action of food restriction in mice. J Gerontol Biol Sci

1995;50A:B78–B82.

18 Rattan SIS: Aging, anti-aging, and hormesis. Mech Ageing Dev 2004;125:285–289.

19 Toussaint O, Remacle J, Dierick J-F, Pascal T, Frippiat C, Royer V, Chainiaux F: Approach of evo-

lutionary theories of aging stress, senescence-like phenotypes, dietary restriction, and hormesis

from the viewpoint of far-from equilibrium thermodynamics. Mech Ageing Dev 2002;123:

937–946.

20 Heydari AR, Wu B, Takahashi R, Strong R, Richardson A: Expression of heat shock protein 70 is

altered by age and diet at the level of transcription. Mol Cell Biol 1993;13:2909–2918.

21 Duffy PH, Feuers FJ, Pipkin JL, Berg TF, Leakey JEA, Turturro A, Hart RW: The effect of dietary

restriction and aging on physiological response to drugs; in Hart RW, Neuman DA, Robertson RT

(eds): Dietary Restriction: Implications for the Design and Interpretation of Toxicity and

Carcinogenicity Studies. Washington, ILSI Press, 1995, pp 125–140.

22 Keenan KP, Ballam GC, Dixit R, Soper KA, Laroque P, Mattson BA, Adams SP, Coleman JB: The

effect of diet, overfeeding, and moderate dietary restriction on Sprague-Dawley rat survival, dis-

ease, and toxicology. J Nutr 1997;127(suppl):851S–856S.

23 Berg TF, Breen PJ, Feuers RJ, Oriaku ET, Chen FX, Hart RW: Acute toxicity of ganciclovir: effect

of dietary restriction and chronobiology. Food Chem Toxicol 1994;32:45–50.

24 Kirkwood TBL: Evolution of ageing. Nature 1977;270:301–304.

25 Maynard Smith J: Prolongation of life of Drosophila subobscura by brief exposure of adults to

high temperature. Nature 1958;181:496–497.

26 Khazaeli AA, Tatar M, Pletcher SD, Curtsinger JW: Heat-induced longevity extension in Drosophila.

I. Heat treatment, mortality, and thermotolerance. J Gerontol Biol Sci 1997;52A:B48–B52.

27 Shama S, Lai CY, Antoniazzi JM, Jiang JC, Jazwinski CM: Heat stress-induced life span exten-

sion in yeast. Exp Cell Res 1998;245:379–388.

Masoro 14

28 Lithgow GJ, White TM, Melov S, Johnson TE: Thermotolerance and extended life-span conferred by

single gene mutations and induced by thermal stress. Proc Natl Acad Sci USA 1995;92:7540–7544.

29 Yashin A, Cypser JR, Johnson TE, Michalski AI, Boyko SI, Novoseltsev VN: Ageing and survival

after different doses of heat shock: the results of analysis of data from stress experiments with the

nematode worm Caenorhabditis elegans. Mech Ageing Dev 2001;122:1477–1495.

30 Cypser JR, Johnson TE: Muliple stressors in Caenorhabditis elegans induce stress hormesis and

extended longevity. J Gerontol Biol Sci 2002;57:B109–B114.

31 Johnson TE, Lithgow GJ, Murakami S: Hypothesis: interventions that increase response to stress

offer the potential for effective life prolongations and increased health. J Gerontol Biol Sci

1996;51A:B392–B395.

32 Kapahi P, Boulton ME, Kirkwood TBL: Positive correlation between mammalian life span and

cellular resistance to stress. Free Radic Biol Med 1999;26:495–500.

33 Yu BP, Masoro EJ, McMahan CA: Nutritional influences on aging of Fischer 344 rats. I. Physical,

metabolic, and longevity characteristics. J Gerontol 1985;40:657–670.

34 Le Bourg E, Minois N, Bullens P, Baret P: A mild stress due to hypergravity exposure at young age

increases longevity in Drosophila melanogaster males. Biogerontology 2000;1:145–153.

35 Martin GM, Austad SN, Johnson TE: Genetic analysis of ageing: role of oxidative damage and

environmental stresses. Nat Genet 1996;13:25–34.

36 Lithgow GJ, Walker GA: Stress resistance as a determinant of C. elegans lifespan. Mech Ageing

Dev 2002;123:765–771.

37 Johnson TE, Cypser J, de Castro E, de Castro S, Henderson S, Murakami S, Rikke B, Tedesco P,

Link C: Gerontogenes mediate health and longevity in nematodes through increasing resistance to

environmental toxins and stressors. Exp Gerontol 2000;35:687–694.

38 Fabrizio P, Pozza F, Pletcher SD, Gendron CM, Longo VD: Regulation of longevity and stress

resistance by Sch9 in yeast. Science 2001;292:288–290.

39 Lin Y-J, Seroude L, Benzer S: Extended life-span and stress resistance in the Drosophila mutant

Methuselah. Science 1998;283:943–946.

40 Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Sinclair DA: Nicotinamide and PNC1 govern

lifespan extension by dietary restriction in Saccharomyces cerevisiae. Nature 2003;423:181–185.

41 Lamming DW, Latorre-Esteves M, Medvedik O, Wong SN, Tsang FA, Wang C, Lin S-J, Sinclair

DA: HST2 mediates SIR2-independent life-span extension by calorie restriction. Science

2005;309:1861–1864.

42 Masoro EJ: Role of sirtuin proteins in life extension by dietary restriction. Mech Ageing Dev

2004;125:591–594.

43 Rogina B, Helfand SL: Sir2 mediates longevity in the fly through a pathway related to calorie

restriction. Proc Natl Acad Sci USA 2004;101:15998–16003.

44 Tissenbaum HA, Guarente L: Increased dosage of a SIR-2 gene extends lifespan of

Caenorhabditis elegans. Nature 2001;410:227–230.

45 Lakowski B, Hekimi S: The genetics of dietary restriction in Caenorhabditis elegans. Proc Natl

Acad Sci USA 1998;95:13091–13096.

46 Fabrizio P, Gattazzo C, Battisella L, Wei M, Cheng C, McGrew L, Longo VD: Sir2 blocks extreme

life-span extension. Cell 2005;123:655–667.

47 Kaeberlein M, Powers RW III, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO, Kirkland KT,

Fields S, Kennedy BK: Regulation of yeast replicative life span by Tor and Sch9 in response to

nutrients. Science 2005;310:1193–1196.

48 Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S: Regulation of lifespan in Drosophila

by modulation of genes in the TOR signaling pathway. Curr Biol 2004;14:885–890.

49 Vellai T, Takacs K, Zhang V, Kovacs AL, Orosz L, Miller F: Influence of TOR kinase on lifespan

in C. elegans. Nature 2003;620:426.

50 Papaconstantinou J, Reisner PD, Liu L, Kuniger DT: Mechanisms of altered gene expression with

aging; in Schneider EL, Rowe JW (eds): Handbook of the Biology of Aging, ed 4. San Diego,

Academic Press, 1996, pp 150–183.

51 Aly KB, Pipkin JL, Hinson WG, Feuers RJ, Duffy PH, Lyn-Cook L, Hart R: Chronic dietary

restriction induces stress proteins in the hypothalamus of rats. Mech Ageing Dev 1994;76:11–23.


52 Pipkin JL, Hinson WG, Feuers RJ, Lyn-Cook LE, Burns ER, Duffy PH, Hart R, Casciano D: The

temporal relationship of synthesis and phosporylation in stress proteins 70 and 90 in aged caloric

restricted rats exposed to bleomycin. Aging Clin Exp Res 1994;6:125–132.

53 Heydari AR, Conrad CC, Richardson A: Expression of heat shock genes in hepatocytes is affected

by age and diet at the level of transcription. J Nutr 1995;125:410–418.

54 Moore SA, Lopez A, Richardson A, Pahlavani MS: Effect of age and dietary restriction on the

expression of heat shock protein 70 in rat alveolar macrophages. Mech Ageing Dev

1998;104:59–73.

55 Heydari A, You S, Takahashi R, Gutsmann A, Sarge KD, Richardson A: Effect of dietary restric-

tion on the expression of heat shock protein 70 and the activation of heat shock. Dev Genet

1996;18:114–124.

56 Tatar M, Khazaeli AA, Curtsinger JW: Chaperoning extended life. Nature 1997;390:30.

57 Garigan D, Hsu A-L, Fraser AG, Kamath J, Kenyon C: Genetic analysis of tissue aging in

Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics

2002;161:1101–1112.

58 Hsu AL, Murphy CT, Kenyon C: Regulation of aging and age-related disease by DAF-16 and heat-

shock factor. Science 2003;300:1142–1145.

59 Yokoyama K, Fukumoto K, Murakami T, Harada S, Hosono R, Wadhwa R, Mitsui Y, Ohkuma S:

Extended longevity of Caenorhabditis elegans by knocking in extra copies of hsp70F, a homolog

of mot-2 (mortalin)/mthsp70/Grp 75. FEBS Lett 2002;516:53–57.

60 Morley JF, Morimoto RI: Regulation of longevity in Caenorhabditis elegans by heat shock factor

and molecular chaperones. Mol Biol Cell 2004;15:657–664.

61 Walker GA, Lithgow GJ: Lifespan extension of C. elegans by a molecular chaperone dependent

upon insulin-like signals. Aging Cell 2003;2:131–140.

62 Guo ZM, Heydari A, Richardson A: Nucleotide excision repair of actively transcribed versus non-

transcribed DNA in rat hepatocytes: effect of age and dietary restriction. Exp Cell Res

1998;245:228–238.

63 Guo ZM, Van Remmen H, Wu WT, Richardson A: Effect of cAMP-induced transcription on the

repair of the phosphoenolpyruvate carboxykinase gene by hepatocytes isolated from young and

old rats. Mutat Res 1998;409:37–48.

64 Lewis SE, Goldspink DF, Phillips JG, Merry BJ, Holehan AM: The effects of aging and chronic

dietary restriction on whole body growth and protein turnover in the rat. Exp Gerontol

1985;20:253–263.

65 Goto S, Takahashi R, Araki S, Nakamoto H: Dietary restriction initiated in late adulthood can

reverse age-related alterations of protein and protein metabolism. Ann NY Acad Sci

2002;959:50–56.

66 Youngman LD, Park JY, Ames BN: Protein oxidation associated with aging is reduced by dietary

restriction of protein or calories. Proc Natl Acad Sci USA 1991;89:9112–9116.

67 Chen LH, Snyder DL: Effect of dietary restriction and germ-free environment on glutathione-

related enzymes in Lobund-Wistar rats. Arch Gerontol Geriatr 1992;14:17–26.

68 Chung MH, Kin HJ, Nishimura S, Yu BP: Protection of DNA damage by dietary restriction. Free

Radic Biol Med 1992;12:523–525.

69 Sohal RS, Agarwal S, Candas M, Forster M, Lal H: Effect of age and dietary restriction on

DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76:

215–224.

70 Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H: Oxidative damage, mitochondrial oxidant gener-

ation, and antioxidant defenses during aging and in response to food restriction in the mouse.

Mech Ageing Dev 1994;74:121–133.

71 Dubey A, Forster MJ, Lal H, Sohal RS: Effect of age and caloric intake on protein oxidation in dif-

ferent brain regions and on behavioral functions of the mouse. Arch Biochem Biophys 1996;333:

189–197.

72 Aksenova MV, Aksenov MY, Carney JM, Rutterfield DA: Protein oxidation and enzyme activity

decline in old Brown Norway rats are reduced by dietary restriction. Mech Ageing Dev

1998;100:157–168.

Masoro 16

73 Leakey JE, Chen S, Manjgaladze M, Turturro A, Duffy PH, Pipkin JL, Hart RW: Role of gluco-

corticoids and ‘caloric stress’ in modulating the effects of dietary restriction in rodents. Ann NY

Acad Sci 1994;719:171–194.

74 Pashko LL, Schwartz AG: Reversal of food restriction induced inhibition of mouse skin tumor

promotion by adrenalectomy. Carcinogenesis 1992;10:1925–1928.

75 Schwartz AG, Pashko LL: Role of adrenocortical steroids in mediating cancer-prevention and age-

retarding effects of food restriction in laboratory rodents. J Gerontol Biol Sci 1994;49:B37–B41.

76 Birt DF, Yatkins A, Duysen E: Glucocorticoid mediation of dietary energy restriction inhibition of

mouse skin carcinogenesis. J Nutr 1999;129(suppl):571S–586S.

77 Neafsey PJ: Longevity hormesis: a review. Mech Ageing Dev 1990;51:1–31.

78 Pletcher SD, Khaezaeli AA, Curtsinger JA, Why do life spans differ? Partitioning mean longevity

differences in terms of age-specific mortality parameters. J Gerontol Biol Sci 2000;55A:

B381–B389.

79 Pugh TD, Oberly TD, Weindruch R: Caloric restriction but not dehydroepiandrosterone sulfate

increase lifespan and lifetime cancer incidence in mice. Cancer Res 1999;59:1642–1648.

80 Mair W, Goymer P, Pletcher SD, Partridge L: Demography of dietary restriction and death in

Drosophila. Science 2003;301:1731–1733.

81 Driver C: A further comment on why the gompertzian plot does not measure aging. Biogerontology

2003;4:325–327.

82 Masoro EJ: Caloric restriction and aging: controversial issues. J Gerontol A Biol Sci Med Sci

2006;61:14–19.

83 Harrison DE, Archer JR: Effects of food restriction on aging mice. J Nutr 1987;117:376–382.

84 Reiser K, McGee C, Rucker R, McDonald R: Effects of aging and dietary restriction on extracel-

lular matrix biosynthesis in a model of injury repair in rats. J Gerontol Biol Sci 1995;50A:

B40–B47.

85 Reed MJ, Penn PE, Li Y, Birnbaum R, Vernon RB, Johnson TS, Pendergrass WR, Sage EH, Abrass

IB, Wolf NS: Enhanced cell proliferation and biosynthesis mediate improved wound repair in

refed, calorie-restricted mice. Mech Ageing Dev 1996;89:21–41.

86 Campbell BA, Richardson R: Effect of chronic undernutrition on susceptibility to cold stress in

young adult and aged rats. Mech Ageing Dev 1988;44:193–202.

87 Gerbase-Delima M, Liu RK, Cheney KE, Mickey R, Walford RL: Immune function and survival

in the long-lived mouse strain subjected to undernutrition. Gerontologia 1975;21:184–193.

88 Pahlavani MA: Intervention in the aging of the immune system: influence of dietary restriction,

dehydroepiandrosterone, melatonin, and exercise. Age 1998;21:153–173.

89 Roecker EB, Kemnitz JW, Ershler WB, Weindruch R: Reduced immune responses in rhesus mon-

keys subjected to dietary restriction. J Gerontol Biol Sci 1996;51A:B276–B279.

90 Weindruch R, Lane MA, Ingram DK, Ershler WB, Roth GS: Dietary restriction in rhesus mon-

keys: lymphopenia and reduced mitogen-induced proliferation in peripheral blood mononuclear

cells. Aging Clin Exp Res 1997;9:304–308.

91 Sun D, Muthukumar AR, Lawrence RA, Fernandes G: Effects of calorie restriction on polymicro-

bial peritonitis induced by cecum ligation and puncture in young C57BL/6 mice. Clin Diag Lab

Immunol 2001;8:1003–1008.

92 Effros RB, Walford RL, Weindruch R, Mitcheltree C: Influence of dietary restriction on immunity

to influenza in aged mice. J Gerontol Biol Sci 1991;46:B142–B147.

93 Gardner EM: Caloric restriction decreases survival of aged mice in response to primary influenza

infection. J Gerontol Biol Sci 2005;60A:688–694.

94 Sapolsky RM: Glucocorticoids, stress, and their adverse neurological effects: relevance to aging.

Exp Gerontol 1999;35:721–732.

95 Calebrese EJ: Changing view of dose-response, a personal account of the history and current sta-

tus. Mutat Res 2002;511:181–189.

96 Michalski AI, Johnson TE, Cypser JR, Yoshin AI: Heating stress patterns in Caenorhabditis ele-

gans longevity and survivorship. Biogerontology 2001;2:35–44.

97 Le Bourg E, Valenti P, Lucchetta P, Payre F: Effects of mild heat shock at young age on aging and

longevity of Drosophila melanogaster. Biogerontology 2001;2:155–164.


98 McEwen BS: Protective and damaging effects of stress mediators. N Engl J Med 1998;338:

171–179.

99 Gredilla P, Sanz A, Lopez-Torres M, Barja G: Caloric restriction decreases mitochondial free rad-

ical generation at complex 1 and lowers oxidative damage to mitochondrial DNA. FASEB J

2001;15:1589–1591.

100 Merry BJ: Oxidative stress and mitochondrial function with aging – The effect of calorie restric-

tion. Aging Cell 2004;3:7–12.

101 Masoro EJ, McCarter RJM, Katz MS, McMahan CA: Dietary restriction alters the characteristics

of glucose fuel use. J Gerontol Biol Sci 1992;47:B202–B208.

Edward J. Masoro, PhD

Professor emeritus of Physiology

211/2 Legare Street

Charleston, SC 29401 (USA)

Tel. �1 843 853 3445, E-Mail [email protected]



Metabolic Reprogramming inDietary Restriction

Rozalyn M. Anderson, Richard Weindruch

Department of Medicine, University of Wisconsin, Madison, Wisc., USA

AbstractIt is widely accepted that energy intake restriction without essential nutrient deficiency

delays the onset of aging and extends life span. The mechanism underlying this phenomenon

is still unknown though a number of different, nonmutually exclusive explanations have been

proposed. In each of these, different facets of physiology play the more significant role in the

mechanism of aging retardation. Some examples include the altered lipid composition

model, the immune response model and models describing changes in endocrine function. In

this paper we propose the hypothesis that metabolic reprogramming is the key event in the

mechanism of dietary restriction, and the physiological effects at the cellular, tissue and

organismal level may be understood in terms of this initial event.


Dietary restriction (DR) is the most successful intervention tested to date

in mammals which greatly extends maximum life span and keeps animals

‘younger longer’ [1–3]. Consequently, any hypothesis about the etiology of

aging must reconcile the effects of DR on aging. With increased knowledge of

the mechanism of DR, we stand to gain a considerable insight into the process

of aging.

We propose that a change in the regulation of energy metabolism in

response to DR is the primary step in the retardation of aging (fig. 1). First we

describe the evidence in support of metabolic reprogramming, a switch to an

altered metabolic state, by DR in mice. Next we consider evidence for metabolic

shifts in other model organisms where life span is extended by DR or by genetic

manipulation. Then we focus on changes in mitochondrial energy metabolism

with age and DR in mammals. Next we will explore the effects of altered mito-

chondrial function in the context of reactive oxygen species (ROS) generation

and oxidative stress. Finally we describe the metabolic and morphological

Metabolic Reprogramming in Dietary Restriction 19

changes in white adipose tissue that we believe are a result of altered mitochon-

drial function. We propose that the activation of adipose tissue through meta-

bolic reprogramming is critical to the mechanism of DR and that it leads to the

changes in the animal physiology that are described in the models indicated

above.

Metabolic Reprogramming in Tissues from

Dietary-Restricted Animals

The inverse linear relationship between calorie intake and life span in mice

[4] suggests that genes central to energy metabolism may be critical in the

underlying mechanism of DR in mammals. We have examined transcriptional

changes with age and with DR in multiple tissues and find shifts in the expres-

sion of genes encoding proteins involved in energy metabolism to be a promi-

nent feature of DR. We propose that these shifts directly contribute to the

longevity of the animal. Our studies have primarily focused on postmitotic tis-

sues because these are most vulnerable to the effects of age. Analysis of the

Master

regulators

Effectors

Altered energy

metabolism

LongevityMorbidity and

mortality

Normal

metabolism

Normal aging

cellular damage

dysfunction and loss

Control DR

Reduced

rate of aging

Fig. 1. Master regulators respond to DR and induce shifts in metabolism in the

restricted organism. These regulators may include the transcriptional coactivator PGC-1�

and members of the nuclear receptor family PPAR-� and PPAR-�. Effectors that respond to

the altered metabolic state are involved in tissue-specific changes that ultimately lead to

changes at the organismal level, delaying aging and promoting longevity.

Anderson/Weindruch 20

transcriptional response to DR in these tissues is likely to reveal clues to the

mechanism of aging retardation.

In skeletal muscle, a decrease in metabolic activity with age is suggested

through a reduction in the expression of genes involved in energy metabolism

[5]. This extends to genes associated with mitochondrial function as well as

genes involved in glycolysis and glycogen synthesis, and large reductions in

expression were also observed for genes involved in fatty acid biosynthesis. We

observed a striking attenuation of these age-related changes in gene expression

in skeletal muscle from DR animals compared to age-matched controls. In par-

ticular, we observed a transcriptional shift toward increased energy metabolism

and increased biosynthesis. The expression of genes involved in glycolysis and

gluconeogenesis was increased, as was the expression of transcripts associated

with fatty acid metabolism. The increased expression of peroxisome proliferator-

activated receptor � (PPAR-�) may contribute to the increased insulin sensitivity

in skeletal muscle of the DR animals [6].

In the heart, lipid metabolism and fatty acid oxidation (FAO) are the major

energy source in adults [7]. In old age, genes involved in lipid transport, lipoly-

sis and FAO are downregulated and genes involved in carbohydrate metabolism

are upregulated, resulting in an overall shift in metabolism [8]. These metabolic

alterations, which are also observed in pathological heart conditions [9], are

completely or partially prevented by DR [8]. In addition, we observed a signif-

icant change with DR in the expression of key genes that are not affected with

age. It is important to emphasize that this latter type of DR effect is distinct

from the prevention of age-associated transcriptional changes. Genes that shift

in expression with DR but do not change with age may provide clues to the

mechanism of aging retardation by DR and may lead to the identification of pri-

mary regulators. In the heart, this group includes many nuclear genes encoding

components of the electron transport system (ETS) that show a striking and

coordinated upregulation with DR [unpubl. data].

In our earlier experiments we sought to characterize the tissue-specific

transcriptional changes with age and examine the effect of DR. We identified

two groups of genes that are regulated by DR: one group is regulated by age at

the transcriptional level and DR either partially or completely compensates for

the age-induced changes; the other group is regulated specifically by DR and

does not show age-dependent changes in gene expression. Clearly the genes

that are regulated specifically by DR and not affected with age are reasonable

candidates in the mechanism of life span extension. The potential involvement

of genes that are regulated by age in the mechanism of DR is less easily

deduced. In analyzing these data it became clear that in order to dissect out the

DR-specific transcriptional changes we would be better served looking at

young to mid-age mice where the age-related changes in transcription would be


less significant. Specifically, we examined the transcriptional changes with

fasting, short-term DR (23 days) or long-term DR (9 months) in epididymal

white adipose tissue from 10-month-old mice [10]. Here again we identified

metabolic reprogramming as a prominent feature. White adipose tissue is

remarkably refractory to both fasting and short-term DR but undergoes a dra-

matic transformation in response to long-term DR. This is in contrast to similar

experiments in the liver where many of the DR-induced changes were observed

with short-term DR [11].

In white adipose tissue, long-term DR increases the expression of genes

involved in the glycolytic pathway, the lipolytic pathway, amino acid metabo-

lism and mitochondrial energy metabolism in young mice [10] suggesting an

activation of energy metabolism. Again, these shifts in gene expression are not

compensatory in the delay of aging and may be involved in the mechanism of

aging retardation. One of the more striking findings was the concerted increase

in expression of 26 nuclear genes encoding mitochondrial ETS proteins. We

also identified a dramatic decrease in the expression of genes encoding inflam-

matory molecules (56 genes); these alterations may play an important role in

the protection against inflammation derived from white adipose tissue and in

life span extension by DR [12].

The transcriptional shifts observed in each of these tissues are indicative of

metabolic reprogramming which we believe is a key component of the mecha-

nism of aging retardation by DR (fig. 1). The coordinated increase in expres-

sion of genes encoding components of mitochondrial ETS in both heart and

adipose tissue is striking. Several aging studies in yeast, worms and flies sup-

port a role for metabolic regulation in longevity. We discuss the evidence below,

with a particular focus on mitochondrial energy metabolism. Studies in trans-

genic and wild-type mice further support our hypothesis that shifts in energy

metabolism can affect a broad spectrum of phenotypes and support our pro-

posal that metabolic shifts are key elements in the mechanism of DR.

Evidence for Metabolic Reprogramming in Organisms

with Extended Life Span

In Saccharomyces cerevisiae, life span extension by DR induces an active

regulated response [13] and there is a shift in metabolism in the restricted

organisms toward increased respiration [14]. The increase in respiration is asso-

ciated with a decrease in ROS production and this is thought to be indicative of

increased mitochondrial uncoupling [15]. Although yeasts are facultative anaer-

obes, the influence of mitochondrial perturbations on life span under aerobic

conditions indicates that manipulation of mitochondrial function directly


influences longevity. The retrograde response pathway in yeast provides a

mechanism for communication of changes in mitochondrial function to the

nucleus [16]. This pathway has been linked to adaptive regulation of metabo-

lism and the stress response [17] and its activation induces the expression of

cytoplasmic, mitochondrial and peroxisomal metabolic genes [18] and life span

extension [19]. These studies demonstrate that changes in mitochondrial func-

tion are transmitted to the nucleus and induce pathways that will provide a com-

pensatory metabolic change. The fact that changes in mitochondrial efficiency

can and do exert large-scale changes in gene expression and metabolic regula-

tion supports the idea that a program as complex as life span extension by DR

could conceivably be initiated in such a manner. Interestingly, mitochondrial

signaling seems to converge with the nutrient-sensing TOR (target of

rapamycin) pathway in yeast, where TOR inhibition activates the transcription

factors involved in the retrograde response [20]. There is evidence that this

cross talk may be conserved in mammalian systems where mitochondrial defi-

ciency stabilizes the interaction between TOR and the inhibitory regulatory

associated protein of mTOR (Raptor) protein [21]. These findings demonstrate

that there is an open line of communication between mitochondrial efficiency

and the nutrient-sensing TOR pathway, allowing for an integration of signaling

pathways and a coordinated metabolic response. Inhibition of TOR signaling

extends life span in yeast [22, 23], worms [24] and flies [25]. Reduced TOR sig-

naling in mice by knockout of the TOR effector S6K1 increases FAO and nega-

tively regulates insulin signaling [26]. It will be interesting to see what role

TOR signaling plays in life span extension by DR.

In Caenorhabditis elegans, a systematic RNA interference screen to iden-

tify gene alterations that affect life span has uncovered a complex relationship

between mitochondrial function and longevity [27], and inhibition of mitochon-

drial function early in development extends life span in this organism [28].

More recent studies in worms have clearly identified a role for metabolic regu-

lators in longevity [29, 30]. These studies involve RNA interference knockdown

of specific gene products starting from the egg hatching stage. This is a situa-

tion where the animal must survive in the absence or depletion of the requisite

pathway and while novel factors that influence life span have been discovered,

no information about any metabolic compensatory mechanisms in response to

this inhibition in the targeted animal has been gleaned. It is possible that the

inhibition of mitochondrial oxidative phosphorylation at an early stage induces

alternative energy metabolism pathways in these animals, resetting the

longevity of the animal [31]. This may explain why inhibiting respiratory chain

components in the adult animal does not affect life span [28].

DR does not appear to alter the metabolic rate in C. elegans [32] and

genetic analysis indicates that life span extension by DR is independent of


DAF-16, the forkhead transcription factor involved in the insulin/IGF pathway

[33]. A number of components downstream of DAF-16 have been shown to

influence metabolism and life span. These include the DAF-12 nuclear hor-

mone receptor [34] and its coregulator DIN-1 [35], and the DAF-15 regulator of

TOR (homologue of mammalian Raptor) [36]. These factors conceivably repre-

sent points of convergence of signaling by glucose/insulin, lipophilic factors

and amino acid limitation-sensing factors. Such cross talk between pathways

would permit regulation of the appropriate coordinated metabolic response to

the nutritional status of the animal.

Several studies in Drosophila have also demonstrated that life span may

be extended by genes involved in metabolic regulation. Flies with a mutation

in INDY (‘I’m not dead yet’), a Krebs cycle intermediate transporter, are long-

lived [37]. These animals do not show a change in metabolic rate [38], remi-

niscent of the results of some DR studies [39], although the presumed changes

in metabolism have yet to be characterized. Disruption of ecdysone steroid

hormone signaling in Drosophila also extends life span [40]. In the absence of

this hormone, the ecdysone receptor complex interacts with transcriptional

repressors Rpd3 and Sin3 [41]. What is interesting about this, from our point

of view, is that reduction of Rpd3 extends life span in flies [42] and reduction

in Sin3 causes upregulation of genes involved in the oxidative metabolism of

fatty acid to acetyl-CoA and genes involved in mitochondrial oxidative phos-

phorylation [43]. The similarity between the ecdysone receptor complex and

the nuclear hormone complexes in mammalian systems has led to speculation

that the mammalian counterparts may also participate in the regulation of

aging [44].

As in worms, life span is extended in flies by reduction of insulin signaling

[45, 46]. Here again the mechanism of life span extension in insulin-signaling-

deficient animals is not thought to be the same as that for DR, but the evidence

suggests that there are common elements [47], perhaps downstream of the fork-

head transcription factor dFOXO. The reduction of blood glucose and enhanced

insulin sensitivity in DR mammals hints at the involvement of altered insulin

signaling in the mechanism of aging retardation. We do not dispute this but sug-

gest that the changes in insulin signaling and sensitivity are secondary to the

metabolic shift in these animals. Studies on long-lived transgenic mouse mod-

els indicate that the effects of DR are not fully explained by reduced growth

hormone/IGF-1 axis activity [48–51].

Overexpression dFOXO in the fat body of flies extends life span [52, 53].

Forkhead transcription factors are downstream of the insulin signaling pathway

and in worms regulate metabolism and the stress response [54, 55]. These find-

ings point to the importance of the fat body in whole-body regulation of metab-

olism and longevity and indicate that secreted factors are involved in the


mechanism of life span extension in these transgenic animals. Even though

there is evidence to suggest that DR acts independently of FOXO transcription

factors [33], factors downstream of FOXO appear to be common to both

insulin/IGF longevity pathways and life span extension by DR, in particular

members of the nuclear receptor family and possibly factors influencing the

TOR nutrient signaling pathway. There is now mounting evidence that signals

from white adipose tissue in mammals can influence whole-body metabolism

and life span. These quantitative and qualitative changes in adipose tissue may

be critically involved in the mechanism of aging retardation by DR (see below).

Taken together, the evidence presented here confirms that life span may be

influenced by regulation of metabolism, that mitochondrial efficiency influ-

ences the metabolic state and that a communication network exists to coordi-

nate changes in mitochondrial function with regulation of metabolism. These

data support the concept that metabolic reprogramming could be an initial event

in the mechanism of life span extension by DR and that many genetic manipu-

lations that extend life span may also be viewed in this way.

Mitochondria in Aging and Dietary Restriction

Mitochondria are the key organelle in substrate utilization and energy pro-

duction. DR directly affects mitochondrial function, increasing the expression

of components of the ETS as well as genes involved in fatty acid transport and

�-oxidation [8, 10] and there is a clear reduction in the production of ROS

[56–61]. DR enhances mitochondrial oxidative capacity in liver and skeletal

muscle in rats [62]. ROS are generated continuously as part of normal mito-

chondrial function [63]. One inbuilt mechanism to combat ROS accumulation

is through uncoupling of the mitochondrial membrane potential by proton leak

[64]. However, studies with mitochondria isolated from liver and skeletal mus-

cle from age-matched control and restricted rats demonstrate that ROS produc-

tion in mitochondria is reduced even though there is no change in proton leak

[65, 66]. These studies touch on the role of mitochondrial uncoupling proteins

UCP2 and UCP3 and raise questions as to how mitochondrial function is

altered by DR. The role of ROS in aging and DR will be discussed in the fol-

lowing section.

In heart, skeletal muscle and white adipose tissue, expression of the mito-

chondrial uncoupling protein UCP3 is elevated by DR [8, 10, 66]. Studies in

mice overexpressing UCP3 support a role for this protein in energy balance and

lipid metabolism [67]. Although the physiological role of UCP3 is controversial

[68–70], increased UCP3 expression augments FAO and decreases ROS pro-

duction without uncoupling respiration [71] and enhances the capacity for fatty


acid transport and FAO in skeletal muscle [72]. These data argue that UCP3 is

not merely a mitochondrial uncoupling protein involved in the regulation of the

proton leak. Elevated free fatty acids induce UCP3 expression consistent with

UCP3 playing a role in the use of free fatty acid as a fuel [73]. In one model

[68], UCP3 works with the carnitine palmitoyl transferases, CPT-1 and CPT-2,

to cycle fatty acid anions through the mitochondria. The increase in CPT-1

expression in restricted tissues supports a role for UCP3 in fatty acid transport,

providing increased capacity for fatty acid metabolism.

Studies in type 2 diabetes have implicated mitochondrial dysfunction in

this disorder [74, 75] providing a link between mitochondrial function and

whole-body endocrine signaling. Mitochondrial abnormalities lead to neuro-

muscular disorders known as mitochondrial myopathies and encephalomy-

opathies [76], as well as heart disease [77]. Mitochondrial function declines

with age in humans [78]; however, the extent of the contribution of mitochondr-

ial function to the onset of age-related pathologies like diabetes and heart dis-

ease is not yet clear. Tissue-specific disruption of the respiratory chain in

mouse hearts causes a switch from fatty acid to glucose metabolism that pre-

cedes the inevitable heart failure in these animals [79]. This demonstrates that

changes in mitochondrial function are sufficient to implement large-scale meta-

bolic changes in mice in vivo.

Recent studies have demonstrated that mice with a mitochondrial mutator

phenotype develop several age-associated disorders providing strong support

for a model in which mitochondrial function is a determinant of aging [80, 81].

Finally, analyses of individual mice have revealed a positive association

between metabolic intensity and life span [82]. One issue that complicates stud-

ies involving isolated mitochondria is that the experimental setup measures the

maximum capacity of the isolated mitochondria in an environment that is

experimentally determined, but cannot reveal the actual in vivo differences in

mitochondrial function where the intracellular environment may not be equiva-

lent in the organism as a whole. Nevertheless, together these data support a key

role for mitochondrial energy metabolism in the control of life span.

Stress, Oxidative Stress and Longevity

Mice [83], rats [84] and monkeys [85] subjected to DR demonstrate

decreased body temperatures indicative of altered energy balance. Reduction in

oxidative stress is a feature of DR in rodents [57, 86] and may be a direct result

of this metabolic reprogramming. DR attenuates the age-associated increase in

rates of mitochondrial ROS generation in multiple tissues and reduces the

accrual of oxidative damage [58–61, 87, 88]. Mitochondrial function is


preserved with age in DR animals, and the loss of mitochondrial membrane flu-

idity is delayed [89] compared to control animals. Reduction of mitochondrial

H2O2 production and oxidative damage to mtDNA in rat gastrocnemius muscle

with DR has been described [90] and we have reported that DR in monkeys

lowers oxidative damage in skeletal muscle [91]. More recently, the role of ROS

has broadened to encompass the control of normal cellular functions (e.g. tran-

scriptional control, signal transduction) and cell death pathways [92]. These

data reveal a potential role of ROS in aging that is independent of damage

induction. It has been proposed that DR, by lowering ROS, attenuates age-

associated increases in the binding activities of redox-sensitive transcriptional

factors (e.g. HIF-1, NF-�B, AP-1) [93]. These factors may be important in the

mechanism of aging retardation by DR where reduced ROS production pre-

vents these signaling molecules from implementing the changes we see in the

transcriptional profiles of aging animals.

In the course of our analysis of mouse microarray databases, a number of

interesting candidates were identified including PPAR coactivator 1a (PGC-1�,

and the redox-sensitive transcription factors HIF-1� and NF-�B. Surprisingly,

RT-PCR analysis demonstrated that DR has little effect on genes from the sir-

tuin and forkhead transcription factor families, genes that have been associated

with longevity in lower organisms [unpubl. data]. Instead, SIRT1 and FOXO3

are regulated post-transcriptionally by DR [94] [unpubl. data]. SIRT1 has sub-

sequently been associated with DR in cell culture models [94] and activation of

SIRT1 is thought to be a key feature in the mechanism of DR, although this has

yet to be conclusively shown in mice. FOXO3 is a homologue of the worm

longevity factor DAF-16, a component of the insulin signaling pathway.

FOXO3 has been linked to cell survival and the stress response in mice and is

associated with both SIRT1 and p53 [95, 96].

The regulation and activation of factors associated with the stress response

has led us to ask if other elements associated with the stress response might also

play a role in DR’s action. We performed a screen to identify kinases activated

by DR in the mouse heart. We used tissue from 10-month-old animals to elimi-

nate the influence of age-dependent changes. Interestingly, we identified a

number of kinases that are regulated by DR both in terms of total protein levels

and degree of modification. Among these are JNK and GSK3�, which are

respectively regulated by DR in multiple tissues [unpubl. data].

JNK signaling enhances resistance to oxidative stress and extends life span

in worms and flies [97, 98]. In mice, JNK plays a role in insulin signaling and

obesity [99, 100], and affects insulin resistance in the liver and insulin produc-

tion in the pancreas [101]. Factors downstream of JNK include FOXO [97, 98],

which is required for JNK-dependent life span extension in worms and flies, and

PPAR-� [102]. Interestingly, activation of JNK under conditions of oxidative


stress is initiated in the mitochondria [103] suggesting that there is direct com-

munication between this organelle and effectors of the stress response. These

findings prompt further investigation into a role for JNK in the mechanism of

life span extension by DR. It is noted that JNK-dependent life span extension in

worms requires DAF-16/FOXO [98] and that life span extension by DR does not

[33]; however, constitutive activation of JNK is not a genetic mimic of DR.

Regulation of JNK by DR potentially provides a link between stress resistance

pathways and longevity, perhaps by influencing factors downstream of FOXO.

The mitogen-activated protein kinase p38 is responsive to numerous stim-

uli, including environmental stress and cytokine signaling [104]. Activation of

p38 increases insulin sensitivity in skeletal muscle in a manner that is indepen-

dent of contractile induced insulin sensitivity [105]. p38 activates the transcrip-

tional coactivator PGC-1� by phosphorylation, thereby regulating the induction

of mitochondrial respiration in muscle [106, 107] where PGC-1� plays a role in

fiber type switching [108]. The yeast homologue of GSK3� is involved in nutri-

ent sensing and the stress response [109]. In mammals, GSK3� is a negative

regulator of JNK [110] and is involved in insulin sensitivity in skeletal muscle

[111]. Interestingly both JNK and p38 are activated by ROS signaling from the

mitochondria [112]. It is unclear how kinases usually associated with the stress

response are activated by DR; however, longevity and stress resistance have

been linked in most genetic studies performed to date.

The Role of Adipose Tissue in Aging Retardation by

Dietary Restriction

Recent studies have highlighted the importance of white adipose tissue in

overall metabolic regulation and data from our laboratory and others suggest

that the changes in white adipose tissue observed in animals on DR are of par-

ticular significance. In mice, long-term DR induces morphological and tran-

scriptional alterations. The mass of epididymal white adipose tissue is reduced

by 75%, which appears to be due to a reduction in cell size [10]. DR suppresses

the expression of over 50 genes in inflammation and promotes structural

remodeling of the cytoskeleton, extracellular matrix and vasculature [12]. It is

probable that reductions in systemic inflammatory tone caused by DR may

underlie its ability to oppose a broad spectrum of age-associated diseases

including cancers and cardiovascular disease. We contend that a key conse-

quence of the metabolic reprogramming induced by DR is the alteration in adi-

pose tissue physiology and metabolism.

Aging is associated with alterations in body fat distribution, obesity and

insulin resistance [113, 114]. High levels of leptin are observed with obesity in


humans and rodents [115, 116]. DR reduces plasma insulin and leptin levels

[117, 118], and opposes the development of age-related insulin and leptin resis-

tance [119, 120]. Transgenic mice lacking the insulin receptor in adipose tissue

have reduced adiposity and display a modestly extended longevity compared to

DR [121]. These data argue that disruption of IGF signaling in adipose tissue

alone is sufficient to extend life span and mirrors the experiments in worms and

flies where fat-body-specific knockdown or overexpression of components of

the insulin signaling pathway affect life span [52, 53, 122].

The concept of adipose tissue as an endocrine organ has come into focus

recently [123]. Elevated serum levels of adipose tissue secretory products have

been associated with numerous pathologies including cardiovascular disease,

insulin resistance and diabetes. Resistin and adiponectin are adipocyte secre-

tory proteins that negatively and positively regulate insulin sensitivity, respec-

tively [124–126]. Resistin expression was upregulated by DR [10] and

adiponectin was not significantly altered. While the significance of the DR-

induced changes is not yet clear, the fact that adipocyte-derived signaling mol-

ecules are directly affected by DR lends support to the idea that changes in

adipose tissue by DR can be transmitted throughout the organism.

DR-induced transcriptional alterations in white adipose tissue included

increased expression of genes involved in adipocyte differentiation. Both

PPAR-� and SIRT1 have previously been implicated in this process [127] but it

is as yet unclear if either is playing a role in DR-induced changes observed in

white adipose tissue. Histological examination of white adipose tissue from

mice on DR confirmed the presence of multilocular adipocytes which may rep-

resent an intermediate phenotype between white and brown adipocytes [10].

The metabolic shifts observed are consistent with this, including the increased

expression of the �3-adrenergic receptor and UCP3. In white adipose tissue,

activation of the �-adrenergic receptors leads to mobilization of fat stores and

regulates the release of several adipokines [128]. In brown fat, �-adrenergic

receptor activation leads to increased expression of the thermogenic uncoupler

UCP1 via p38 and the transcriptional coactivator PGC-1� [129]. Ordinarily,

PGC-1� protein levels are barely detectable in white adipose tissue but are

increased in white adipose tissue from DR animals [unpubl. data]. The increase

in PGC-1� may be critical in the activation of adipose tissue by DR.

Adenovirus-driven expression of PGC-1� increased the expression of ETS

components and FAO enzymes in human adipocytes, and transcription profil-

ing indicated a metabolic activation of the fat cells [130]. Adenovirus-induced

hyperleptinemia reduces fat stores in normal rats and increased the capacity for

fat oxidation [131]. In these animals, expression of PGC-1� was dramatically

increased, as was the expression of gene targets of PGC-1�, and electron

microscopy revealed changes in mitochondrial number and morphology. These


data describe a striking similarity between the effect of DR and the effect of

upregulation of PGC-1� on adipose tissue.

We believe that the changes observed in white adipose tissue are funda-

mental to the mechanism of life span extension by DR. Age-related changes in

adiposity correlate with systemic oxidative stress in humans and mice, and in

cultured adipocytes, elevation of fatty acids increased oxidative stress and

caused dysregulated production of adipokines [132]. The pharmacological

induction of �-oxidation is currently being explored as a treatment for obesity

and diabetes [133]; both of these disorders are prevented by DR. We suggest

that the DR-induced shift in metabolism in white adipose tissue provides an

increased capacity for FAO and permits the mobilization of fat stores without

increasing oxidative damage through altered mitochondrial function and the

induction of UCP3. The activation of white adipose tissue in this manner influ-

ences whole-body physiology in a manner that promotes longevity: in our

model we predict that changes in levels of adipokines and other adipose secre-

tory factors systemically influence metabolism, endrocine and immune func-

tion and that quantitative and qualitative changes in serum lipids affect nuclear

receptor signaling in multiple tissues and influence lipid composition through-

out the organism.

PGC-1�� is a Candidate Factor in the Mechanism

of Aging Retardation by Dietary Restriction

We have presented evidence that DR induces metabolic shifts in multiple

tissues and that the influence of metabolism on longevity is conserved across

species. Based on these observations and because mitochondrial function has

been linked to aging and life span extension by DR [134], we examined our

heart microarray data set and looked for regulators of mitochondrial function as

potential effectors of DR in mice. PGC-1� is a critical transcriptional coactiva-

tor of mitochondrial function that is responsive to changes in energy demands

[135, 136]. It induces mitochondrial biogenesis and the expression of genes

involved in multiple mitochondrial pathways. Overexpression of PGC-1� stim-

ulates the mitochondrial antioxidant defense system in vascular endothelial

cells [137]. PGC-1� has been associated with glucose regulation, the insulin

signaling pathway and has been implicated in diabetes [138, 139] and obesity

[140], conditions that are prevented by DR.

Microarray analysis demonstrates that expression of PGC-1� is increased

in hearts from DR mice and that there is a coordinated increase in expression of

targets of PGC-1� activity [unpubl. data]. In both heart [8] and adipose tissue

[10], we observe a clear trend of upregulation of nuclear genes encoding


components of the ETS, many of which are targets of PGC-1�. Expression of

PGC-1� is also elevated in epididymal white adipose tissue of young/mid-age

DR animals [unpubl. data], indicating that induction of PGC-1� is part of a reg-

ulated metabolic response to DR.

Apart from its role in mitochondrial regulation, PGC-1� acts as a tran-

scriptional coactivator of the PPAR nuclear receptor family. The PPARs have

been linked to obesity and metabolic regulation and play a central role in the

cross talk between glucose and lipid homeostasis [141]. Metabolic integration

of FAO, carbohydrate metabolism, energy uncoupling and whole-body insulin

sensitivity is attained through the coordinated activity of PPAR-�, PPAR-�

and PPAR-� in adipose and liver tissues where PGC-1� levels are elevated by

DR. Transcriptional analysis of the effect of DR in wild-type and PPAR-�

knockout mice has revealed that 19% of the transcriptional changes in the liver

are dependent on PPAR-� [142] stressing the importance of this nuclear recep-

tor in the mechanism of DR.

Studies in PGC-1� null mice confirm the role of PGC-1� in adaptive

energy metabolism [143, 144]. In the liver, PGC-1� is associated with FOXO1,

one of the mammalian DAF-16/dFOXO homologues, and is involved in hepatic

insulin signaling [145]. FOXO1 is involved in PPAR-� regulation in adipocytes,

and there is a complex interplay between these factors in adipocyte differentia-

tion [146]. PPAR-� is also regulated by mTOR, and PPAR-� activity is depen-

dent on amino acid sufficiency [147]. Another factor that appears to provide a

connection between PGC-1�, FOXO and PPARs is SIRT1. SIRT1 regulates

PPAR-� in adipocytes [148] and is involved in PGC-1� activation in the insulin

signaling pathway in the liver [149, 150]. Control of gene expression in this

manner, where transcriptional coactivators and repressor factors are the targets

for numerous signaling pathways, provides a strategy that permits the func-

tional integration of multiple distinct biological programs [151].

Conclusion

The central role of energy metabolism in longevity has been a unifying

feature in our work and in aging research. Here we propose a model for the

mechanism of DR where metabolic reprogramming, the coordinate induction of

an altered metabolic state, is an early event in the mechanism of life span exten-

sion by DR. We predict that tissue-specific changes in energy metabolism occur

through PGC-1� and the PPAR nuclear receptor family. These shifts in energy

metabolism induce a move from fat storage to fat mobilization, influence stress

pathway signaling and ROS production. Activation of adipose tissue is a critical

event in the mechanism of life span extension and leads to altered adipokine


and lipid signaling and reduced systemic inflammation. The influence of meta-

bolic reprogramming on endocrine and immune function leads to a reduced rate

of aging. It is clear from the data described here that this model is highly sim-

plified. Many if not all of the pathways and factors described here have been

shown to be interconnected and are influenced through multiple inputs. The key

to our model is the initial event, which is the shift in how energy is generated

and how fuel is utilized, and that this occurs through small changes in activity

of metabolic regulators to influence the balance of fuel utilization without

deregulating nutrient homeostasis in the animal as a whole. In thinking of the

mechanism of DR in this way, it is possible to extrapolate and understand some

transgenic models of longevity in the context of metabolic regulation and also

to see where the effect of nonphysiological genetic manipulations on life span

could be misleading.

References

1 Weindruch RH, Walford RL: The Retardation of Aging and Disease by Dietary Restriction.

Springfield, Thomas, 1988.

2 Masoro EJ: Caloric restriction. Aging (Milano) 1998;10:173–174.

3 Masoro EJ: Caloric restriction: a key to understanding and modulating aging; in Vijg J (ed):

Research Profiles in Aging. Amsterdam, Elsevier, 2002, vol 1.

4 Weindruch R, Walford RL, Fligiel S, Guthrie D: The retardation of aging in mice by dietary

restriction: longevity, cancer, immunity and lifetime energy intake. J Nutr 1986;116:641–654.

5 Lee CK, Klopp RG, Weindruch R, Prolla TA: Gene expression profile of aging and its retardation

by caloric restriction. Science 1999;285:1390–1393.

6 Kintscher U, Law RE: PPARgamma-mediated insulin sensitization: the importance of fat versus

muscle. Am J Physiol Endocrinol Metab 2005;288:E287–E291.

7 Huss JM, Kelly DP: Nuclear receptor signaling and cardiac energetics. Circ Res 2004;95:

568–578.

8 Lee CK, Allison DB, Brand J, Weindruch R, Prolla TA: Transcriptional profiles associated with

aging and middle age-onset caloric restriction in mouse hearts. Proc Natl Acad Sci USA

2002;99:14988–14993.

9 Finck BN, Lehman JJ, Barger PM, Kelly DP: Regulatory networks controlling mitochondrial

energy production in the developing, hypertrophied, and diabetic heart. Cold Spring Harb Symp

Quant Biol 2002;67:371–382.

10 Higami Y, Pugh TD, Page GP, Allison DB, Prolla TA, Weindruch R: Adipose tissue energy metab-

olism: altered gene expression profile of mice subjected to long-term caloric restriction. FASEB J

2004;18:415–417.

11 Cao SX, Dhahbi JM, Mote PL, Spindler SR: Genomic profiling of short- and long-term caloric

restriction effects in the liver of aging mice. Proc Natl Acad Sci USA 2001;98:10630–10635.

12 Higami Y, Barger JL, Page GP, Allison DB, Smith SR, Prolla TA, Weindruch R: Energy restriction

lowers the expression of genes linked to inflammation, the cytoskeleton, the extracellular matrix,

and angiogenesis in mouse adipose tissue. J Nutr 2006;136:343–352.

13 Anderson RM, Bitterman KJ, Wood JG, Medvedik O, Sinclair DA: Nicotinamide and PNC1 gov-

ern lifespan extension by calorie restriction in Saccharomyces cerevisiae. Nature 2003;423:

181–185.

14 Lin SJ, Kaeberlein M, Andalis AA, Sturtz LA, Defossez PA, Culotta VC, Fink GR, Guarente L:

Calorie restriction extends Saccharomyces cerevisiae lifespan by increasing respiration. Nature

2002;418:344–348.


15 Barros MH, Bandy B, Tahara EB, Kowaltowski AJ: Higher respiratory activity decreases mito-

chondrial reactive oxygen release and increases life span in Saccharomyces cerevisiae. J Biol

Chem 2004;279:49883–49888.

16 Jazwinski SM: The retrograde response links metabolism with stress responses, chromatin-dependent

gene activation, and genome stability in yeast aging. Gene 2005;354:22–27.

17 Butow RA, Avadhani NG: Mitochondrial signaling: the retrograde response. Mol Cell 2004;14:

1–15.

18 Epstein CB, Waddle JA, Hale WT, Dave V, Thornton J, Macatee TL, Garner HR, Butow RA:

Genome-wide responses to mitochondrial dysfunction. Mol Biol Cell 2001;12:297–308.

19 Jazwinski SM: Metabolic control and gene dysregulation in yeast aging. Ann NY Acad Sci

2000;908:21–30.

20 Komeili A, Wedaman KP, O’Shea EK, Powers T: Mechanism of metabolic control: target of

rapamycin signaling links nitrogen quality to the activity of the Rtg1 and Rtg3 transcription fac-

tors. J Cell Biol 2000;151:863–878.

21 Kim DH, Sarbassov DD, Ali SM, King JE, Latek RR, Erdjument-Bromage H, Tempst P, Sabatini

DM: mTOR interacts with raptor to form a nutrient-sensitive complex that signals to the cell

growth machinery. Cell 2002;110:163–175.

22 Kaeberlein M, Powers RW 3rd, Steffen KK, Westman EA, Hu D, Dang N, Kerr EO, Kirkland KT,

Fields S, Kennedy BK: Regulation of yeast replicative life span by TOR and Sch9 in response to

nutrients. Science 2005;310:1193–1196.

23 Powers RW 3rd, Kaeberlein M, Caldwell SD, Kennedy BK, Fields S: Extension of chronological

life span in yeast by decreased TOR pathway signaling. Genes Dev 2006;20:174–184.

24 Vellai T, Takacs-Vellai K, Zhang Y, Kovacs AL, Orosz L, Muller F: Genetics: influence of TOR

kinase on lifespan in C. elegans. Nature 2003;426:620.

25 Kapahi P, Zid BM, Harper T, Koslover D, Sapin V, Benzer S: Regulation of lifespan in Drosophila

by modulation of genes in the TOR signaling pathway. Curr Biol 2004;14:885–890.

26 Um SH, Frigerio F, Watanabe M, Picard F, Joaquin M, Sticker M, Fumagalli S, Allegrini PR,

Kozma SC, Auwerx J, Thomas G: Absence of S6K1 protects against age- and diet-induced obesity

while enhancing insulin sensitivity. Nature 2004;431:200–205.

27 Lee SS, Lee RY, Fraser AG, Kamath RS, Ahringer J, Ruvkun G: A systematic RNAi screen identi-

fies a critical role for mitochondria in C. elegans longevity. Nat Genet 2003;33:40–48.

28 Dillin A, Hsu AL, Arantes-Oliveira N, Lehrer-Graiwer J, Hsin H, Fraser AG, Kamath RS, Ahringer J,

Kenyon C: Rates of behavior and aging specified by mitochondrial function during development.

Science 2002;298:2398–2401.

29 Hamilton B, Dong Y, Shindo M, Liu W, Odell I, Ruvkun G, Lee SS: A systematic RNAi screen for

longevity genes in C. elegans. Genes Dev 2005;19:1544–1555.

30 Hansen M, Hsu AL, Dillin A, Kenyon C: New genes tied to endocrine, metabolic, and dietary reg-

ulation of lifespan from a Caenorhabditis elegans genomic RNAi screen. PLos Genet 2005;1:e17.

31 Rea S, Johnson TE: A metabolic model for life span determination in Caenorhabditis elegans.

Dev Cell 2003;5:197–203.

32 Houthoofd K, Braeckman BP, Lenaerts I, Brys K, De Vreese A, Van Eygen S, Vanfleteren JR: No

reduction of metabolic rate in food restricted Caenorhabditis elegans. Exp Gerontol 2002;37:

1359–1369.

33 Houthoofd K, Braeckman BP, Johnson TE, Vanfleteren JR: Life extension via dietary restriction is

independent of the Ins/IGF-1 signalling pathway in Caenorhabditis elegans. Exp Gerontol

2003;38:947–954.

34 Gerisch B, Weitzel C, Kober-Eisermann C, Rottiers V, Antebi A: A hormonal signaling pathway influ-

encing C. elegans metabolism, reproductive development, and life span. Dev Cell 2001;1:841–851.

35 Ludewig AH, Kober-Eisermann C, Weitzel C, Bethke A, Neubert K, Gerisch B, Hutter H, Antebi A:

A novel nuclear receptor/coregulator complex controls C. elegans lipid metabolism, larval devel-

opment, and aging. Genes Dev 2004;18:2120–2133.

36 Jia K, Chen D, Riddle DL: The TOR pathway interacts with the insulin signaling pathway to regu-

late C. elegans larval development, metabolism and life span. Development 2004;131:3897–3906.

37 Rogina B, Reenan RA, Nilsen SP, Helfand SL: Extended life-span conferred by cotransporter gene

mutations in Drosophila. Science 2000;290:2137–2140.


38 Marden JH, Rogina B, Montooth KL, Helfand SL: Conditional tradeoffs between aging and

organismal performance of Indy long-lived mutant flies. Proc Natl Acad Sci USA 2003;100:

3369–3373.

39 Ramsey JJ, Harper ME, Weindruch R: Restriction of energy intake, energy expenditure, and aging.

Free Radic Biol Med 2000;29:946–968.

40 Simon AF, Shih C, Mack A, Benzer S: Steroid control of longevity in Drosophila melanogaster.

Science 2003;299:1407–1410.

41 Tsai CC, Kao HY, Yao TP, McKeown M, Evans RM: SMRTER, a Drosophila nuclear receptor

coregulator, reveals that EcR-mediated repression is critical for development. Mol Cell

1999;4:175–186.

42 Rogina B, Helfand SL, Frankel S: Longevity regulation by Drosophila Rpd3 deacetylase and

caloric restriction. Science 2002;298:1745.

43 Pile LA, Spellman PT, Katzenberger RJ, Wasserman DA: The SIN3 deacetylase complex

represses genes encoding mitochondrial proteins: implications for the regulation of energy metab-

olism. J Biol Chem 2003;278:37840–37848.

44 Tatar M, Bartke A, Antebi A: The endocrine regulation of aging by insulin-like signals. Science

2003;299:1346–1351.

45 Clancy DJ, Gems D, Harshman LG, Oldham S, Stocker H, Hafen E, Leevers SJ, Partridge L:

Extension of life-span by loss of CHICO, a Drosophila insulin receptor substrate protein. Science

2001;292:104–106.

46 Tatar M, Kopelman A, Epstein D, Tu MP, Yin CM, Garofalo RS: A mutant Drosophila insulin

receptor homolog that extends life-span and impairs neuroendocrine function. Science

2001;292:107–110.

47 Clancy DJ, Gems D, Hafen E, Leevers SJ, Partridge L: Dietary restriction in long-lived dwarf flies.

Science 2002;296:319.

48 Shimokawa I, Higami Y, Tsuchiya T, Otani H, Komatsu T, Chiba T, Yamaza H: Life span extension

by reduction of the growth hormone-insulin-like growth factor-1 axis: relation to caloric restric-

tion. Faseb J 2003;17:1108–1109.

49 Bartke A, Wright JC, Mattison JA, Ingram DK, Miller RA, Roth GS: Extending the lifespan of

long-lived mice. Nature 2001;414:412.

50 Masternak MM, Al-Regaiey K, Bonkowski MS, Panici J, Sun L, Wang J, Przybylski GK, Bartke A:

Divergent effects of caloric restriction on gene expression in normal and long-lived mice.

J Gerontol A Biol Sci Med Sci 2004;59:784–788.

51 Masternak MM, Al-Regaiey KA, Del Rosario Lim MM, Jimenez-Ortega V, Panici JA, Bonkowski

MS, Bartke A: Effects of caloric restriction on insulin pathway gene expression in the skeletal

muscle and liver of normal and long-lived GHR-KO mice. Exp Gerontol 2005;40:679–684.

52 Hwangbo DS, Gersham B, Tu MP, Palmer M, Tatar M: Drosophila dFOXO controls lifespan and

regulates insulin signalling in brain and fat body. Nature 2004;429:562–566.

53 Giannakou ME, Goss M, Junger MA, Hafen E, Leevers SJ, Partridge L: Long-lived Drosophila

with overexpressed dFOXO in adult fat body. Science 2004;305:361.

54 Murphy CT, McCarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, Kenyon C:

Genes that act downstream of DAF-16 to influence the lifespan of Caenorhabditis elegans. Nature

2003;424:277–283.

55 Lee SS, Kennedy S, Tolonen AC, Ruvkun G: DAF-16 target genes that control C. elegans life-span

and metabolism. Science 2003;300:644–647.

56 Masoro EJ, Shimokawa I, Yu BP: Retardation of the aging processes in rats by food restriction.

Ann NY Acad Sci 1991;621:337–352.

57 Sohal RS, Weindruch R: Oxidative stress, caloric restriction, and aging. Science 1996;273:59–63.

58 Yu BP, Chen JJ, Kang CM, Choe M, Maeng YS, Kristal BS: Mitochondrial aging and lipoperox-

idative products. Ann NY Acad Sci 1996;786:44–56.

59 Lass A, Sohal BH, Weindruch R, Forster MJ, Sohal RS: Caloric restriction prevents age-associated

accrual of oxidative damage to mouse skeletal muscle mitochondria. Free Radic Biol Med

1998;25:1089–1097.

60 Merry BJ: Calorie restriction and age-related oxidative stress. Ann NY Acad Sci 2000;908:

180–198.


61 Nagai M, Takahashi R, Goto S: Dietary restriction initiated late in life can reduce mitochondrial

protein carbonyls in rat livers: Western blot studies. Biogerontology 2000;1:321–328.

62 Barazzoni R, Zanetti M, Bosutti A, Biolo G, Vitali-Serdoz L, Stebel M, Guarnieri G: Moderate

caloric restriction, but not physiological hyperleptinemia per se, enhances mitochondrial oxidative

capacity in rat liver and skeletal muscle-tissue-specific impact on tissue triglyceride content and

AKT activation. Endocrinology 2005;146:2098–2106.

63 Balaban RS, Nemoto S, Finkel T: Mitochondria, oxidants, and aging. Cell 2005;120:483–495.

64 Brand MD: Uncoupling to survive? The role of mitochondrial inefficiency in ageing. Exp

Gerontol 2000;35:811–820.

65 Hagopian K, Harper ME, Ram JJ, Humble SJ, Weindruch R, Ramsey JJ: Long-term calorie restric-

tion reduces proton leak and hydrogen peroxide production in liver mitochondria. Am J Physiol

Endocrinol Metab 2005;288:E674–E684.

66 Bevilacqua L, Ramsey JJ, Hagopian K, Weindruch R, Harper ME: Long-term caloric restriction

increases UCP3 content but decreases proton leak and reactive oxygen species production in rat

skeletal muscle mitochondria. Am J Physiol Endocrinol Metab 2005;289:E429–E438.

67 Clapham JC, Arch JR, Chapman H, Haynes A, Lister C, Moore GB, Piercy V, Carter SA, Lehner I,

Smith SA, Beeley LJ, Godden RJ, Herrity N, Skehel M, Changani KK, Hockings PD, Reid DG,

Squires SM, Hatcher J, Trail B, Latcham J, Rastan S, Harper AJ, Cadenas S, Buckingham JA,

Brand MD, Abuin A: Mice overexpressing human uncoupling protein-3 in skeletal muscle are

hyperphagic and lean. Nature 2000;406:415–418.

68 Himms-Hagen J, Harper ME: Physiological role of UCP3 may be export of fatty acids from mito-

chondria when fatty acid oxidation predominates: a hypothesis. Exp Biol Med (Maywood)

2001;226:78–84.

69 Schrauwen P, Hesselink MK: The role of uncoupling protein 3 in fatty acid metabolism: protection

against lipotoxicity? Proc Nutr Soc 2004;63:287–292.

70 Brand MD, Esteves TC: Physiological functions of the mitochondrial uncoupling proteins UCP2

and UCP3. Cell Metab 2005;2:85–93.

71 MacLellan JD, Gerrits MF, Gowing A, Smith PJ, Wheeler MB, Harper ME: Physiological

increases in uncoupling protein 3 augment fatty acid oxidation and decrease reactive oxygen

species production without uncoupling respiration in muscle cells. Diabetes 2005;54:2343–2350.

72 Bezaire V, Spriet LL, Campbell S, Sabet N, Gerrits M, Bonen A, Harper ME: Constitutive UCP3

overexpression at physiological levels increases mouse skeletal muscle capacity for fatty acid

transport and oxidation. Faseb J 2005;19:977–979.

73 Weigle DS, Selfridge LE, Schwartz MW, Seeley RJ, Cummings DE, Havel PJ, Kuijper JL, Beltran

del Rio H: Elevated free fatty acids induce uncoupling protein 3 expression in muscle: a potential

explanation for the effect of fasting. Diabetes 1998;47:298–302.

74 Petersen KF, Dufour S, Befroy D, Garcia R, Shulman GI: Impaired mitochondrial activity in the

insulin-resistant offspring of patients with type 2 diabetes. N Engl J Med 2004;350:664–671.

75 Lowell BB, Shulman GI: Mitochondrial dysfunction and type 2 diabetes. Science 2005;307:384–387.

76 Wallace DC: A mitochondrial paradigm of metabolic and degenerative diseases, aging, and can-

cer: a dawn for evolutionary medicine. Annu Rev Genet 2005;39:359–407.

77 Rosenberg P: Mitochondrial dysfunction and heart disease. Mitochondrion 2004;4:621–628.

78 Short KR, Bigelow ML, Kahl J, Singh R, Coenen-Schimke J, Raghavakaimal S, Nair KS: Decline

in skeletal muscle mitochondrial function with aging in humans. Proc Natl Acad Sci USA

2005;102:5618–5623.

79 Hansson A, Hance N, Dufour E, Rantanen A, Hultenby K, Clayton DA, Wibom R, Larsson NG:

A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-defi-

cient mouse hearts. Proc Natl Acad Sci USA 2004;101:3136–3141.

80 Trifunovic A, Wredenberg A, Falkenberg M, Spelbrink JN, Rovio AT, Bruder CE, Bohlooly YM,

Gidlof S, Oldfors A, Wibom R, Tornell J, Jacobs HT, Larsson NG: Premature ageing in mice

expressing defective mitochondrial DNA polymerase. Nature 2004;429:417–423.

81 Kujoth GC, Hiona A, Pugh TD, Someya S, Panzer K, Wohlgemuth SE, Hofer T, Seo AY, Sullivan R,

Jobling WA, Morrow JD, Van Remmen H, Sedivy JM, Yamasoba T, Tanokura M, Weindruch R,

Leeuwenburgh C, Prolla TA: Mitochondrial DNA mutations, oxidative stress, and apoptosis in

mammalian aging. Science 2005;309:481–484.


82 Speakman JR, Talbot DA, Selman C, Snart S, McLaren JS, Redman P, Krol E, Jackson DM,

Johnson MS, Brand MD: Uncoupled and surviving: individual mice with high metabolism have

greater mitochondrial uncoupling and live longer. Aging Cell 2004;3:87–95.

83 Koizumi A, Tsukada M, Wada Y, Masuda H, Weindruch R: Mitotic activity in mice is suppressed

by energy restriction-induced torpor. J Nutr 1992;122:1446–1453.

84 Duffy PH, Feuers R, Nakamura KD, Leakey J, Hart RW: Effect of chronic caloric restriction on the

synchronization of various physiological measures in old female Fischer 344 rats. Chronobiol Int

1990;7:113–124.

85 Lane MA, Baer DJ, Rumpler WV, Weindruch R, Ingram DK, Tilmont EM, Cutler RG, Roth GS:

Calorie restriction lowers body temperature in rhesus monkeys, consistent with a postulated anti-

aging mechanism in rodents. Proc Natl Acad Sci USA 1996;93:4159–4164.

86 Merry BJ: Oxidative stress and mitochondrial function with aging – The effects of calorie restric-


87 Sohal RS, Agarwal S, Candas M, Forster MJ, Lal H: Effect of age and caloric restriction on

DNA oxidative damage in different tissues of C57BL/6 mice. Mech Ageing Dev 1994;76:

215–224.

88 Sohal RS, Ku HH, Agarwal S, Forster MJ, Lal H: Oxidative damage, mitochondrial oxidant gener-

ation and antioxidant defenses during aging and in response to food restriction in the mouse. Mech

Ageing Dev 1994;74:121–133.

89 Chen JJ, Yu BP: Alterations in mitochondrial membrane fluidity by lipid peroxidation products.


90 Drew B, Phaneuf S, Dirks A, Selman C, Gredilla R, Lezza A, Barja G, Leeuwenburgh C: Effects

of aging and caloric restriction on mitochondrial energy production in gastrocnemius muscle and

heart. Am J Physiol Regul Integr Comp Physiol 2003;284:R474–R480.

91 Zainal TA, Oberley TD, Allison DB, Szweda LI, Weindruch R: Caloric restriction of rhesus mon-

keys lowers oxidative damage in skeletal muscle. Faseb J 2000;14:1825–1836.

92 Droge W: Free radicals in the physiological control of cell function. Physiol Rev 2002;82:47–95.

93 Kim HJ, Jung KJ, Yu BP, Cho CG, Choi JS, Chung HY: Modulation of redox-sensitive transcrip-

tion factors by calorie restriction during aging. Mech Ageing Dev 2002;123:1589–1595.

94 Cohen HY, Miller C, Bitterman KJ, Wall NR, Hekking B, Kessler B, Howitz KT, Gorospe M, de

Cabo R, Sinclair DA: Calorie restriction promotes mammalian cell survival by inducing the

SIRT1 deacetylase. Science 2004;305:390–392.

95 Motta MC, Divecha N, Lemieux M, Kamel C, Chen D, Gu W, Bultsma Y, McBurney M, Guarente L:

Mammalian SIRT1 represses forkhead transcription factors. Cell 2004;116:551–563.

96 Brunet A, Sweeney LB, Sturgill JF, Chua KF, Greer PL, Lin Y, Tran H, Ross SE, Mostoslavsky R,

Cohen HY, Hu LS, Cheng HL, Jedrychowski MP, Gygi SP, Sinclair DA, Alt FW, Greenberg ME:

Stress-dependent regulation of FOXO transcription factors by the SIRT1 deacetylase. Science

2004;303:2011–2015.

97 Wang MC, Bohmann D, Jasper H: JNK extends life span and limits growth by antagonizing cellu-

lar and organism-wide responses to insulin signaling. Cell 2005;121:115–125.

98 Oh SW, Mukhopadhyay A, Svrzikapa N, Jiang F, Davis RJ, Tissenbaum HA: JNK regulates lifes-

pan in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription fac-

tor/DAF-16. Proc Natl Acad Sci USA 2005;102:4494–4499.

99 Hirosumi J, Tuncman G, Chang L, Gorgun CZ, Uysal KT, Maeda K, Karin M, Hotamisligil GS:

A central role for JNK in obesity and insulin resistance. Nature 2002;420:333–336.

100 Ozcan U, Cao Q, Yilmaz E, Lee AH, Iwakoshi NN, Ozdelen E, Tuncman G, Gorgun C, Glimcher

LH, Hotamisligil GS: Endoplasmic reticulum stress links obesity, insulin action, and type 2 dia-

betes. Science 2004;306:457–461.

101 Kaneto H, Nakatani Y, Kawamori D, Miyatsuka T, Matsuoka TA, Matsuhisa M, Yamasaki Y: Role

of oxidative stress, endoplasmic reticulum stress, and c-Jun N-terminal kinase in pancreatic beta-

cell dysfunction and insulin resistance. Int J Biochem Cell Biol 2005;37:1595–1608.

102 Park KS, Lee RD, Kang SK, Han SY, Park KL, Yang KH, Song YS, Park HJ, Lee YM, Yun YP, Oh

KW, Kim DJ, Yun YW, Hwang SJ, Lee SE, Hong JT: Neuronal differentiation of embryonic mid-

brain cells by upregulation of peroxisome proliferator-activated receptor-gamma via the JNK-

dependent pathway. Exp Cell Res 2004;297:424–433.


103 Dougherty CJ, Kubasiak LA, Frazier DP, Li H, Xiong WC, Bishopric NH, Webster KA:

Mitochondrial signals initiate the activation of c-Jun N-terminal kinase (JNK) by hypoxia-

reoxygenation. Faseb J 2004;18:1060–1070.

104 Brancho D, Tanaka N, Jaeschke A, Ventura JJ, Kelkar N, Tanaka Y, Kyuuma M, Takeshita T, Flavell

RA, Davis RJ: Mechanism of p38 MAP kinase activation in vivo. Genes Dev 2003;17:1969–1978.

105 Geiger PC, Wright DC, Han DH, Holloszy JO: Activation of p38 MAP kinase enhances sensitiv-

ity of muscle glucose transport to insulin. Am J Physiol Endocrinol Metab 2005;288:E782–E788.

106 Barger PM, Browning AC, Garner AN, Kelly DP: p38 mitogen-activated protein kinase activates

peroxisome proliferator-activated receptor alpha: a potential role in the cardiac metabolic stress

response. J Biol Chem 2001;276:44495–44501.

107 Fan M, Rhee J, St-Pierre J, Handschin C, Puigserver P, Lin J, Jaeger S, Erdjument-Bromage H,

Tempst P, Spiegelman BM: Suppression of mitochondrial respiration through recruitment of p160

myb binding protein to PGC-1alpha: modulation by p38 MAPK. Genes Dev 2004;18:278–289.

108 Lin J, Wu H, Tarr PT, Zhang CY, Wu Z, Boss O, Michael LF, Puigserver P, Isotani E, Olson EN,

Lowell BB, Bassel-Duby R, Spiegelman BM: Transcriptional co-activator PGC-1 alpha drives the

formation of slow-twitch muscle fibres. Nature 2002;418:797–801.

109 Hirata Y, Andoh T, Asahara T, Kikuchi A: Yeast glycogen synthase kinase-3 activates Msn2p-

dependent transcription of stress responsive genes. Mol Biol Cell 2003;14:302–312.

110 Liu S, Yu S, Hasegawa Y, Lapushin R, Xu HJ, Woodgett JR, Mills GB, Fang X: Glycogen synthase

kinase 3beta is a negative regulator of growth factor-induced activation of the c-Jun N-terminal

kinase. J Biol Chem 2004;279:51075–51081.

111 Dokken BB, Sloniger JA, Henriksen EJ: Acute selective glycogen synthase kinase-3 inhibition

enhances insulin signaling in prediabetic insulin-resistant rat skeletal muscle. Am J Physiol


112 Horbinski C, Chu CT: Kinase signaling cascades in the mitochondrion: a matter of life or death.


113 Enzi G, Gasparo M, Biondetti PR, Fiore D, Semisa M, Zurlo F: Subcutaneous and visceral fat dis-

tribution according to sex, age, and overweight, evaluated by computed tomography. Am J Clin

Nutr 1986;44:739–746.

114 Fraze E, Chiou YA, Chen YD, Reaven GM: Age-related changes in postprandial plasma glucose,

insulin, and free fatty acid concentrations in nondiabetic individuals. J Am Geriatr Soc

1987;35:224–228.

115 Ma XH, Muzumdar R, Yang XM, Gabriely I, Berger R, Barzilai N: Aging is associated with resis-

tance to effects of leptin on fat distribution and insulin action. J Gerontol A Biol Sci Med Sci

2002;57:B225–B231.

116 Wang ZW, Pan WT, Lee Y, Kakuma T, Zhou YT, Unger RH: The role of leptin resistance in the

lipid abnormalities of aging. Faseb J 2001;15:108–114.

117 Masoro EJ: Caloric restriction and aging: an update. Exp Gerontol 2000;35:299–305.

118 Shimokawa I, Higami Y: A role for leptin in the antiaging action of dietary restriction: a hypothe-

sis. Aging (Milano) 1999;11:380–382.

119 Barzilai N, Banerjee S, Hawkins M, Chen W, Rossetti L: Caloric restriction reverses hepatic

insulin resistance in aging rats by decreasing visceral fat. J Clin Invest 1998;101:1353–1361.

120 Fernandez-Galaz C, Fernandez-Agullo T, Perez C, Peralta S, Arribas C, Andres A, Carrascosa JM,

Ros M: Long-term food restriction prevents ageing-associated central leptin resistance in Wistar

rats. Diabetologia 2002;45:997–1003.

121 Barger JL, Walford RL, Weindruch R: The retardation of aging by caloric restriction: its signifi-

cance in the transgenic era. Exp Gerontol 2003;38:1343–1351.

122 Libina N, Berman JR, Kenyon C: Tissue-specific activities of C. elegans DAF-16 in the regulation

of lifespan. Cell 2003;115:489–502.

123 Kershaw EE, Flier JS: Adipose tissue as an endocrine organ. J Clin Endocrinol Metab

2004;89:2548–2556.

124 Steppan CM, Bailey ST, Bhat S, Brown EJ, Banerjee RR, Wright CM, Patel HR, Ahima RS, Lazar

MA: The hormone resistin links obesity to diabetes. Nature 2001;409:307–312.

125 Berg AH, Combs TP, Scherer PE: ACRP30/adiponectin: an adipokine regulating glucose and lipid

metabolism. Trends Endocrinol Metab 2002;13:84–89.


126 Mora S, Pessin JE: An adipocentric view of signaling and intracellular trafficking. Diabetes Metab

Res Rev 2002;18:345–356.

127 Picard F, Guarente L: Molecular links between aging and adipose tissue. Int J Obes (Lond)

2005;29(suppl 1):S36–S39.

128 Collins S, Cao W, Robidoux J: Learning new tricks from old dogs: beta-adrenergic receptors teach

new lessons on firing up adipose tissue metabolism. Mol Endocrinol 2004;18:2123–2131.

129 Cao W, Daniel KW, Robidoux J, Puigserver P, Medvedev AV, Bai X, Floering LM, Spiegelman

BM, Collins S: p38 mitogen-activated protein kinase is the central regulator of cyclic AMP-dependent

transcription of the brown fat uncoupling protein 1 gene. Mol Cell Biol 2004;24:3057–3067.

130 Tiraby C, Tavernier G, Lefort C, Larrouy D, Bouillaud F, Ricquier D, Langin D: Acquirement of

brown fat cell features by human white adipocytes. J Biol Chem 2003;278:33370–33376.

131 Orci L, Cook WS, Ravazzola M, Wang MY, Park BH, Montesano R, Unger RH: Rapid transfor-

mation of white adipocytes into fat-oxidizing machines. Proc Natl Acad Sci USA 2004;101:

2058–2063.

132 Furukawa S, Fujita T, Shimabukuro M, Iwaki M, Yamada Y, Nakajima Y, Nakayama O, Makishima M,

Matsuda M, Shimomura I: Increased oxidative stress in obesity and its impact on metabolic syn-

drome. J Clin Invest 2004;114:1752–1761.

133 Bogacka I, Ukropcova B, McNeil M, Gimble JM, Smith SR: Structural and functional conse-

quences of mitochondrial biogenesis in human adipocytes in vitro. J Clin Endocrinol Metab

2005;90:6650–6656.

134 Merry BJ: Molecular mechanisms linking calorie restriction and longevity. Int J Biochem Cell

Biol 2002;34:1340–1354.

135 Finck BN, Kelly DP: Peroxisome proliferator-activated receptor alpha (PPARalpha) signaling in

the gene regulatory control of energy metabolism in the normal and diseased heart. J Mol Cell

Cardiol 2002;34:1249–1257.

136 Puigserver P, Spiegelman BM: Peroxisome proliferator-activated receptor-gamma coactivator 1

alpha (PGC-1 alpha): transcriptional coactivator and metabolic regulator. Endocr Rev 2003;24:

78–90.

137 Valle I, Alvarez-Barrientos A, Arza E, Lamas S, Monsalve M: PGC-1alpha regulates the mitochon-

drial antioxidant defense system in vascular endothelial cells. Cardiovasc Res 2005;66:562–573.

138 Mootha VK, Lindgren CM, Eriksson KF, Subramanian A, Sihag S, Lehar J, Puigserver P, Carlsson E,

Ridderstrale M, Laurila E, Houstis N, Daly MJ, Patterson N, Mesirov JP, Golub TR, Tamayo P,

Spiegelman B, Lander ES, Hirschhorn JN, Altshuler D, Groop LC: PGC-1alpha-responsive genes

involved in oxidative phosphorylation are coordinately downregulated in human diabetes. Nat

Genet 2003;34:267–273.

139 Patti ME, Butte AJ, Crunkhorn S, Cusi K, Berria R, Kashyap S, Miyazaki Y, Kohane I, Costello M,

Saccone R, Landaker EJ, Goldfine AB, Mun E, DeFronzo R, Finlayson J, Kahn CR, Mandarino

LJ: Coordinated reduction of genes of oxidative metabolism in humans with insulin resistance and

diabetes: potential role of PGC1 and NRF1. Proc Natl Acad Sci USA 2003;100:8466–8471.

140 Semple RK, Crowley VC, Sewter CP, Laudes M, Christodoulides C, Considine RV, Vidal-Puig A,

O’Rahilly S: Expression of the thermogenic nuclear hormone receptor coactivator PGC-1alpha is

reduced in the adipose tissue of morbidly obese subjects. Int J Obes Relat Metab Disord

2004;28:176–179.

141 Evans RM, Barish GD, Wang YX: PPARs and the complex journey to obesity. Nat Med 2004;10:

355–361.

142 Corton JC, Apte U, Anderson SP, Limaye P, Yoon L, Latendresse J, Dunn C, Everitt JI, Voss KA,

Swanson C, Kimbrough C, Wong JS, Gill SS, Chandraratna RA, Kwak MK, Kensler TW, Stulnig

TM, Steffensen KR, Gustafsson JA, Mehendale HM: Mimetics of caloric restriction include ago-

nists of lipid-activated nuclear receptors. J Biol Chem 2004;279:46204–46212.

143 Lin J, Wu PH, Tarr PT, Lindenberg KS, St-Pierre J, Zhang CY, Mootha VK, Jager S, Vianna CR,

Reznick RM, Cui L, Manieri M, Donovan MX, Wu Z, Cooper MP, Fan MC, Rohas LM, Zavacki

AM, Cinti S, Shulman GI, Lowell BB, Krainc D, Spiegelman BM: Defects in adaptive energy

metabolism with CNS-linked hyperactivity in PGC-1alpha null mice. Cell 2004;119:121–135.

144 Leone TC, Lehman JJ, Finck BN, Schaeffer PJ, Wende AR, Boudina S, Courtois M, Wozniak DF,

Sambandam N, Bernal-Mizrachi C, Chen Z, Holloszy JO, Medeiros DM, Schmidt RE, Saffitz JE,


Abel ED, Semenkovich CF, Kelly DP: PGC-1alpha deficiency causes multi-system energy meta-

bolic derangements: muscle dysfunction, abnormal weight control and hepatic steatosis. PLos

Biol 2005;3:e101.

145 Puigserver P, Rhee J, Donovan J, Walkey CJ, Yoon JC, Oriente F, Kitamura Y, Altomonte J, Dong H,

Accili D, Spiegelman BM: Insulin-regulated hepatic gluconeogenesis through FOXO1-PGC-1alpha

interaction. Nature 2003;423:550–555.

146 Dowell P, Otto TC, Adi S, Lane MD: Convergence of peroxisome proliferator-activated receptor

gamma and Foxo1 signaling pathways. J Biol Chem 2003;278:45485–45491.

147 Kim JE, Chen J: Regulation of peroxisome proliferator-activated receptor-gamma activity by

mammalian target of rapamycin and amino acids in adipogenesis. Diabetes 2004;53:2748–2756.

148 Picard F, Kurtev M, Chung N, Topark-Ngarm A, Senawong T, Machado De Oliveira R, Leid M,

McBurney MW, Guarente L: Sirt1 promotes fat mobilization in white adipocytes by repressing

PPAR-gamma. Nature 2004;429:771–776.

149 Rodgers JT, Lerin C, Haas W, Gygi SP, Spiegelman BM, Puigserver P: Nutrient control of glucose

homeostasis through a complex of PGC-1alpha and SIRT1. Nature 2005;434:113–118.

150 Nemoto S, Fergusson MM, Finkel T: SIRT1 functionally interacts with the metabolic regulator

and transcriptional coactivator PGC-1alpha. J Biol Chem 2005;280:16456–16460.

151 Spiegelman BM, Heinrich R: Biological control through regulated transcriptional coactivators.

Cell 2004;119:157–167.

Rozalyn M. Anderson, PhD

GRECC, D5209

VA Hospital, 2500 Overlook Terrace

Madison, WI 53705 (USA)

Tel. �1 608 256 1901 (ext. 11591), Fax �1 608 280 7291, E-Mail [email protected]



Secrets of the lac Operon

Glucose Hysteresis as a Mechanism in Dietary Restriction,

Aging and Disease

Charles V. Mobbs, Jason W. Mastaitis, Minhua Zhang,

Fumiko Isoda, Hui Cheng, Kelvin Yen

Departments of Neuroscience and Geriatrics, Mount Sinai School of Medicine,

New York, N.Y., USA

AbstractElevated blood glucose associated with diabetes produces progressive and apparently

irreversible damage to many cell types. Conversely, reduction of glucose extends life span in

yeast, and dietary restriction reduces blood glucose. Therefore it has been hypothesized that

cumulative toxic effects of glucose drive at least some aspects of the aging process and, con-

versely, that protective effects of dietary restriction are mediated by a reduction in exposure

to glucose. The mechanisms mediating cumulative toxic effects of glucose are suggested by

two general principles of metabolic processes, illustrated by the lac operon but also observed

with glucose-induced gene expression. First, metabolites induce the machinery of their own

metabolism. Second, induction of gene expression by metabolites can entail a form of mole-

cular memory called hysteresis. When applied to glucose-regulated gene expression, these

two principles suggest a mechanism whereby repetitive exposure to postprandial excursions

of glucose leads to an age-related increase in glycolytic capacity (and reduction in �-oxida-

tion of free fatty acids), which in turn leads to an increased generation of oxidative damage

and a decreased capacity to respond to oxidative damage, independent of metabolic rate.

According to this mechanism, dietary restriction increases life span and reduces pathology

by reducing exposure to glucose and therefore delaying the development of glucose-induced

glycolytic capacity.


Masoro et al. [1] proposed that ‘dietary restriction retards the aging

processes by altering the characteristics of fuel use’. Similarly, on the basis of a

large-scale analysis of gene expression, Lee et al. [2] concluded that ‘aging was

associated with transcriptional alterations consistent with a metabolic shift

from fatty acid to carbohydrate metabolism’ and that dietary restriction

Mobbs/Mastaitis/Zhang/Isoda/Cheng/Yen 40

‘resulted in alterations in gene expression consistent with preserved fatty acid

metabolism’ through ‘transcriptional reprogramming’ (see also Anderson and

Weindruch in this volume). Indeed, life span in yeast is increased simply by

reducing glucose concentrations which, interestingly, actually increases meta-

bolic rate [3]. In the present review we extend these concepts and propose a

specific mechanism by which a cumulative toxic effect of glucose drives at

least some aspects of the aging process, reduction in which mediates protective

effects of dietary restriction.

Glucose Increases Glycolysis and Inhibits Alternative

Metabolic Pathways, Including ��-Oxidation of Free Fatty Acids

A general feature of metabolic regulation is that substrates typically induce

the metabolic machinery necessary for their own metabolism. The classic

example of this phenomenon is the lac operon, in which lactose induces both

the activity and gene expression of �-galactosidase, the rate-limiting enzyme

for the degradation of lactose [4]. In yeast, whose natural history entails cyclic

utilization of glucose followed by ethanol as energy sources, glucose depletion

inhibits glycolysis and activates the pathways for ethanol metabolism [5]. In

mammals, glucose induces the coordinated expression of glycolytic enzymes

including phosphofructokinase, the rate-limiting enzyme for glycolysis [6].

Conversely, glucose inhibits the expression of pyruvate dehydrogenase kinase 4

(PDK-4), which is an inhibitor of pyruvate dehydrogenase [7]. Thus glucose

induces the utilization of glucose carbons in both glycolysis and in the tricar-

boxylic acid cycle. In insulin-sensitive tissues, glucose acts in concert with

insulin to increase glycolytic activity [8]. Cognate induction of its own metabo-

lism is particularly salient for glucose, probably because, as the unique product

of photosynthesis and the ultimate source of biological energy, metabolism of

glucose has been particularly optimized through evolution. Thus, although lac-

tose induces the lac operon, the lac operon is robustly inhibited by a similar

concentration of glucose. Similar glucose repression of alternative metabolic

pathways is observed in yeast [5, 9] and fungi [10]. As with the lac operon,

induction of glycolytic capacity by glucose is accompanied by suppression of

the utilization of alternate fuels such as oxidation of fatty acids [11]. It should

be noted that fatty acids conversely induce the metabolic machinery for their

own metabolism, especially through the regulation of members of the peroxiso-

mal proliferator receptor family of transcription factors, which by inducing

PDK-4 [7] can conversely reduce pyruvate dehydrogenase activity and there-

fore glucose metabolism. Thus in mammals glucose and free fatty acids are

constantly engaged in substrate competition.

Secrets of the lac Operon 41

Metabolic Regulation of Gene Expression

Exhibits Hysteresis

Of particular relevance to aging, the effects of metabolites on their own

metabolic fate may not be entirely reversible, i.e. metabolic reprogramming

may exhibit hysteresis. Hysteresis refers to a phenomenon by which a system

exhibits memory. For example, a classic example of hysteresis is the Schmidt

trigger switch encountered in electrical engineering. If the switch is off and the

voltage increases from 0 to 5 mV, for example, the switch is not activated until

reaching 5 mV. However, if the trigger begins in the activated position at 5 mV

and voltage decreases from 5 to 0 mV, the switch does not turn off until the

voltage reaches 0 mV. Therefore at 3 mV, the trigger will be either on or off,

depending on the history of the circuit. Although not as widely appreciated,

gene expression also exhibits hysteresis, also sometimes called ‘gene memory’

or ‘priming’ [12–16]. For example, the first time the ovalbumin gene is

exposed to estrogen the induction is sluggish and subtle, but subsequent expo-

sures to estrogen produce much more rapid and robust inductions, a phenome-

non associated with permanent changes in chromatin structure produced by

the initial exposure to estrogen [17]. Indeed, ‘cellular memory’ has been

invoked to describe the phenomenon of permanent change in gene expression

after transient exposure to inducers during development [18]. Metabolic regu-

lation of gene expression also exhibits hysteresis. For example, induction of

the lac operon exhibits a history-dependent dose dependency that is precisely

analogous to a Schmidt trigger [19, 20]. Thus if the system begins with the lac

operon inactive at low levels of inducer, the operon does not become active

until exposed to high levels of inducer. On the other hand, if the system begins

with the lac operon active at high levels of inducer, it stays active until exposed

to very low levels of inducer. Thus, depending on the history of the system, the

operon would be either on or off at modest levels of inducer. In fact, this

behavior has led investigators to refer to the lac operon as a multistable

‘switch’ [19, 20].

Accumulating evidence suggests that the regulation of gene expression by

glucose and other factors also exhibits hysteresis [15]. For example, glucose-

induced expression of fibronectin appears to be remarkably persistent even

after reduction of glucose concentration [21]. Furthermore, it is increasingly

clear that deleterious effects of elevated glucose persist for years after correc-

tion of blood glucose in diabetes [22]. As described below, based on our analy-

sis of glucose-regulated gene expression and studies of the mechanism of

neuroendocrine glucose sensing, we propose that hysteresis of glucose-regulated

gene expression entails persistent self-induction of cytoplasmic NADH, the sig-

nature metabolite of glycolysis.


Mitochondrial Complex II Promotes Longevity, Other

Mitochondrial Complexes Reduce Longevity

The fact that diabetes accelerates many age-related pathologies, especially

cardiovascular pathologies, suggests that diabetes and aging may share common

pathological mechanisms. Certainly this is clear for yeast, in which reducing

glucose concentration is sufficient to increase life span [3]. A role for glucose

metabolism in determining life span is also suggested by examination of the

role of specific complexes of the mitochondrial electron transport chain (ETC)

in determining longevity. Genome-wide screening studies have demonstrated

that genes coding for mitochondrial functions constitute possibly the most con-

spicuous single class of ‘senescence assurance genes’, ablation of which

increases life span [23, 24]. Almost all of these life-span-limiting mitochondrial

genes code for proteins in mitochondrial complexes I, III, IV or V [23–26]. For

example, of 23 genes discovered in an exhaustive genome-wide screen whose

inhibition increased life span [26], 12 were genes coding for proteins in mito-

chondrial (ETC) complexes I, III, IV or V, and one gene coded for a key enzyme

in glycolysis, glucose-6-phosphate isomerase. An independent screen from

another laboratory obtained very similar results, though discovering a different

glycolytic enzyme whose inhibition increases life span [25]. Strikingly absent

from these screens were genes for proteins in mitochondrial complex II [25,

26]. Indeed, classic genetic screens had already identified that mutations caus-

ing impairments in complex II reduce life span [27]. Thus proteins in mito-

chondrial complexes I, III, IV and V and at least some glycolytic enzymes

function to limit life span, whereas genes for proteins in mitochondrial complex

II function to increase life span. As described below, this pattern also implicates

glucose in regulating life span, because glucose catabolism preferentially uti-

lizes complex I, whereas lipid catabolism preferentially utilizes complex II.

Complex II Produces Fewer Reactive Oxygen

Species than Other Complexes

The effects of mitochondrial impairments to increase life span are not due

to reduced production of ATP (oxidative phosphorylation) since mutations in

complex II function to reduce respiratory function [28], as do impairments in

complexes I, III, IV and V [23]. Conversely, other studies demonstrate that

simply reducing production of ATP is not sufficient to increase life span [23]. In

fact, in yeast, reducing exposure to glucose increased metabolic rate while also

increasing longevity [3]. The most likely mechanism for the striking dichotomy

of mitochondrial complex function with respect to life span is that reactive


oxygen species are normally produced in complex I, III, IV and V, but much

less so in complex II [29–31]. This basic fact about mitochondrial function

explains why inactivating mutations in complex II produce profound sensitivity

to oxidative damage as well as reduced life span [28, 32]. They also suggest the

hypothesis that disruption of mitochondrial complexes I, III, IV and V increase

life span by producing a relative increase in the utilization of complex II over

the other complexes. While such a pattern of fuel use might or might not reduce

ATP synthesis, the actual mechanism extending life span would be, according

to this hypothesis, reduced production of reactive oxygen species due to rela-

tively increased utilization of complex II.

Reduced Complex I Activity Is Associated with

Increased Life Span in Worms, Mice and Humans

As indicated above, genome-wide screening revealed that RNA-interference-

mediated reduction in complex I activity increases life span in Caenorhabditis

elegans [25, 26]. Furthermore, classic genetic screens had previously identified

that mutations in the clk-1 gene [33], which also influences mitochondrial func-

tion [34], increase life span. This gene codes for a protein that converts Q8

ubiquinone, supplied by bacteria, to Q9 and other ubiquinone species, used in

oxidative phosphorylation [34]. Although it is not precisely yet clear how muta-

tions in the clk-1 gene increase life span, the increased life span does not appear

to be due to reduced respiration [35, 36] or increased superoxide dismutase

(SOD) or catalase [36]. However, it has now been demonstrated that the clk-1

mutation depresses complex I activity while sparing complex II activity [37],

thus favoring the utilization of complex II at the expense of complex I.

Similarly, in mice, ablation of the p66shc gene increases life span and reduces

the production of reactive oxygen species, apparently by reducing NADH levels

and thus the utilization of complex I [38]. Interestingly, a mutation in NADH

dehydrogenase, a key enzyme in complex I, is also associated with increased

longevity in humans [39].

Dietary Restriction May Increase Life Span by

Reducing Oxidative Stress through a clk-1-Like Mechanism

Dietary restriction may increase life span through a clk-1-like mechanism,

since effects of dietary restriction and the clk-1 mutants on life span are

reported to be nonadditive [40]. This hypothetical common mechanism does

not appear to require a reduction in metabolic rate, since neither the clk-1 muta-

tion [35, 36] nor dietary restriction [41, 42] reduce mass-specific oxygen


consumption in C. elegans. Similarly, the mechanism by which dietary restric-

tion increases life span in yeast [3] and mammals [1, 43] appears not to require

a reduction in mass-specific oxygen consumption. On the other hand, several

lines of evidence suggest that a final common mechanism by which life-extending

mutations [44, 45] and dietary restriction [46, 47] increase life span entails

reducing oxidative damage. Furthermore, dietary restriction reduces the activ-

ity of complexes I, III and IV [48]; ATP production is presumably maintained

by elevation of the relative complex II function. Similarly, dietary restriction

decreases the production of reactive oxygen species in complex I without

reducing mitochondrial oxygen consumption or reducing the production of

reactive oxygen species in complex II [49–51]. The authors of these elegant

studies concluded that ‘caloric restricted mitochondria released less reactive

oxygen species per unit electron flow, due to a decrease in the reduction degree

of the complex I generator’ [50]. Since mitochondrial oxygen consumption did

not change, this mechanism likely involves an increase in the relative utilization

of complex II. These data suggest that dietary restriction, possibly like the clk-1

mutation, may increase life span by causing a relative reduction in the utiliza-

tion of complex I and a relative enhancement in the utilization of complex II,

without necessarily reducing the overall metabolic rate.

Dietary Restriction Reduces Glycolysis and Increases

Oxidation of Lipids and Amino Acids; Aging Produces

the Opposite Profile

The mechanism by which dietary restriction might produce this relative

shift toward the utilization of complex II is suggested by studies examining

effects of dietary restriction on the expression of metabolic genes and levels of

metabolic products. Dietary restriction in most tissues produces a metabolic

profile indicating a striking shift away from glycolysis and toward lipid metab-

olism, whereas aging produces the opposite profile relative to the young ad libi-

tum profile [2]. The metabolic shift away from glycolysis in dietary restriction

is illustrated by the effect of dietary restriction to increase PDK-4 [52]. PDK-4

is a key inhibitor of pyruvate dehydrogenase, which controls the rate-limiting

step in the recruitment of glucose-derived carbons for complete oxidation in the

Krebs cycle [53]. Conversely, the metabolic shift toward lipid metabolism is

illustrated by the effect of dietary restriction to increase expression of carnitine

palmitoyl transferase 1, the rate-limiting enzyme for the transfer of lipids to the

mitochondria [2, 54]. Similarly, direct analysis of glycolytic enzyme activities

and metabolic products in young and old liver from ad libitum fed and dietary-

restricted mice elegantly demonstrated the same phenomenon, that chronic


dietary restriction strikingly reduces glycolysis while apparently increasing

reliance on lipid (and possibly amino acid) mitochondrial oxidation [55]. While

reducing glycolysis, dietary restriction apparently increases the metabolism of

glucose through the alternative pentose pathway, as evidenced by the induction

of transketolase [2]. Thus dietary restriction produces a shift away from glucose

metabolism in glycolysis, but increased metabolism in the pentose pathway, and

toward lipid and amino acid oxidation, whereas aging produces the opposite

effect. The significance of this metabolic profile for the regulation of life span

is suggested by the observation that antioxidants produced many of the same

effects as did dietary restriction, but failed to prevent the age-related shift

toward glycolysis and also failed to increase life span [56]. As indicated above

and as also described below, the effect of dietary restriction on metabolic gene

expression is highly similar to the effect of hypoglycemia, i.e. the opposite of

the effect of elevated glucose. This similarity further supports that in mammals

effects of dietary restriction on life span are mediated by a reduction in expo-

sure to glucose [1], as is the case in yeast [3].

It should be noted of course that dietary restriction also reduces insulin

secretion, and genetic ablation of insulin-like pathways increases life span in

worms [57] (see Houthoofd et al. in this volume), mice [58] (see Bartke et al. in

this volume) and flies [59] (see chapter by Tatar in this volume). Furthermore,

insulin produces metabolic effects (e.g. increased glycolysis and reduced �-oxi-

dation) that are similar to, and thus potentially similarly as toxic as, those pro-

duced by glucose. These observations initially suggested that dietary restriction

might increase life span by reducing insulin secretion. However, subsequently it

has been shown that dietary restriction and ablation of the insulin-like pathway

produce additive effects in worms [60] and mice [61], leading many investiga-

tors to conclude that dietary restriction increases life span through a mechanism

other than reducing insulin secretion. This conclusion was questioned by

elegant studies in flies [62], but as discussed in the chapter by Tatar in this

volume, even those studies are not definitive. On the other hand, it should be

noted that not all tissues are equally insulin sensitive, and in fact about half of

the glucose disposal under normal conditions is not regulated by insulin.

Therefore we hypothesize that aging is driven by the proglycolytic gene profile

produced by both insulin and glucose, but that dietary restriction increases life

span only by reducing glucose exposure because the reduction in insulin secre-

tion is balanced by increased insulin sensitivity. Thus dietary restriction is addi-

tive with ablation of the insulin-like pathway because further reduction of

glycolysis in insulin-sensitive tissues is additive with the reduction in glycolysis

produced by dietary restriction. In short, we propose that the glucose switch

with hysteresis is a final common pathway between dietary restriction and the

insulin-like pathways.


Because diabetes accelerates many age-related pathologies, especially car-

diovascular diseases, hyperglycemia has long been considered a model for

aging, thus leading to the hypothesis that exposure to glucose drives the aging

process [63, 64]. Among many commonalities between diabetes and aging is

that both conditions entail mitochondrial impairments that probably drive many

of the pathologies associated with those conditions [65, 66]. An interesting

model of aging has been developed in which mitochondrial function is impaired

by genetic deletion of a mitochondrial transcription factor in specific tissues

[67]. This model is characterized by progressive failure of function and pro-

gressive reduction in ATP synthesis in a variety of tissues, and, as has been sug-

gested for aging, the reduced metabolic capacity was initially thought to be

the cause of the progressive pathology. However, detailed examination of gene

expression demonstrated that before significant pathology developed, genes

that stimulated glycolysis were induced and genes that stimulated �-oxidation

were inhibited [67]. The authors concluded that ‘at least some of the secondary

gene expression alterations in mitochondrial cardiomyopathy do not compen-

sate but rather directly contribute to heart failure progression’ [67]. In view of

the evidence discussed above, it is clear why this profile of gene expression

would produce pathologies and supports the hypothesis that the impairments in

mitochondrial function observed during aging in many studies and tissues [66]

could be secondary to the switch in metabolism toward enhanced glycolysis and

reduction in �-oxidation.

Glucose Oxidation Favors Complex I, Lipid/Amino Acid

Oxidation Favors Complex II

The significance of the shift in source of carbon atoms for oxidation pro-

duced by dietary restriction may be that the oxidation of lipids and amino acids

depends much more on mitochondrial complex II than on (free-radical generat-

ing) complex I, whereas glucose oxidation depends much more on complex I

than on complex II. When glucose is broken down by glycolysis, the only

reducing equivalents it makes are in the form of NADH. When the final carbon

product of glucose, pyruvate, is metabolized in the Krebs cycle, almost all the

reducing equivalents are produced in the form of NADH, except for one step

at complex II (succinate dehydrogenase) that makes (then oxidizes) FADH2.

Ultimately the metabolism of one molecule of glucose produces an NADH:

FADH2 ratio of 5:1 [53, p. 20]. In contrast, when lipids are broken down by

�-oxidation (fatty acid counterpart to glycolysis), an equal number of NADH

and FADH2 molecules are formed. When the lipid-derived carbons are metabo-

lized in the Krebs cycle, reducing equivalents are produced in the ratio of


3 NADH molecules per FADH2 molecule. Therefore ultimately lipid metabo-

lism yields an NADH:FADH2 ratio of about 2:1 [53, p. 38] or even less if the

fatty acid contains enough carbon atoms. For example, when one molecule of

palmitate is oxidized, it produces 15 molecules of FADH2 and 31 molecules of

NADH, which are ultimately oxidized to produce a net total of 129 ATP mole-

cules. In contrast, production of the same number of ATP molecules from glu-

cose would entail producing then oxidizing 8.66 FADH2 and 43.3 NADH

molecules. Amino acid oxidation also proceeds by a similar 2-step mechanism

yielding an NADH:FADH2 ratio between that of lipids and that of glucose, the

precise number depending on the specific amino acid. The significance of this

shift in the NADH:FADH2 ratio is that NADH is oxidized only at mitochondr-

ial complex I, whereas FADH2 is oxidized only at complex II [53, p. 17]. Thus

palmitate oxidation entails utilizing complex II at roughly twice the (FADH2-

dependent) rate as glucose oxidation entails. Therefore shifting away from glu-

cose utilization toward lipid and amino acid utilization would be expected to

substantially reduce the production of reactive oxygen species, without neces-

sarily reducing ATP production. As described below, other beneficial effects

also occur as a result of this altered pattern of glucose fuel use, including a shift

toward producing antioxidizing NADPH and increased protein and lipid turn-

over, which reduces the accumulation of oxidized protein and lipids.

Potential Cumulative Toxic Effect of Glucose on

Neuroendocrine Neurons Regulating Metabolic Function

A guiding hypothesis for our research program for almost 20 years has

been that longevity is governed in part by a cumulative toxic effect of glucose,

particularly on glucose-sensitive neuroendocrine cells in the hypothalamus and

pancreas, a phenomenon we referred to as glucose hysteresis [13, 14]. Similarly

Masoro et al. [1] proposed the hypothesis that ‘dietary restriction retards the

aging processes by altering the characteristics of (glucose) fuel use’. Our ‘glucose

hysteresis’ hypothesis and Masoro’s ‘glucose fuel use’ hypothesis are potentially

complementary and could represent two aspects of the same overall mechanism,

but exactly how these mechanisms are related has been unclear until recently

(see below).

Our hypothesis suggested that key peptides expressed in glucose-stimulated

hypothalamic neurons would: (1) be reduced during aging; (2) be reduced by

fasting and in genetic obesity; (3) be reduced by treatment with the glucose

toxin gold thioglucose; (4) be stimulated by glucose and other nutritional fac-

tors; (5) exert catabolic neuroendocrine effects, and impairment in the function

of such peptides would exert anabolic effects; (6) be irreversibly impaired by


prolonged exposure to elevated glucose. In a series of studies we have found

that hypothalamic �-melanocyte-stimulating hormone, which is produced from

the proopiomelanocortin (POMC) precursor and is preferentially reduced dur-

ing aging [68–73], is reduced by fasting and in genetic obesity [74], and is

reduced by gold thioglucose [75]. POMC neurons are stimulated by glucose

and other nutritional factors [76], and transgenic correction of reduced hypo-

thalamic POMC corrects impairments in glucose homeostasis in obese mice

[77]. Thus age-related impairments in hypothalamic POMC could plausibly

contribute to age-related obesity and metabolic impairments. However,

although we hypothesized that cumulative toxic effects of glucose cause the

age-related reduction in hypothalamic POMC, the mechanism by which glu-

cose would cause such a toxic effect has until recently been elusive.

Glucose Metabolism Is Necessary for Effects of Glucose on

Glucose-Regulated Neurons: Key Role for the

Production of NADH

To further assess the hypothesis that cumulative exposure to glucose may

drive hypothalamic impairments during aging, it was necessary to assess in

more detail the mechanisms by which these neurons sense glucose. Such stud-

ies have been useful for two reasons. First, determination of key elements of the

glucose-sensing apparatus has provided useful markers to test the generality

that glucose-sensitive neurons are specifically sensitive to aging and to protec-

tive effects of dietary restriction. Second, assessment of the sensing mecha-

nisms suggested mechanisms, described below, mediating the vulnerability of

such neurons to glucose toxicity.

Our working hypothesis was that hypothalamic neurons sense glucose

through a mechanism similar to that utilized by pancreatic �-cells. It is well estab-

lished that the pancreatic form of the enzyme glucokinase constitutes a key com-

ponent of the glucose-sensing mechanism in pancreatic �-cells [78–82]. We

observed that the pancreatic form of glucokinase is expressed in hypothalamic

neurons, but not significantly in the rest of the brain [83], consistent with results

by Jetton et al. [84] and corroborated in detail by Lynch et al. [85]. Based on these

results, we examined in greater detail the extent to which hypothalamic neurons

sense glucose through mechanisms similar to those of pancreatic �-cells.

Extensive examination with both metabolic inhibitors and glycolytic inter-

mediates demonstrated that, like pancreatic �-cells, hypothalamic neurons

sense glucose through glucose metabolism [83]. In particular, inhibitors of glu-

cokinase blocked the response of hypothalamic neurons. However, surprisingly

a key step was the conversion of NAD� to NADH and not, as expected, the


production of ATP [83]. The significance of this observation became clear

when it was found that the NADH shuttle system plays a key role in mediating

effects of pancreatic �-cells to glucose [86]. Of even greater potential signifi-

cance, these studies suggest that glucose metabolism may drive specific neu-

roendocrine age-related impairments, through the conversion of NAD� to

NADH, a mechanism similar to that reported in yeast [87].

Glucose Regulates Its Own Metabolic Fate:

The Glucose Switch Gene Profile

As indicated above, several lines of evidence suggested that protective

effects of dietary restriction could be mediated by reduction of glucose [13]

leading to changes in glucose utilization [1] through ‘metabolic reprogram-

ming’ [2], but the mechanism mediating these effects has remained unclear. To

address this question we sought to discover genes regulated by glucose using

DNA microarray analysis. In our studies of the regulation of POMC by leptin,

we had found that gene regulation often occurs more robustly to ablation of a

signal than to enhancement of a signal [74]. Therefore to discover genes regu-

lated by glucose, we examined molecular responses to low glucose (hypo-

glycemia) compared to normal glucose (euglycemia) [88]. We examined

responses in the hypothalamus since we hypothesized that neuroendocrine

responses to glucose would be particularly important in mediating effects of

glucose on life span [13]. Hypoglycemia was produced by injecting mice that

had been food deprived for 48 h with insulin; therefore initially we could not

determine if the regulation was due to fasting or hypoglycemia; however, we

subsequently demonstrated with RT-PCR that almost all genes regulated by

fasting alone were also regulated similarly by hypoglycemia alone [88].

In our initial study, using a small cDNA microarray of our own fabrication,

we observed only a fairly small number of genes induced in association with

hypoglycemia [88]. Among these genes were the glucose transporter GLUT-1,

and the transcription factor CITED-1, also known as p300/CBP-interacting pro-

tein. The induction of GLUT-1 by hypoglycemia suggested that reduced glucose

would produce a compensatory increase in glucose utilization, which would

limit the importance of this mechanism in mediating effects of dietary restric-

tion. However, it seemed unlikely that this analysis provided a comprehensive

view of the molecular effects of glucose, since only about 1,000 genes were rep-

resented on this cDNA microarray. We therefore used the much more extensive

U74 microarray chip to analyze the same RNA samples as were analyzed in the

cDNA microarray study. Focusing specifically on genes coding for intermedi-

ary metabolism function (representing several hundred genes), only 26 met


criteria that we had established as highly predictive of true regulation verifiable

by RT-PCR [89].

Examination of this select group of genes provided a strikingly clear mech-

anism activated by hypoglycemia, a mechanism we call the ‘glucose switch’.

Specifically, hypoglycemia was associated with reduced expression of genes

that stimulate glycolysis (e.g. phosphofructokinase) and mitochondrial utiliza-

tion of carbons derived from glucose (e.g. by inducing PDK-4, which inhibits

pyruvate dehydrogenase). Nevertheless, this study confirmed that GLUT-1 was

induced by hypoglycemia, implying that glucose carbons were being utilized in

nonglycolytic pathways. Several genes whose products stimulate the pentose

pathway were also induced, suggesting that hypoglycemia leads to a shunting of

glucose carbons away from glycolysis towards the pentose pathway. In contrast,

genes coding for peroxisomal proteins and genes involved in protein degrada-

tion and utilization of amino acids were induced by hypoglycemia. Furthermore,

a rate-limiting enzyme for the tricarboxylic acid cycle, NADP-dependent isoci-

trate dehydrogenase, was also induced by hypoglycemia, suggesting that overall

respiration might actually be increased, not decreased, by hypoglycemia. It

should be noted that, as indicated above, this general metabolic profile, away

from glycolysis and toward the pentose pathway, �-oxidation and protein

turnover is very similar to that produced by chronic dietary restriction [2, 54,

55, 90].

This profile of gene expression implies a strikingly clear response to low

glucose that could serve as the mechanistic basis of Masoro’s ‘alternate fuel

use’ and the ‘reprogramming’ hypothesis (see Anderson and Weindruch in this

volume) to explain effects of dietary restriction on life span. This response to

low glucose involves extensive rerouting of glucose and other carbons without

reducing (indeed, possibly elevating) the respiration rate. Hence we refer to this

response as the ‘glucose switch’ profile. In this response to low glucose, glu-

cose carbon atoms are shunted away from production of NADH through glycol-

ysis and toward synthesis of NADPH through the pentose pathway and by the

NADP-dependent form of isocitrate dehydrogenase. Since NADPH is the only

major source of reducing equivalents for antioxidant defense, this profile would

be expected to enhance antioxidant defenses. For example, elevation of

glucose-6-phosphate dehydrogenase, the rate-limiting step in the pentose path-

way, produces dramatic resistance to oxidative damage without changing levels

of catalase or SOD [91, 92], whereas reduction of this enzyme greatly enhances

cellular sensitivity to oxidative stress [93]. The pentose pathway absolutely

requires carbons derived from glucose, so for this essential source of cytoplas-

mic NADPH to function in the presence of low glucose, alternative metabolic

pathways for glucose carbons must be inhibited by low glucose, which they

robustly are at several rate-limiting steps. Indeed, polymorphisms in the gene


for glucose-6-phosphate dehydrogenase are strikingly correlated with life span

across strains of Drosophila: the longest-lived strain exhibited a 64% higher

activity of glucose-6-phosphate dehydrogenase than the shortest-lived strain

[94]. Similarly, the NADP-dependent form of isocitrate dehydrogenase is a

major source of NADPH in mitochondria, and elevation of this enzyme protects

against oxidative stress in vitro [95] and even increases replicative life span

[95]. Furthermore, expression of this enzyme decreases with age [96] and vari-

ants in NADP-dependent isocitrate dehydrogenase were associated with life

span in Drosophila [97].

Interestingly, however, this ‘alternate pattern of fuel use’ produces two

other possibly coincidental antioxidative effects. First, as indicated above, �-

oxidation of lipids, the lipid equivalent of glycolysis to prepare lipid carbons of

oxidative phosphorylation in the Krebs cycle, produces reducing equivalents in

the form of FADH2, rather than the NADH produced by glycolysis. FADH2 is

oxidized at mitochondrial complex II, whereas NADH is oxidized at mitochon-

drial complex I. As reviewed above, reactive oxygen species are produced at

much greater rates in complex I than in complex II, and indeed mutations in

complex I extend life span whereas mutations in complex II reduce life span.

Therefore a shift toward lipid oxidation (and to a lesser extent amino acid oxi-

dation) would be expected to produce a lower rate of reactive oxygen species

compared to deriving energy from glucose. Second, by enhancing the turnover

rate of proteins and lipids, the average half-life of these macromolecules will be

reduced, thus reducing the cellular burden of oxidatively damaged macromole-

cules. A final effect of this ‘alternate fuel use’ would be to dramatically shift the

redox state of the NAD system away from NADH toward NAD�, though the

overall redox state of the cell would be shifted toward a reduced state by eleva-

tion of NADPH and FADH2. It has been reported that the effect of dietary

restriction to increase life span (in yeast) requires the activity of the silencing

protein SIR2 which is dependent on (oxidized) NAD� [87]. Although the pre-

cise role of NAD� in regulating SIR2 activity has been disputed, considerable

evidence supports that the NAD�:NADH ratio serves as a key signal for the

metabolic state of the cell, as we showed in our own studies [83]. The signifi-

cance of this shift has therefore to be fully elucidated.

It should be noted that this antioxidant profile is potentially independent of

the classic antioxidant system involving SOD and catalase, although we did

find that hypoglycemia induced several isoforms of SOD, glutathione peroxi-

dase and glutathione reductase (though not catalase), consistent with effects of

dietary restriction [98, 99]. In fact, we have found that hypoglycemia decreases,

and hyperglycemia increases, expression of other isoforms of SOD and that

ablation of specific isoforms of SOD has no effect on life span in C. elegans

[Yen et al., unpubl. data], consistent with studies in mice [100]. Furthermore,


expression levels of SOD either did not correlate with life span or even corre-

lated negatively with life span across 5 strains of mice (see below). On the other

hand, even to the extent that classic antioxidant defenses play a role in the aging

process, it should be noted that glutathione, a key metabolite in antioxidant

defenses, ultimately derives its reducing potential from NADPH and thus

mainly from glucose-6-phosphate dehydrogenase and NADP-dependent isoci-

trate dehydrogenase.

Based on these observations, we propose that glucose regulates its own

metabolic fate, promoting glycolysis and reducing the relative activity of the

pentose pathway, �-oxidation and amino acid oxidation. Thus glucose produces

a metabolic profile that produces NADH at the expense of NADPH and FADH2.

The net effect of elevated glucose would therefore be to decrease antioxidant

capacity (by decreasing NADPH), decrease the activity of Sir-type histone

acetyltransferases (by converting NAD� to NADH), increase the production of

free radicals (by promoting the oxidation of NADH at complex I, which is the

main site of free radical production, at the expense FADH2 oxidation at complex

II, which is a minor site of free radical generation) and decrease the turnover of

oxidized lipids and proteins (by decreasing �- and amino acid oxidation).

While the ‘glucose switch’ hypothesis clearly suggests a mechanism for

diabetic complications, which are currently thought to be due to oxidative dam-

age [101], by itself it would be insufficient to explain the aging process, since

glucose levels in general do not increase with age. Even for diabetic complica-

tions, however, the glucose switch mechanism by itself does not explain why, as

with aging, impairments develop progressively and are apparently irreversible.

To explain the progressive nature of aging and diabetic complications, we pro-

pose that the glucose switch transcriptional machinery exhibits hysteresis, as

demonstrated with the highly analogous lac operon [19]. As described above,

the lac operon exhibits hysteresis, in that sensitivity to the inducer depends

on the history of exposure: if previously exposed to a high concentration of

inducer, the operon is highly sensitive to inducer, whereas if previously exposed

to a low concentration of inducer, the operon is relatively insensitive to inducer.

Also as described above, there is evidence that the regulation of gene expres-

sion by glucose also exhibits hysteresis [21, 22]. Indeed, to the extent that tran-

scriptional effects of glucose are mediated through NADH, the glucose switch

mechanism directly predicts glucose hysteresis: since glucose induces the

machinery to produce NADH, prior exposure to elevated glucose would subse-

quently lead to more NADH production per glucose molecule, i.e. greater sen-

sitivity to glucose.

Thus two features of the lac operon lead to a comprehensive mechanism

that accounts for key features of aging, dietary restriction and diabetic compli-

cations: that substrates induce the machinery of their own metabolism (in the


case of glucose especially, at the expense of alternative pathways), and that this

self-induction entails hysteresis. Therefore we propose that postprandial excur-

sions of glucose produce a glucose switch response, inducing glycolysis at the

expense of �-oxidation, a state that would tend to persist even when glucose

levels return to their preprandial levels. It should be noted that hysteresis in the

lac operon is observed stochastically across cells so that the lac operon is either

completely on or completely off. Thus we propose that the effect of successive

exposures to (postprandial) glucose would be to produce progressively more

cells in the proglycolytic glucose switch position. It should be noted, however,

that as glycolysis increases and produces monotonic increases in oxidative

damage, especially in mitochondria, and a greater reliance on glycolysis, this

could eventually lead to a reduction in mitochondrial metabolic capacity in the

late phases of senescence, which could be a final precipitating event leading to

mortality, as appears to be the case with mitochondrial impairments produced

by genetic modification [67]. The elevation of insulin secretion early during the

prediabetic phase of type 2 diabetes, followed by a reduction in insulin secre-

tion as the precipitating event of diabetes itself, might be considered an analo-

gous process, and indeed may well be produced by exactly the same mechanism

(increased glycolysis leading to increased secretion, followed by oxidative-

stress-induced ‘burnout’, followed by hyposecretion of insulin and diabetes).

Genetic Correlation between Gene Expression

and Life Span

Other than two studies linking life span to expression levels of glucose-6-

phosphate dehydrogenase [94] or to variants in NADP-dependent isocitrate

dehydrogenase [97], very few studies have directly linked variations in levels of

glucose-sensitive genes to life span. We therefore examined the correlation

between hypothalamic expression of glucose-sensitive genes and average life

span across 5 strains of mice. This analysis was validated by the observation that,

in our analysis, hypothalamic expression of sirtuin 2, the mammalian homolog

of yeast SIR2, was positively correlated with life span (r2 � 74%) and inhibited

by 10 versus 2 mM glucose (p � 0.01) as well as leptin, thus behaving quite sim-

ilarly to yeast Sirt-2 [87]. Over 90% of the genes whose expression correlated

positively with life span (and with an r2 greater than 70%) were inhibited by glu-

cose in vitro. Most of these genes were transcription factors or involved in neu-

roendocrine signaling. For example, expression of polycomb group ring finger 5

correlated positively with life span (r2 � 75%) and was significantly and inde-

pendently inhibited by glucose, leptin and insulin (p � 0.05 for all three).

Interestingly, polycomb transcription factors primarily function to inhibit gene


expression [102], as is the case for genes in the sirtuin family. Conversely, genes

that correlated negatively with life pan were generally induced by glucose. For

example, the transcription factor hypoxia-induced factor 1� is negatively corre-

lated with life span (r2 � 83%) and induced by glucose (p � 0.01). The signifi-

cance of the relationship between hypoxia-induced factor 1� and life span may

be that this gene is an important stimulator of glycolysis [103]. Indeed, pyruvate

dehydrogenase expression is negatively correlated with life span and stimulated

by glucose. Also of some interest in view of the hypothesis of Andersen and

Weindruch (in this volume), the expression of the peroxisome proliferation-

activated receptor � coactivator 1� is also highly and positively correlated with

life span (r2 � 83%). Similarly, hypothalamic expression of the gene for stearoyl

coenzyme A desaturase 1 is positively correlated with life span (r2 � 83%). The

significance of these latter two genes is that they are induced by free fatty acids

(which of course are elevated during fasting and dietary restriction for prolonged

periods of time) and act primarily to increase fat metabolism. Thus across

5 strains of mice, elevated expression of glucose-stimulated genes predicts a

reduced life span and elevated expression of glucose-inhibited genes predicts an

increased life span; expression of genes induced by free fatty acids also predicts

a longer life span.

Problems

The glucose hysteresis hypothesis suggests, though does not require, that

the respiratory quotient (RQ) should increase with age, reflecting more carbo-

hydrate utilization and less �-oxidation, whereas dietary restriction should pro-

duce the opposite effect. While some studies have reported an increase in RQ

with age [104], others have not observed this effect. Similarly, while one study

reported that dietary restriction reduces 24-hour RQ [43] (though not specifi-

cally indicating the significance of this effect), in a different study, although the

same effect of dietary restriction to reduce RQ was observed, the effect was not

significant [1]. In our own studies (unpublished) we observed that chronic

dietary restriction in young mice significantly reduced 24-hour RQ by about the

same amount as reported by McCarter et al. [43] in rats. Although the effect

was small, it was about the same magnitude as observed for the effect of a high-

fat diet. We therefore conclude that while measurements of RQ are not uni-

formly supportive of the glucose switch hypothesis, neither are they uniformly

contradictory. Further studies should clarify this issue. Nevertheless, even if the

reduction of total 24-hour RQ by dietary restriction is not robust, we would sug-

gest that the robust alteration in temporal pattern, in which lipid is the dominant

fuel for a much longer part of the day with dietary restriction than with ad


libitum food intake, would still produce a protective effect by reducing the total

time glucose metabolism is the main source of energy.

The glucose hysteresis hypothesis also predicts that glycolysis relative to

alternate sources of fuel should increase with age, which is observed in the

heart [2], brain [54], and liver [55], but in mouse muscle at least some genes for

glycolysis as well as many other genes coding for mitochondrial function were

reported to decrease with age [105]. On the other hand, this decrease in gly-

colytic gene expression was not observed in muscle tissue from nonhuman pri-

mates [52]. Therefore we hypothesize that even though expression of some

glycolytic genes may decrease with age in mouse muscle, the utilization of

other fuels (e.g. �-oxidation) decreases more so that the net effect is still a shift

toward glycolysis. Similarly, the hypothesis predicts that dietary restriction

should decrease glycolysis relative to the use of other fuels, which is observed

in yeast [5], flies [106], the heart [2], liver [55] and brain [54], but in adipose

tissue, long-term dietary restriction was reported to increase the expression of

glycolytic genes [107]. However, dietary restriction also robustly increased

many other metabolic pathways in adipose tissue, including �-oxidation and the

synthesis of fatty acids. As with mouse muscle in aging, therefore, it is difficult

to determine simply from gene expression whether glycolysis is actually

increased or decreased relative to other metabolic pathways.

A possibly more serious problem with the emphasis on glucose metabo-

lism is that restriction of methionine only is reported to increase life span in rats

[108] and mice [109]. Furthermore, restriction of yeast is reported to extend life

span in flies more robustly than restriction of glucose [110]. On the other hand,

dietary restriction without reduction in protein extends life span as well as

dietary restriction with reduction of protein [111]. Furthermore, reduction of

lipids only without reduction of caloric intake failed to increase life span [112].

Therefore while the effect of (extreme) methionine restriction is indeed inter-

esting, it is unlikely to mediate the effects of dietary restriction on life span, at

least in rats. With regard to the methionine effect in mice, it is of some interest

that methionine restriction reduced plasma glucose, raising the possibility that

restriction of methionine actually increases life span by reducing blood glucose,

as we propose for dietary restriction [109]. Regarding the role of glucose versus

yeast in flies, there are a number of complexities in the design of that study

which precludes definitive interpretation, as described by Tatar in the present

volume. Perhaps the best position to take at the moment is that across species it

is not clear how much of the effect of dietary restriction is due to reduced expo-

sure to glucose. To the extent that the effect is due to reduction in exposure to

glucose, we propose that glucose hysteresis is a mechanism that could explain

these toxic effects of glucose during aging. We further propose that at least with

respect to one major disease of aging, diabetes, glucose hysteresis constitutes


the major mechanism mediating the effects of hyperglycemia to induce diabetic

complications.

Implications: Oxidative Stress and Tumor Burden

As described above, glucose hysteresis is a relatively simple unitary mech-

anism that potentially explains the following apparently otherwise unrelated

phenomena: (i) glycolysis increases with age (in at least some tissues); (ii)

oxidative damage increases with age; (iii) reducing glycolysis or the activity of

ETC complexes I, III, IV and V increases life span, whereas reducing the activ-

ity of ETC complex II reduces life span; (iv) dietary restriction increases the

relative utilization of complex II; (v) intermittent dietary restriction that does

not reduce the average caloric intake nevertheless produces beneficial effects of

dietary restriction, associated with reduced plasma glucose [113]. Thus glucose

hysteresis can plausibly account for the majority of age-related impairments

arising from oxidative stress and their attenuation by dietary restriction.

However, the attenuation by dietary restriction of at least one major age-

related pathology probably does not arise by decreasing oxidative stress: tumor

burden. As described by Klebanov in this volume, dietary restriction primarily if

not exclusively reduces tumor burden by inhibiting the promotion phase of

tumor growth. However, it is difficult to envision a mechanism by which reduc-

ing oxidative damage would inhibit the promotion phase. If anything, it would be

the initiation phase that would probably be sensitive to oxidative stress. These

considerations would seem to suggest that dietary restriction reduces oxidative

stress and tumor burden by two distinct mechanisms and, conversely, that aging

promotes these two pathological processes through distinct mechanisms.

Nevertheless, the glucose switch hypothesis suggests a unifying mechanism:

increased glycolysis during aging. Tumor cells are characterized by a unique

dependency on glycolysis, a phenomenon known as the Warburg effect [114,

115]. This unique dependency on glycolysis possibly arises from mitochondrial

damage, but has the effect of allowing tumor cells to thrive at relatively low

oxygen tensions typically observed in tumors. Of particular interest, this unique

dependency on glycolysis makes tumor cells highly sensitive to the toxic effects

of glycolysis inhibitors, which have therefore been proposed as a promising class

of antitumor agents [116]. It is therefore plausible that tumor cells are particu-

larly sensitive to the low levels of glucose, and the resulting shift away from

glycolysis, that are at least transiently produced by dietary restriction. Indeed,

even a transient (3-hour) reduction in ATP produces robust apoptosis in trans-

formed cells, 48 h later [117]. Furthermore, glycolytic inhibitors have been

shown to produce many of the protective effects of dietary restriction [118]. Thus


reduction in glycolysis reduces both oxidative stress and tumor burden, thereby

potentially accounting for most or all beneficial effects of dietary restriction.

A final implication of glucose hysteresis is its relevance to the genetic con-

trol of longevity. A key aspect of the theory is that it is largely independent of the

classic antioxidant enzymes catalase and SOD, and focuses instead on the pro-

duction of reactive oxygen species in complex I, as well as the role of increased

protein and lipid turnover. However, it is by now well established that across

species, ‘the longer the life span, the lower the rate of mitochondrial oxygen rad-

ical production. This is true even in animal groups that do not conform to the rate

of living theory of aging, such as birds’ [119–121]. Similarly, as described

above, genes involved in the glucose switch mechanism constitute the main set

of genes that limit life span, according to nonbiased genome-wide screening

[26]. In contrast, activities of the classic antioxidant defense enzymes do not

correlate with life span across species [122]. Furthermore, it is now clear that

impairments in classic antioxidant defenses do not reduce life span, even when such

impairments increase oxidative damage [100]. Thus we propose that genetic

influences on the regulation of gene expression by glucose could constitute key

genetic influences that regulate life span. On the other hand, there are at least two

relevant potentially distinct genetic influences, those that influence acute control

of gene expression by glucose and those that influence the development of hys-

teretic effects. We suggest that genetic effects on both of these mechanisms must

play a role in determining life span, since even at a young age species differ in

their rate of production of reactive oxygen species [119] (possibly reflecting at

least in part the acute effects of glucose on complex I activity), but on the other

hand, the age-related increase in oxidative damage, reflecting hysteresis, scales

with life span [123–125]. Thus we suggest that the rate of hysteresis may also be

higher in short-lived than in long-lived species [119].

Context

It is of some interest to place the mechanism of glucose hysteresis into the

context of current approaches being developed in the field of dietary restriction,

as reflected in the present volume. First, Masoro has elegantly developed the

concept that dietary restriction acts through hormesis, a protective mechanism

activated by low-level stressors. Our proposal is quite consistent with this

hypothesis, since hypoglycemia is a classic inducer of the stress response. Thus,

for example, reduction of blood glucose stimulates secretion of glucocorticoids

(a classic stress response) in mice [126], rats [127] and humans [128]. Simi-

larly, reduction of blood glucose activates the sympathetic nervous system, also

a classic stress response, in mice [129], rats [127] and humans [128]. Indeed,


hypoglycemia even produces homotypic densitization, a classic feature of stress

responses, in mice [129], rats [127] and humans [130].

Anderson and Weindruch in the present volume extend the concept, already

articulated by Weindruch and colleagues previously [2], that protective effects of

dietary restriction may be mediated through ‘transcriptional reprogramming’.

Clearly glucose hysteresis represents precisely an example of transcriptional

reprogramming, and indeed the work of Weindruch and colleagues represents

some of the most important evidence for the mechanism. Bartke et al. in the pre-

sent volume examine the allied concept that effects of the insulin-like pathway

and dietary restriction are mediated by a common set of genes. While so far the

evidence does not support this hypothesis, as argued above, it may well be the

case that the proglycolytic effects of insulin drive senescence in insulin-sensitive

tissues, whereas the proglycolytic effects of glucose drive senescence in insulin-

insensitive tissues.

Morgan et al. in the present volume develop an elegant argument that many

age-related pathologies arise from age-related increases in inflammatory

processes and that dietary restriction attenuates age-related impairments in part

by reducing inflammation. Morgan et al. also speculate about mechanisms

mediating effects of dietary restriction to reduce inflammation, including a role

for glucose acting through the receptor for advanced glycation end products.

However, while this is a plausible explanation for the effect of dietary restric-

tion, this mechanism fails to explain why inflammation increases with age,

since in general, certainly in rodents, plasma glucose does not increase with

age. Furthermore, elevated glucose induces cytokines very rapidly (within a

few hours) through a mechanism requiring glucose metabolism [131], whereas

advanced glycation end products develop far too slowly to account for such

rapid effects. However, as emphasized by Morgan et al., at least some cytokines

are induced by reactive oxygen species. We therefore suggest that inflammation

increases with age because of increased reactive oxygen species, secondary to

glucose hysteresis, and that dietary restriction retards this age-related increase

in inflammation by reducing exposure to glucose.

Houthoofd et al. in the present volume describe the value of C. elegans as

a model organism. We completely agree that C. elegans constitutes possibly the

most powerful system to study mechanisms of aging, and indeed much of the

most compelling support for the mechanism of glucose hysteresis comes from

studies in C. elegans. Houthoofd et al. also argue strongly against the rate of

living theory and indicate that dietary restriction increases life span without

reducing metabolic rate, a conclusion also drawn by investigators studying

rodents [1, 43]. In our view one of the most compelling aspects of the glucose

hysteresis model is that, by invoking substrate competition, it can explain how

the oxidative damage can decrease even without reducing ATP production.


Interestingly, Tatar in the present volume reviews the use of Drosophila in aging

research and carefully analyzes difficulties in interpreting studies that suggest a

minimal role for glucose mediating the effects of dietary restriction [110]. As

discussed in some detail elsewhere [132], a number of results suggest that

mechanisms mediating effects of dietary restriction in Drosophila, and indeed

mechanisms of senescence themselves, may be distinct to some extent from

those in other species. It should be noted that of the many organisms and exper-

imental paradigms described in the present review in support of the glucose

hysteresis hypothesis, none have involved Drosophila. This corroborates the

importance of studying these mechanisms in a wide variety of organisms to

probe for the generality of those mechanisms. On the other hand, as described

by Mattison et al. in the present volume, so far dietary restriction appears to

produce similar results in nonhuman primates, including a reduction in mean

blood glucose. We take this as evidence that dietary restriction is therefore

likely to actually increase life span in nonhuman primates.

In the final two chapters of the present volume, effects of dietary restriction

on specific disease processes, Alzheimer’s disease and cancer, are discussed. We

should note that we have no plausible mechanism through which the specific

effects of dietary restriction on �-amyloid peptide generation described by

Pasinetti et al. in the present volume can be accounted for by glucose hysteresis,

and agree with them that insulin, rather than glucose, probably plays a key role.

On the other hand, in view of the critical role of oxidative stress in mediating

�-amyloid peptide toxicity in particular [133] and neurodegeneration in

Alzheimer’s disease in general [134], it is plausible that glucose hysteresis plays a

role in the age-related neurodegeneration associated with Alzheimer’s disease.

Finally, in the last chapter Klebanov makes a strong case that dietary restriction

reduces cancer burden specifically by interfering with the promotion phase of

tumor growth, not the initiation phase. As noted above, the promotion phase is

unlikely to be dependent on oxidative stress, in contrast to the initiation phase.

However, also as indicated above, tumor cells become increasingly dependent on

glycolysis as they progress, due to the Warburg effect [114]. Therefore, the effect

of dietary restriction to interfere with the promotion phase is plausibly due

directly to the reduction of glycolytic capacity, rather than the reduction of oxida-

tive stress.

Other Age-Related Diseases

One of the most challenging problems in gerontology is to account for the

age dependency of diseases whose incidence increases with age. As indicated

above, at least for Alzheimer’s disease, the age dependency can be explained at


least in part by the increase in oxidative stress with age, which in our view can

in turn be accounted for by glucose hysteresis. In fact, it is plausible to hypothesize

that the dependence of most age-related diseases (except, as indicated above,

possibly cancer) can be accounted for by an age-related increase in oxidative

stress. For example, Huntington’s disease appears to be due to a reduction in the

utilization of ETC complex II [135]. Furthermore, dietary restriction substan-

tially ameliorates symptoms in a mouse transgenic model of Huntington’s dis-

ease [136]. We therefore propose that the increased penetrance of the

Huntington’s disease gene with age is due to a gradual reduction in the utiliza-

tion of complex II in neurons during aging, secondary to glucose hysteresis,

which in patients with Huntington’s disease becomes lethal due to a preexisting

impairment in complex II.

With regard to metabolic diseases, while it is clear how glucose hysteresis

would account for age-related increases in diabetic complications, it is not so

evident how this mechanism would account for the increased incidence in (type

2) diabetes with age. Type 2 diabetes is caused by the development of insulin

resistance, usually accompanied by increased insulin secretion, eventually fol-

lowed by pancreatic decompensation [12]. Oxidative stress plays a causal role

in multiple forms of insulin resistance, many of which can be ameliorated by

antioxidant treatments [138]. Similarly oxidative stress is implicated in pancre-

atic �-cell impairments [139]. Thus it is clear that increased oxidative stress

during aging, secondary to glucose hysteresis, could plausibly account for the

increased incidence of type II diabetes.

Testing the Hypothesis: Reversibility of Senescence

Although a substantial array of evidence supports that glucose hysteresis

mediates at least part of the effects of dietary restriction, the hypothesis is never-

theless still subject to falsifiability. For example, the hypothesis predicts that fac-

tors which mediate effects of glucose on gene expression also mediate at least

some of the effects of dietary restriction on gene expression. The hypothesis fur-

ther predicts that manipulations that block effects of hypoglycemia on gene

expression would also block at least some effects of dietary restriction on gene

expression, and, more importantly, would attenuate beneficial effects of dietary

restriction. For example, it may be possible to block effects of hypoglycemia on

gene expression by ablating specific transcriptional factors or cofactors [140]. If

blocking hypoglycemia-induced gene expression fails to block beneficial effects

of dietary restriction, this would effectively falsify the glucose switch hypothesis.

Another obvious prediction is that reduction of plasma glucose by, for exam-

ple, transgenic expression of a glucose transporter, to the same extent as is


observed with dietary restriction, should mimic effects of dietary restriction with-

out caloric restriction. While this would indeed constitute a test of the hypothesis,

there would be two caveats in interpreting these results. First, as indicated above,

considerable evidence suggests that glucose signaling as well as the toxic effects

of glucose are mediated by the production of NADH. If reduction of plasma glu-

cose is accompanied by an increase in lactate, and lactate is used as an alternative

fuel for glucose, lactate would plausibly produce the same levels of NADH as

glucose, and therefore might prevent the beneficial effects of reduced glucose.

This could be assessed by examining the expression of genes regulated by glu-

cose: if hypoglycemia-induced genes are not induced by a reduction in plasma

glucose, this would suggest that some compensatory mechanism, likely lactate, is

preventing the beneficial effects of lower plasma glucose. Second, as also

described above, some evidence suggests that the total 24-hour RQ is not reduced

by dietary restriction and, if so, we would argue that the protective effect arises

from reducing the total amount of time that tissues are predominantly using glu-

cose as a fuel. If so, simply reducing total exposure to glucose may not be ade-

quate to reproduce the extended period of time that cells are protected by

predominant �-oxidation. If so, a test of the theory would require reproducing the

pattern of RQ produced by dietary restriction, possibly using inducible promot-

ers.

Another implication of the hypothesis is that it may be possible to reverse

age-related impairments by producing carefully controlled hypoglycemia at

levels lower than can be produced by optimum dietary restriction. Dietary

restriction at 50% ad libitum levels, about the most restriction compatible with

extended life, only reduces plasma glucose to around 4.5 mM. However, coun-

terregulatory and other protective responses are not activated until lower glu-

cose levels of 3.6 mM or possibly even lower are reached, whereas cognitive

impairments are not observed until much lower levels yet, around 2.6 mM.

Therefore there is a potentially safe range of plasma glucose that would allow

the production of even more potent beneficial effects than are produced by

dietary restriction. The hysteretic behavior of the lac operon suggests that even

transient repetitive reductions in glucose could reverse the proglycolytic glu-

cose switch transcriptional state, thereby reversing the increase in glycolysis,

oxidative damage and tumor burden. Studies to assess this hypothesis are cur-

rently under way.

References

1 Masoro EJ, McCarter RJ, Katz MS, McMahan CA: Dietary restriction alters characteristics of glu-

cose fuel use. J Gerontol 1992;47:B202–B208.


2 Lee CK, Allison DB, Brand J, Weindruch R, Prolla TA: Transcriptional profiles associated with

aging and middle age-onset caloric restriction in mouse hearts. Proc Natl Acad Sci USA 2002;

99:14988–14993.



2002;418:344–348.

4 Lewis M: The lac repressor. C R Biol 2005;328:521–548.

5 DeRisi JL, Iyer VR, Brown PO: Exploring the metabolic and genetic control of gene expression on

a genomic scale. Science 1997;278:680–686.

6 Roche E, Assimacopoulos-Jeannet F, Witters LA, Perruchoud B, Yaney G, Corkey B, Asfari M,

Prentki M: Induction by glucose of genes coding for glycolytic enzymes in a pancreatic beta-cell

line (INS-1). J Biol Chem 1997;272:3091–3098.

7 Abbot EL, McCormack JG, Reynet C, Hassall DG, Buchan KW, Yeaman SJ: Diverging regulation

of pyruvate dehydrogenase kinase isoform gene expression in cultured human muscle cells. FEBS

J 2005;272:3004–3014.

8 Pilkis SJ, Granner DK: Molecular physiology of the regulation of hepatic gluconeogenesis and

glycolysis. Annu Rev Physiol 1992;54:885–909.

9 Carlson M: Glucose repression in yeast. Curr Opin Microbiol 1999;2:202–207.

10 Ronne H: Glucose repression in fungi. Trends Genet 1995;11:12–17.

11 Ruderman NB, Saha AK, Vavvas D, Witters LA: Malonyl-CoA, fuel sensing, and insulin resis-

tance. Am J Physiol 1999;276:E1–E18.

12 Mobbs CV: Neurohumoral hysteresis as a mechanism for senescence: comparative aspects; in

Scanes CG, Schriebman MP (eds): Development, Maturation, and Senescence of the Neuroendocrine

System. New York, Academic Press, 1989, pp 223–252.

13 Mobbs CV: Neurotoxic effects of estrogen, glucose, and glucocorticoids: neurohumoral hysteresis

and its pathological consequences during aging. Rev Biol Res Aging 1990;4:201–228.

14 Mobbs CV: Genetic influences on glucose neurotoxicity, aging, and diabetes: a possible role for

glucose hysteresis. Genetica 1993;91:239–253.

15 Mobbs CV: Molecular hysteresis: residual effects of hormones and glucose on genes during aging.

Neurobiol Aging 1994;15:523–534.

16 Pfaff DW, Brooks PJ, Funabashi T, Pfaus JG, Mobbs CV: Gene memory in neuroendocrine and

behavioural systems. Ciba Found Symp 1992;168:165–183.

17 Burch JB, Weintraub H: Temporal order of chromatin structural changes associated with activation

of the major chicken vitellogenin gene. Cell 1983;33:65–76.

18 Ringrose L, Paro R: Epigenetic regulation of cellular memory by the Polycomb and Trithorax

group proteins. Annu Rev Genet 2004;38:413–443.

19 Ozbudak EM, Thattai M, Lim HN, Shraiman BI, Van Oudenaarden A: Multistability in the lactose

utilization network of Escherichia coli. Nature 2004;427:737–740.

20 Laurent M, Charvin G, Guespin-Michel J: Bistability and hysteresis in epigenetic regulation of the

lactose operon: since Delbruck, a long series of ignored models. Cell Mol Biol (Noisy-le-Grand)

2005;51:583–594.

21 Roy S, Sala R, Cagliero E, Lorenzi M: Overexpression of fibronectin induced by diabetes or high

glucose: phenomenon with a memory. Proc Natl Acad Sci USA 1990;87:404–408.

22 Nathan DM, Cleary PA, Backlund JY, Genuth SM, Lachin JM, Orchard TJ, Raskin P, Zinman B:

Intensive diabetes treatment and cardiovascular disease in patients with type 1 diabetes. N Engl J

Med 2005;353:2643–2653.

23 Dillin A, Hsu AL, Arantes-Oliveira N, Lehrer-Graiwer J, Hsin H, Fraser AG, Kamath RS, Ahringer J,

Kenyon C: Rates of behavior and aging specified by mitochondrial function during development.

Science 2002;298:2398–2401.

24 Lee SS, Lee RY, Fraser AG, Kamath RS, Ahringer J, Ruvkun G: A systematic RNAi screen identi-

fies a critical role for mitochondria in C. elegans longevity. Nat Genet 2003;33:40–48.

25 Hamilton B, Dong Y, Shindo M, Liu W, Odell I, Ruvkun G, Lee SS: A systematic RNAi screen for

longevity genes in C. elegans. Genes Dev 2005;19:1544–1555.

26 Hansen M, Hsu AL, Dillin A, Kenyon C: New genes tied to endocrine, metabolic, and dietary regula-

tion of lifespan from a Caenorhabditis elegans genomic RNAi screen. PLos Genet 2005;1:119–128.


27 Ishii N, Fujii M, Hartman PS, Tsuda M, Yasuda K, Senoo-Matsuda N, Yanase S, Ayusawa D,

Suzuki K: A mutation in succinate dehydrogenase cytochrome b causes oxidative stress and age-

ing in nematodes. Nature 1998;394:694–697.

28 Senoo-Matsuda N, Hartman PS, Akatsuka A, Yoshimura S, Ishii N: A complex II defect affects

mitochondrial structure, leading to ced-3- and ced-4-dependent apoptosis and aging. J Biol Chem

2003;278:22031–22036.

29 Herrero A, Barja G: Localization of the site of oxygen radical generation inside the complex I

of heart and nonsynaptic brain mammalian mitochondria. J Bioenerg Biomembr 2000;32:

609–615.

30 Lenaz G: The mitochondrial production of reactive oxygen species: mechanisms and implications

in human pathology. IUBMB Life 2001;52:159–164.

31 Barja G: Aging in vertebrates, and the effect of caloric restriction: a mitochondrial free radical

production-DNA damage mechanism? Biol Rev Camb Philos Soc 2004;79:235–251.

32 Senoo-Matsuda N, Yasuda K, Tsuda M, Ohkubo T, Yoshimura S, Nakazawa H, Hartman PS, Ishii N:

A defect in the cytochrome b large subunit in complex II causes both superoxide anion overpro-

duction and abnormal energy metabolism in Caenorhabditis elegans. J Biol Chem 2001;276:

41553–41558.

33 Lakowski B, Hekimi S: Determination of life-span in Caenorhabditis elegans by four clock genes.

Science 1996;272:1010–1013.

34 Jonassen T, Larsen PL, Clarke CF: A dietary source of coenzyme Q is essential for growth of long-

lived Caenorhabditis elegans clk-1 mutants. Proc Natl Acad Sci USA 2001;98:421–426.

35 Branicky R, Benard C, Hekimi S: clk-1, mitochondria, and physiological rates. Bioessays 2000;

22:48–56.

36 Braeckman BP, Houthoofd K, Brys K, Lenaerts I, De Vreese A, Van Eygen S, Raes H, Vanfleteren

JR: No reduction of energy metabolism in Clk mutants. Mech Ageing Dev 2002;123:1447–1456.

37 Kayser EB, Sedensky MM, Morgan PG, Hoppel CL: Mitochondrial oxidative phosphorylation is

defective in the long-lived mutant clk-1. J Biol Chem 2004;279:54479–54486.

38 Nemoto S, Combs CA, French S, Ahn BH, Fergusson MM, Balaban RS, Finkel T: The mammalian

longevity-associated gene product p66shc regulates mitochondrial metabolism. J Biol Chem

2006;281:10555–10560.

39 Tanaka M, Gong J, Zhang J, Yamada Y, Borgeld HJ, Yagi K: Mitochondrial genotype associated

with longevity and its inhibitory effect on mutagenesis. Mech Ageing Dev 2000;116:65–76.

40 Lakowski B, Hekimi S: The genetics of caloric restriction in Caenorhabditis elegans. Proc Natl

Acad Sci USA 1998;95:13091–13096.



1359–1369.

42 Houthoofd K, Braeckman BP, Lenaerts I, Brys K, De Vreese A, Van Eygen S, Vanfleteren JR:

Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span

in Caenorhabditis elegans. Exp Gerontol 2002;37:1371–1378.

43 McCarter R, Masoro EJ, Yu BP: Does food restriction retard aging by reducing the metabolic rate?

Am J Physiol 1985;248:E488–E490.

44 Johnson TE, Cypser J, de Castro E, de Castro S, Henderson S, Murakami S, Rikke B, Tedesco P,

Link C: Gerontogenes mediate health and longevity in nematodes through increasing resistance to

environmental toxins and stressors. Exp Gerontol 2000;35:687–694.

45 Obici S, Feng Z, Tan J, Liu L, Karkanias G, Rossetti L: Central melanocortin receptors regulate

insulin action. J Clin Invest 2001;108:1079–1085.

46 Sohal RS, Weindruch R: Oxidative stress, caloric restriction, and aging. Science 1996;273:59–63.

47 Lass A, Sohal BH, Weindruch R, Forster MJ, Sohal RS: Caloric restriction prevents age-associated

accrual of oxidative damage to mouse skeletal muscle mitochondria. Free Radic Biol Med 1998;25:

1089–1097.

48 Desai VG, Weindruch R, Hart RW, Feuers RJ: Influences of age and dietary restriction on gastroc-

nemius electron transport system activities in mice. Arch Biochem Biophys 1996;333:145–151.


49 Gredilla R, Sanz A, Lopez-Torres M, Barja G: Caloric restriction decreases mitochondrial free

radical generation at complex I and lowers oxidative damage to mitochondrial DNA in the rat

heart. Faseb J 2001;15:1589–1591.

50 Lopez-Torres M, Gredilla R, Sanz A, Barja G: Influence of aging and long-term caloric restriction

on oxygen radical generation and oxidative DNA damage in rat liver mitochondria. Free Radic

Biol Med 2002;32:882–889.

51 Sanz A, Caro P, Ibanez J, Gomez J, Gredilla R, Barja G: Dietary restriction at old age lowers mito-

chondrial oxygen radical production and leak at complex I and oxidative DNA damage in rat

brain. J Bioenerg Biomembr 2005;37:83–90.

52 Kayo T, Allison DB, Weindruch R, Prolla TA: Influences of aging and caloric restriction on the

transcriptional profile of skeletal muscle from rhesus monkeys. Proc Natl Acad Sci USA 2001;98:

5093–5098.

53 Salway JG: Metabolism at a Glance. Oxford, Blackwell, 1994.

54 Lee CK, Weindruch R, Prolla TA: Gene-expression profile of the ageing brain in mice. Nat Genet

2000;25:294–297.

55 Hagopian K, Ramsey JJ, Weindruch R: Influence of age and caloric restriction on liver glycolytic

enzyme activities and metabolite concentrations in mice. Exp Gerontol 2003;38:253–266.

56 Lee CK, Pugh TD, Klopp RG, Edwards J, Allison DB, Weindruch R, Prolla TA: The impact of �-

lipoic acid, coenzyme Q10 and caloric restriction on life span and gene expression patterns in mice.


57 Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G: daf-2, an insulin receptor-like gene that regulates

longevity and diapause in Caenorhabditis elegans. Science 1997;277:942–946.

58 Brown-Borg HM, Borg KE, Meliska CJ, Bartke A: Dwarf mice and the ageing process. Nature

1996;384:33.


2003;299:1346–1351.


independent of the Ins/IGF-1 signalling pathway in Caenorhabditis elegans. Exp Gerontol 2003;

38:947–954.




Science 2002;296:319.

63 Cerami A: Hypothesis: glucose as a mediator of aging. J Am Geriatr Soc 1985;33:626–634.

64 Asif M, Egan J, Vasan S, Jyothirmayi GN, Masurekar MR, Lopez S, Williams C, Torres RL, Wagle D,

Ulrich P, Cerami A, Brines M, Regan TJ: An advanced glycation endproduct cross-link breaker

can reverse age-related increases in myocardial stiffness. Proc Natl Acad Sci USA 2000;97:

2809–2813.

65 Rolo AP, Palmeira CM: Diabetes and mitochondrial function: role of hyperglycemia and oxidative

stress. Toxicol Appl Pharmacol 2006;212:167–178.

66 Wallace DC: A mitochondrial paradigm of metabolic and degenerative diseases, aging, and can-

cer: a dawn for evolutionary medicine. Annu Rev Genet 2005;39:359–407.

67 Hansson A, Hance N, Dufour E, Rantanen A, Hultenby K, Clayton DA, Wibom R, Larsson NG:

A switch in metabolism precedes increased mitochondrial biogenesis in respiratory chain-deficient

mouse hearts. Proc Natl Acad Sci USA 2004;101:3136–3141.

68 Gruenewald DA, Matsumoto AM: Age-related decrease in proopiomelanocortin gene expression

in the arcuate nucleus of the male rat brain. Neurobiol Aging 1991;12:113–121.

69 Lloyd JM, Scarbrough K, Weiland NG, Wise PM: Age-related changes in proopiomelanocortin

(POMC) gene expression in the periarcuate region of ovariectomized rats. Endocrinology

1991;129:1896–1902.

70 Nelson JF, Bender M, Schacter BS: Age-related changes in proopiomelanocortin messenger

ribonucleic acid levels in hypothalamus and pituitary of female C57Bl/6J mice. Endocrinology

1988;123:340–344.

71 Abel TW, Rance NE: Proopiomelanocortin gene expression is decreased in the infundibular

nucleus of postmenopausal women. Brain Res Mol Brain Res 1999;69:202–208.


72 Abel TW, Rance NE: Stereologic study of the hypothalamic infundibular nucleus in young and

older women. J Comp Neurol 2000;424:679–688.

73 Abel TW, Voytko ML, Rance NE: The effects of hormone replacement therapy on hypothalamic

neuropeptide gene expression in a primate model of menopause. J Clin Endocrinol Metab 1999;84:

2111–2118.

74 Mizuno TM, Kleopoulos SP, Bergen HT, Roberts JL, Priest CA, Mobbs CV: Hypothalamic pro-

opiomelanocortin mRNA is reduced by fasting and in ob/ob and db/db mice, but is stimulated by

leptin. Diabetes 1998;47:294–297.

75 Bergen HT, Mizuno TM, Taylor J, Mobbs CV: Hyperphagia and weight gain after gold-thioglucose:

relation to hypothalamic neuropeptide Y and proopiomelanocortin. Endocrinology 1998;139:

4483–4488.

76 Ibrahim N, Bosch MA, Smart JL, Qiu J, Rubinstein M, Ronnekleiv OK, Low MJ, Kelly MJ:

Hypothalamic proopiomelanocortin neurons are glucose responsive and express K(ATP) chan-

nels. Endocrinology 2003;144:1331–1340.

77 Mizuno TM, Kelly K, Pasinetti GM, Roberts JL, Mobbs CV: Transgenic neuronal expression of

proopiomelanocortin attenuates fasting-induced hyperphagia and reverses metabolic impairments

in leptin-deficient obese mice. Diabetes 2003;52:2675–2683.

78 Matschinsky FM: Banting Lecture 1995 – A lesson in metabolic regulation inspired by the

glucokinase glucose sensor paradigm. Diabetes 1996;45:223–241.

79 Matschinsky FM, Collins HW: Essential biochemical design features of the fuel-sensing system in

pancreatic �-cells. Chem Biol 1997;4:249–257.

80 Matschinsky FM, Glaser B, Magnuson MA: Pancreatic �-cell glucokinase: closing the gap

between theoretical concepts and experimental realities. Diabetes 1998;47:307–315.

81 Matschinsky F, Liang Y, Kesavan P, Wang L, Froguel P, Velho G, Cohen D, Permutt MA, Tanizawa Y,

Jetton TL, et al: Glucokinase as pancreatic beta cell glucose sensor and diabetes gene. J Clin Invest

1993;92:2092–2098.

82 Matschinsky FM: Glucokinase as glucose sensor and metabolic signal generator in pancreatic

�-cells and hepatocytes. Diabetes 1990;39:647–652.

83 Yang XJ, Kow LM, Funabashi T, Mobbs CV: Hypothalamic glucose sensor: similarities to and dif-

ferences from pancreatic �-cell mechanisms. Diabetes 1999;48:1763–1672.

84 Jetton TL, Liang Y, Pettepher CC, Zimmerman EC, Cox FG, Horvath K, Matschinsky FM,

Magnuson MA: Analysis of upstream glucokinase promoter activity in transgenic mice and iden-

tification of glucokinase in rare neuroendocrine cells in the brain and gut. J Biol Chem 1994;269:

3641–3654.

85 Lynch RM, Tompkins LS, Brooks HL, Dunn-Meynell AA, Levin BE: Localization of glucokinase

gene expression in the rat brain. Diabetes 2000;49:693–700.

86 Eto K, Tsubamoto Y, Terauchi Y, Sugiyama T, Kishimoto T, Takahashi N, Yamauchi N, Kubota N,

Murayama S, Aizawa T, Akanuma Y, Aizawa S, Kasai H, Yazaki Y, Kadowaki T: Role of NADH

shuttle system in glucose-induced activation of mitochondrial metabolism and insulin secretion.

Science 1999;283:981–985.

87 Lin SJ, Defossez PA, Guarente L: Requirement of NAD and SIR2 for life-span extension by calo-

rie restriction in Saccharomyces cerevisiae. Science 2000;289:2126–2128.

88 Mastaitis JW, Cheng H, Sealfon SC, Mobbs CV: Acute induction of gene expression in brain and

liver by insulin-induced hypoglycemia. Diabetes 2005;54:952–958.

89 Mobbs CV, Yen K, Mastaitis J, Nguyen H, Watson E, Wurmbach E, Sealfon SC, Brooks A, Salton

SR: Mining microarrays for metabolic meaning: nutritional regulation of hypothalamic gene

expression. Neurochem Res 2004;29:1093–1103.

90 Ward WF: Food restriction enhances the proteolytic capacity of the aging rat liver. J Gerontol

1988;43:B121–B124.

91 Salvemini F, Franze A, Iervolino A, Filosa S, Salzano S, Ursini MV: Enhanced glutathione levels

and oxidoresistance mediated by increased glucose-6-phosphate dehydrogenase expression. J Biol

Chem 1999;274:2750–2757.

92 Ursini MV, Parrella A, Rosa G, Salzano S, Martini G: Enhanced expression of glucose-6-phosphate

dehydrogenase in human cells sustaining oxidative stress. Biochem J 1997;323:801–806.


93 Filosa S, Fico A, Paglialunga F, Balestrieri M, Crooke A, Verde P, Abrescia P, Bautista JM, Martini G:

Failure to increase glucose consumption through the pentose-phosphate pathway results in the

death of glucose-6-phosphate dehydrogenase gene-deleted mouse embryonic stem cells subjected

to oxidative stress. Biochem J 2003;370:935–943.

94 Luckinbill LS, Riha V, Rhine S, Grudzien TA: The role of glucose-6-phosphate dehydrogenase in

the evolution of longevity in Drosophila melanogaster. Heredity 1990;65:29–38.

95 Jo SH, Son MK, Koh HJ, Lee SM, Song IH, Kim YO, Lee YS, Jeong KS, Kim WB, Park JW, Song

BJ, Huh TL, Huhe TL: Control of mitochondrial redox balance and cellular defense against oxidative

damage by mitochondrial NADP�-dependent isocitrate dehydrogenase. J Biol Chem 2001;276:

16168–16176.

96 Kil IS, Lee YS, Bae YS, Huh TL, Park JW: Modulation of NADP(�)-dependent isocitrate dehy-

drogenase in aging. Redox Rep 2004;9:271–277.

97 Da Cunha GL, de Oliveira AK: Citric acid cycle: a mainstream metabolic pathway influencing life

span in Drosophila melanogaster? Exp Gerontol 1996;31:705–715.

98 Sreekumar R, Unnikrishnan J, Fu A, Nygren J, Short KR, Schimke J, Barazzoni R, Nair KS:

Effects of caloric restriction on mitochondrial function and gene transcripts in rat muscle. Am J

Physiol Endocrinol Metab 2002;283:E38–E43.

99 Agarwal S, Sharma S, Agrawal V, Roy N: Caloric restriction augments ROS defense in S. cerevisiae,

by a Sir2p independent mechanism. Free Radic Res 2005;39:55–62.

100 Van Remmen H, Ikeno Y, Hamilton M, Pahlavani M, Wolf N, Thorpe SR, Alderson NL, Baynes

JW, Epstein CJ, Huang TT, Nelson J, Strong R, Richardson A: Life-long reduction in MnSOD

activity results in increased DNA damage and higher incidence of cancer but does not accelerate

aging. Physiol Genomics 2003;16:29–37.

101 Brownlee M: Biochemistry and molecular cell biology of diabetic complications. Nature 2001;414:

813–820.

102 Zhang Y, Cao R, Wang L, Jones RS: Mechanism of Polycomb group gene silencing. Cold Spring

Harb Symp Quant Biol 2004;69:309–317.

103 Acker T, Plate KH: Hypoxia and hypoxia inducible factors (HIF) as important regulators of tumor

physiology. Cancer Treat Res 2004;117:219–248.

104 Rising R, Tataranni PA, Snitker S, Ravussin E: Decreased ratio of fat to carbohydrate oxidation

with increasing age in Pima Indians. J Am Coll Nutr 1996;15:309–312.

105 Lee CK, Klopp RG, Weindruch R, Prolla TA: Gene expression profile of aging and its retardation

by caloric restriction. Science 1999;285:1390–1393.

106 Zinke I, Schutz CS, Katzenberger JD, Bauer M, Pankratz MJ: Nutrient control of gene expression

in Drosophila: microarray analysis of starvation and sugar-dependent response. Embo J 2002;21:

6162–6173.

107 Higami Y, Pugh TD, Page GP, Allison DB, Prolla TA, Weindruch R: Adipose tissue energy metab-

olism: altered gene expression profile of mice subjected to long-term caloric restriction. Faseb J

2004;18:415–417.

108 Orentreich N, Matias JR, DeFelice A, Zimmerman JA: Low methionine ingestion by rats extends

life span. J Nutr 1993;123:269–274.

109 Miller RA, Buehner G, Chang Y, Harper JM, Sigler R, Smith-Wheelock M: Methionine-deficient

diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and

insulin levels, and increases hepatocyte MIF levels and stress resistance. Aging Cell 2005;4:

119–125.

110 Mair W, Piper MD, Partridge L: Calories do not explain extension of life span by dietary restric-

tion in Drosophila. PLos Biol 2005;3:e223.

111 Masoro EJ, Iwasaki K, Gleiser CA, McMahan CA, Seo EJ, Yu BP: Dietary modulation of the pro-

gression of nephropathy in aging rats: an evaluation of the importance of protein. Am J Clin Nutr

1989;49:1217–1227.

112 Iwasaki K, Gleiser CA, Masoro EJ, McMahan CA, Seo EJ, Yu BP: Influence of the restriction of

individual dietary components on longevity and age-related disease of Fischer rats: the fat com-

ponent and the mineral component. J Gerontol 1988;43:B13–B21.


113 Anson RM, Guo Z, de Cabo R, Iyun T, Rios M, Hagepanos A, Ingram DK, Lane MA, Mattson MP:

Intermittent fasting dissociates beneficial effects of dietary restriction on glucose metabolism and

neuronal resistance to injury from calorie intake. Proc Natl Acad Sci USA 2003;100:6216–6220.

114 Semenza GL, Artemov D, Bedi A, Bhujwalla Z, Chiles K, Feldser D, Laughner E, Ravi R, Simons J,

Taghavi P, Zhong H: ‘The metabolism of tumours’: 70 years later. Novartis Found Symp

2001;240:251–260, discussion 260–264.

115 Altenberg B, Greulich KO: Genes of glycolysis are ubiquitously overexpressed in 24 cancer

classes. Genomics 2004;84:1014–1020.

116 Xu RH, Pelicano H, Zhou Y, Carew JS, Feng L, Bhalla KN, Keating MJ, Huang P: Inhibition of

glycolysis in cancer cells: a novel strategy to overcome drug resistance associated with mitochon-

drial respiratory defect and hypoxia. Cancer Res 2005;65:613–621.

117 Izyumov DS, Avetisyan AV, Pletjushkina OY, Sakharov DV, Wirtz KW, Chernyak BV, Skulachev

VP: ‘Wages of fear’: transient threefold decrease in intracellular ATP level imposes apoptosis.

Biochim Biophys Acta 2004;1658:141–147.

118 Wan R, Camandola S, Mattson MP: Intermittent fasting and dietary supplementation with 2-

deoxy-D-glucose improve functional and metabolic cardiovascular risk factors in rats. Faseb J

2003;17:1133–1134.

119 Perez-Campo R, Lopez-Torres M, Cadenas S, Rojas C, Barja G: The rate of free radical produc-

tion as a determinant of the rate of aging: evidence from the comparative approach. J Comp

Physiol B 1998;168:149–158.

120 Barja G, Herrero A: Localization at complex I and mechanism of the higher free radical produc-

tion of brain nonsynaptic mitochondria in the short-lived rat than in the longevous pigeon. J

Bioenerg Biomembr 1998;30:235–243.

121 Barja G, Herrero A: Oxidative damage to mitochondrial DNA is inversely related to maximum life

span in the heart and brain of mammals. Faseb J 2000;14:312–318.

122 Sohal RS, Sohal BH, Brunk UT: Relationship between antioxidant defenses and longevity in dif-

ferent mammalian species. Mech Ageing Dev 1990;53:217–227.

123 Ishii N, Goto S, Hartman PS: Protein oxidation during aging of the nematode Caenorhabditis ele-

gans. Free Radic Biol Med 2002;33:1021–1025.

124 Nicolle MM, Gonzalez J, Sugaya K, Baskerville KA, Bryan D, Lund K, Gallagher M, McKinney

M: Signatures of hippocampal oxidative stress in aged spatial learning-impaired rodents.

Neuroscience 2001;107:415–431.


keys lowers oxidative damage in skeletal muscle. Faseb J 2000;14:1825–1836.

126 Sindelar DK, Ste Marie L, Miura GI, Palmiter RD, McMinn JE, Morton GJ, Schwartz MW:

Neuropeptide Y is required for hyperphagic feeding in response to neuroglucopenia.

Endocrinology 2004;145:3363–3368.

127 Tkacs NC, Dunn-Meynell AA, Levin BE: Presumed apoptosis and reduced arcuate nucleus

neuropeptide Y and pro-opiomelanocortin mRNA in non-coma hypoglycemia. Diabetes 2000;49:

820–826.

128 Cryer PE: Hierarchy of physiological responses to hypoglycemia: relevance to clinical hypo-

glycemia in type I (insulin dependent) diabetes mellitus. Horm Metab Res 1997;29:92–96.

129 Jacobson L, Ansari T, McGuinness OP: Counterregulatory deficits occur within 24 h of a single

hypoglycemic episode in conscious, unrestrained, chronically cannulated mice. Am J Physiol


130 Cryer PE: Hypoglycemia-associated autonomic failure in insulin-dependent diabetes mellitus.

Adv Pharmacol 1998;42:620–622.

131 Wen Y, Gu J, Li SL, Reddy MA, Natarajan R, Nadler JL: Elevated glucose and diabetes promote

interleukin 12 cytokine gene expression in mouse macrophages. Endocrinology 2006;147:

2518–2525.

132 Masoro EJ: Caloric restriction and aging: controversial issues. J Gerontol A Biol Sci Med Sci

2006;61:14–19.

133 Mohmmad Abdul H, Sultana R, Keller JN, St Clair DK, Markesbery WR, Butterfield DA:

Mutations in amyloid precursor protein and presenilin-1 genes increase the basal oxidative stress

in murine neuronal cells and lead to increased sensitivity to oxidative stress mediated by amyloid


beta-peptide (1–42), HO and kainic acid: implications for Alzheimer’s disease. J Neurochem

2006;96:1322–1335.

134 Moreira PI, Honda K, Liu Q, Santos MS, Oliveira CR, Aliev G, Nunomura A, Zhu X, Smith MA,

Perry G: Oxidative stress: the old enemy in Alzheimer’s disease pathophysiology. Curr Alzheimer

Res 2005;2:403–408.

135 Benchoua A, Trioulier Y, Zala D, Gaillard MC, Lefort N, Dufour N, Saudou F, Elalouf JM, Hirsch E,

Hantraye P, Deglon N, Brouillet E: Involvement of mitochondrial complex II defects in neuronal

death produced by N-terminus fragment of mutated huntingtin. Mol Biol Cell 2006;17:

1652–1663.

136 Duan W, Guo Z, Jiang H, Ware M, Li XJ, Mattson MP: Dietary restriction normalizes glucose

metabolism and BDNF levels, slows disease progression, and increases survival in huntingtin

mutant mice. Proc Natl Acad Sci USA 2003;100:2911–2916.

137 Gandhi S, Wood NW: Molecular pathogenesis of Parkinson’s disease. Hum Mol Genet 2005;14:

2749–2755.

138 Houstis N, Rosen ED, Lander ES: Reactive oxygen species have a causal role in multiple forms of

insulin resistance. Nature 2006;440:944–948.

139 Simmons RA, Suponitsky-Kroyter I, Selak MA: Progressive accumulation of mitochondrial DNA

mutations and decline in mitochondrial function lead to �-cell failure. J Biol Chem 2005;280:

28785–28791.

140 Towle HC: Glucose as a regulator of eukaryotic gene transcription. Trends Endocrinol Metab

2005;16:489–494.

Charles V. Mobbs, PhD

Departments of Neuroscience and Geriatrics

Mount Sinai School of Medicine, Box 1639, 1 Gustave L. Levy Place

New York, NY 10029 (USA)

Tel. �1 212 659 5929, Fax �1 212 849 2510, E-Mail [email protected]



Effects of Dietary Restriction on theExpression of Insulin-Signaling-RelatedGenes in Long-Lived Mutant Mice

Andrzej Bartke, Michal M. Masternak, Khalid A. Al-Regaiey,

Michael S. Bonkowski

Geriatrics Research, Departments of Internal Medicine and Physiology, Southern

Illinois University School of Medicine, Springfield, Ill., USA

AbstractHypopituitary Ames dwarf mice and growth-hormone-resistant (growth hormone

receptor knockout, GHRKO) mice have reduced plasma levels of insulin-like growth factor 1

and insulin, enhanced insulin sensitivity and a remarkably increased life span. This resembles

the phenotypic characteristics of genetically normal animals subjected to dietary restriction

(DR). Interestingly, DR leads to further increases in insulin sensitivity and longevity in Ames

dwarfs but not in GHRKO mice. It was therefore of interest to examine the effects of DR on

the expression of insulin-related genes in these two types of long-lived mutant mice. The

effects of DR partially overlapped but did not duplicate the effects of Ames dwarfism or

GHR deletion on the expression of genes related to insulin signaling and cell responsiveness

to insulin. Moreover, the effects of DR on the expression of the examined genes in different

insulin target organs were not identical. Some of the insulin-related genes were similarly

affected by DR in both GHRKO and normal mice, some were affected only in GHRKO mice

and some only in normal animals. This last category is of particular interest since genes

affected in normal but not GHRKO mice may be related to mechanisms by which DR

extends longevity.


Many of an animal’s physiological responses to nutritional signals are

mediated through insulin, insulin-like growth factor 1 (IGF-1) or homologous

pathways. There is considerable evidence that dietary restriction (DR) reduces

circulating levels of both IGF-1 and insulin, and diminished IGF-1/insulin sig-

naling is among the mechanisms believed to link DR with delayed aging and

extended longevity [1, 2]. In laboratory stocks of house mice (Mus musculus),

Bartke/Masternak/Al-Regaiey/Bonkowski 70

several spontaneous or experimentally produced mutations were shown to

increase longevity. Most of these ‘longevity genes’ cause a disruption of soma-

totropic signaling, leading to reduced peripheral IGF-1 levels or partial IGF-1

resistance (table 1). In at least 3 of these long-lived mutants, reduced soma-

totropic signaling is associated with secondary alterations in insulin release and

responsiveness to insulin actions [8–10]. It was therefore of interest to examine

the interaction of DR with murine longevity genes. In this paper, we will dis-

cuss the effects of DR on the expression of insulin-related genes in 2 types of

long-lived mutant mice: Ames dwarfs and growth hormone receptor knockout

(GHRKO) mice.

Ames Dwarf Mice

Ames dwarfism is due to a loss-of-function mutation at the Prop-1 locus

(df; Prop-1df) that leads to a congenital absence of cells producing growth hor-

mone (GH), prolactin (PRL) and thyroid-stimulating hormone in the anterior

pituitary [11–13]. Consequently, Ames dwarf mice are deficient in GH, PRL

and thyroid-stimulating hormone, small and hypothyroid. Females are infertile

due to PRL deficiency and the resulting luteal failure [14, 15]. In 1996 we

reported that Ames dwarf mice live 45–60% longer than normal animals from

Table 1. Mice with altered somatotropic signaling and prolonged

longevity

Genotype Endocrine effect Reference

Ames dwarf Prop-1df GH deficiency [3]

Snell dwarf Pit-1dw (combined with [4]

PRL and TSH deficiency)

Little GHRHRlit GH deficiency [4]

GHR/GHBP –/– GH resistance [5]

IGFIR �/– partial IGF-1 resistance [6]

�MUPA reduced IGF-1 [7]

(reduced food intake)

GHBP � Growth hormone-binding protein; GHR � growth hormone receptor;

GHRHRlit � growth hormone-releasing hormone receptor, little; IGFIR �

IGF/ insulin receptor; Pit-1dw � pituitary factor-1, Snell dwarf; PRL � prolactin;

Prop-1df � prophet of pit-1, Ames dwarf; �MUPA � urokinase-type plasminogen

activator; TSH � thyroid-stimulating hormone.

Dietary Restriction and Insulin-Signaling-Related Mouse Genes 71

the same line [3] (fig. 1). This observation was confirmed in subsequent studies

[16]. The increased longevity of Ames dwarf mice is associated with retention

of cognitive functions and other indices of delayed aging [17].

Characteristics of Ames dwarf mice which may contribute to their pro-

longed longevity include increased activity and/or level of antioxidant enzymes

[18, 19], reduced oxidative damage [19], reduced plasma glucose and insulin

levels [17], increased responsiveness to insulin [20], reduced body temperature

[21], hypothyroidism [11, 13, 17] and reduced adult body size [22].

Growth Hormone Receptor/Binding Protein

Knockout Mice

Targeted disruption of the GHR/BP gene in GHRKO mice leads to an

absence of GHRs, GH resistance, and reduced plasma IGF-1, postnatal growth

and adult body size in spite of elevated GH levels [23]. Both sexes of GHRKO

mice can reproduce in spite of quantitative deficits in sexual maturation and

fertility [23–25]. GHRKO �/� animals live significantly longer than normal

(�/� or �/�) mice [5]. This has been shown in two laboratories and on three

different genetic backgrounds [26, 27].

The extension of life span in GHRKO animals is striking and statistically

significant, although apparently somewhat smaller than the extension of life in

0 5 10 15 20 25 30 35 40 45 500

10

20

30

40

50

60

70

80

90

100

Months

Su

rviv

al (%

)Normal

Dwarf

Fig. 1. Survival plot for Ames dwarf (Prop-1df) mice.


Ames dwarfs. The reported increases in the average longevity of GHRKO as

compared to normal mice in different studies ranged from 26 to 55%, depend-

ing on genetic background and gender, while in Ames dwarfs they were

between 35 and 70%, depending on gender and diet. The characteristics of

GHRKO mice that might be contributing to their longevity include reduction in

plasma insulin and glucose levels with increased responsiveness to insulin [28],

IGF-1 deficiency and reduced body size [23, 24] and mild hypothyroidism with

slight reduction in body temperature [29].

Comparison of the Effects of Dietary Restriction,

Dwarfism and GHRKO

A comparison of the physiological consequences of DR with the character-

istics of Ames dwarfs and GHRKO animals (table 2) reveals numerous similar-

ities, as well as many quantitative and some qualitative differences. Reductions

in body size and temperature, and lower levels of IGF-1, insulin, glucose and

thyroid hormones are observed in each of the 3 ‘models’ of delayed aging, but

the magnitude of these effects differs, often drastically. For example, plasma

insulin levels are lower in GHRKO than in DR or dwarf mice, while thyroid

hormones and body temperature are lower in Ames dwarfs than in GHRKO

mice. Moreover, Ames dwarfs are completely PRL deficient [15], and DR mice

are hypoprolactinemic [30], while in GHRKO mice, PRL levels are signifi-

cantly elevated [24].

Food consumption per gram of body weight is significantly greater in

Ames dwarfs [16] and GHRKO mice [unpubl. data] than in the corresponding

Table 2. Effects of murine ‘longevity genes’ resemble effects of DR

Ames and GHRKO mice DR

Snell dwarfs

Body weight ↓↓ ↓↓ ↓

Body temperature ↓↓ ↓ ↓↓

Plasma glucose ↓ ↓ ↓

Plasma insulin ↓ ↓↓ ↓

Fertility ↓↓ ↓ ↓↓

Life span ↑↑ ↑↑ ↑↑

Arrows denote statistically significant effects; double arrows mean propor-

tionately larger effects. Arrows pointing up indicate increases; arrows pointing

down indicate decreases.


normal controls. This contrasts with the situation in DR animals in which

reduced food consumption is imposed and eventually becomes approximately

normal, i.e. appropriate for the animal’s body weight. Lastly, middle-aged and

elderly dwarf and GHRKO mice often become obese, while genetically normal

(wild-type) DR animals do not.

Thus, it can be concluded that neither hypopituitary dwarf mice nor

GHRKO mice are DR mimetics, although they share many characteristics.

Effects of Dietary Restriction on Longevity of

Ames Dwarf and GHRKO Mice

In our studies, the DR protocol consisted of feeding the animals every day

70% of the amount consumed daily by mice of the same sex, age and genotype

that were given unlimited access to food (ad libitum). This regimen was intro-

duced gradually, starting at approximately 2 months of age and continued until

the animals died of natural causes or were killed either for experiments or

because of severe illness or apparently imminent death. In Ames dwarf mice,

DR significantly increased the average and the maximal life span resembling

the effects of DR in normal animals from the same stock [31] (fig. 2). This result

0 5 10 15 20 25 30 35 40 45 50

0

10

20

30

40

50

60

70

80

90

100

Months

Su

rviv

al (%

)N-AL

N-DR

Df-AL

Df-DR

Fig. 2. Effects of DR in Ames dwarf mice. N-AL � Normal mice fed ad libitum;

N-DR � normal mice fed under DR; Df-AL � dwarf mice fed ad libitum; Df-DR � dwarf

mice fed under DR.


was unexpected because Ames dwarfs fed ad libitum exhibit many characteristics

of normal animals subjected to DR, and their exceptionally long life span would

seem to make further increases in longevity very unlikely.

Results of our recent study of the effects of identical regimens of DR on the

longevity of GHRKO mice indicate that DR has no effect on the median or aver-

age life span of these animals [51], although it produces the expected robust

increase in longevity of normal animals from the same line.

Microarray analysis of wide profiles of hepatic gene expression in Ames

dwarf, GHRKO and normal mice fed ad libitum or subjected to long-term 30%

DR [32, 33] confirmed our earlier suggestion [31] that the effects of these

‘longevity assurance genes’ (LAGs) and DR are overlapping but not identical.

From a suite of genes included in the Affymetrix U74Av2 mouse array, the

expression of 212 genes was affected by Ames dwarfism only, 77 genes were

affected by DR only, and 100 genes were affected by both dwarfism and DR in

either an additive (n � 95) or interactive (n � 5) fashion [33]. These marked

differences between the effects of a LAG and DR on gene expression were in

excellent agreement with the conclusion from an earlier study in which separate

and interactive effects of a different LAG (GHRKO) and DR were analyzed

using Clontech microarrays [32].

However, comparison of these two studies also revealed interesting differ-

ences. For instance, in comparison to DR, Ames dwarfism affected expression

levels of a greater number of genes, while GHRKO affected much fewer genes.

Moreover, for genes affected by DR in both normal and GHRKO mice,

the effects of DR on the expression level were proportionally smaller in the

mutants [32].

Quantitative Analysis of Expression Levels of

Insulin- and IGF-1-Related Genes

In view of the findings summarized above and our recent evidence that

effects of DR on the longevity of Ames dwarfs and GHRKO mice are substan-

tially different, we became interested in quantitative (real-time PCR) analysis of

the effects of LAGs and DR (singly and combined) on the expression of

selected genes. In these studies we are examining the expression of insulin- and

IGF-1-related genes because of their suspected involvement in the control of

longevity. We are also interested in the comparison of the alterations of gene

expression in different organs, including major targets of insulin action, namely

liver, skeletal muscle and fat. In a series of studies conducted in collaboration

with Drs. Turyn, Dominici and their colleagues, we have obtained evidence that

effects of LAGs on early steps of insulin signaling can be different and in some


cases opposite in the liver as compared to the skeletal muscle [34, 35].

Moreover, the dependence of IGF-1 expression on prevailing GH levels differs

widely between different tissues; therefore the consequences of GH deficiency

(in Ames dwarf mice) or GH resistance (in GHRKO mice) on local IGF-1

biosynthesis are organ specific [36, 37].

The major results of our studies on the expression of insulin- and IGF-1-

related genes are briefly described here. In these experiments, mRNA levels

were quantitated by real-time PCR and levels of the corresponding protein

products were assessed by Western blots.

(1) Effects of Ames dwarfism and GHRKO on the expression of the exam-

ined genes are not identical. Hepatic expression of peroxisome proliferator-

activated receptor (PPAR) � was increased in both Ames dwarf and GHRKO

mice in comparison to the normal animals from the same stock, while expres-

sion of IGF-1 was reduced in both mutants, as expected. However, expression

of insulin receptor (IR), insulin receptor substrate (IRS) 2 and PPAR-a was

increased in GHRKO but not in Ames dwarf mice [38–40].

(2) Effects of DR on gene expression differed between the Ames dwarf and

the GHRKO mice. The levels of PPAR-� mRNA and protein in the liver were

increased by DR in GHRKO mice but were not affected in Ames dwarfs.

Suppression of IRS-1 message levels by DR was also observed in GHRKO but

not in Ames dwarf mice [38, 40, 41]. Further work will be necessary to relate

differences between the effects of DR on gene expression to the differential

effects of DR on longevity in these 2 mutants.

(3) Subjecting normal animals to DR does not reproduce the effects of

Ames dwarfism or GHRKO on gene expression. Several of the alterations in

hepatic gene expression in long-lived mutants resembled the changes measured

in normal animals subjected to DR. These included increase in IR protein in

dwarf mice and increases in IRS-2 message, PPAR-� message and PPAR-� pro-

tein in GHRKO mice. However, increases in IR message, IRS-1 protein, or

PPAR-� message and protein in one or both mutants were not reproduced by

DR in normal mice. Moreover, IGF-1 expression was profoundly suppressed in

both mutants, but was either unaltered or increased by DR in normal mice from

the examined stocks [38, 40, 41].

Studies of hepatic expression of a different set of insulin- and IGF-1-

related genes in GHRKO and normal mice fed ad libitum or subjected to DR

revealed numerous differences [40, 42]. DR failed to reproduce stimulatory

effects of GHRKO on the expression of protein kinase B (Akt-1, Akt-2),

forkhead box class O (Foxo-1), phosphoenolpyruvate carboxykinase (PEPCK),

glucose-6-phosphatase (G6Pase), superoxide dysmutase-2 (SOD-2), PPAR-�,

peroxisome proliferator-activated receptor-� coactivator-1� (PGC-1�) or

PPARs binding partners retinoid X receptor (RxR)-a, -g or -b/d. In the same


studies, both DR and GHRKO increased the expression of PPAR-�, reduced the

levels of phosphorylated Akt protein, and increased the levels of PGC-1� pro-

tein, phosphorylated p38 mitogen-activated protein kinase (MAPK) and phos-

phorylated cAMP response element-binding protein (pCREB). Sirt-1 protein

levels were not affected by GHRKO but were increased by DR in both normal

and GHRKO mice.

(4) Expression of insulin- and IGF-1-related genes in different organs is

differentially affected by LAGs as well as by DR. Results of ongoing studies of

gene expression in the skeletal muscle, heart and adipose tissue revealed

numerous differences from the results obtained in the liver. For example, IR and

IRS-2 message levels were increased in the liver of GHRKO mice but not in

their skeletal muscle, although IGF-1 expression was reduced in both organs.

The response to DR also differed between the organs in both normal and mutant

mice. In GHRKO mice and in normal animals from the same stock, DR did not

affect the expression of IR, IRS-1 or IGF-1 in the liver but reduced expression

of these genes in the skeletal muscle. In normal mice, levels of IRS-1 message

were increased by DR in the liver but reduced in the muscle [40].

(5) Effects of DR on the expression of insulin and IGF-1-related genes dif-

fer between GHRKO and normal mice. Some of the changes produced by DR

were similar in normal and GHRKO animals and thus are unlikely to be related

to differential effects of DR in their longevity. These changes included increases

in the levels of messages for PPAR-�, PGC-1� and Sirt-1 and in the level of

pCREB protein in the liver, reduced levels of mRNAs for IR, IRS-1, IRS-2,

Foxo-1 and Foxo-4, reduced levels of c-Jun N-terminal kinase (pJNK)-2 and

increased levels of adenosine monophosphate-activated protein kinase

(pAMPK) in the skeletal muscle, and increased levels of IRS-1 and Glucose

transporter-4 (Glut-4) mRNAs in the heart [40, 42, 43; unpubl. observations].

However, GHRKO mice exhibited multiple DR-induced alterations in gene

expression that were not observed in normal animals. These changes could be

viewed as potential causes for the failure of these animals to respond to DR

with increased longevity. Among the changes we detected were increased

expression of genes related to gluconeogenesis (PEPCK and G6Pase) in the

liver, reduced levels of Akt-2, PGC-1�, mRNAs and pAkt in the muscle, and

increased levels of PPAR-�, PPAR-�, PPAR-�/� and Glut-4 proteins in the

heart.

Of particular interest were alterations that were induced by DR in normal

mice but not in GHRKO animals and thus correlated with the effects of DR on

longevity. These included increased IRS-2 mRNA and reduced pAkt in the liver,

reduced hepatic levels of PPAR-�/� mRNA and protein, increased levels of

Akt-2 and PGC-1� and reduced levels of JNK-1 in the skeletal muscle, and

increased levels of IGF-1 and IR messages in the heart [40–43].


Effects of DR on the levels of proteins and phosphorylated proteins related

to insulin and IGF-1 signaling in GHRKO and normal mice are compared in

table 3.

Discussion

In mammals, DR reduces plasma insulin levels, enhances insulin sensitiv-

ity and exerts complex, age-related effects on somatotropic signaling. In rats

subjected to DR, GH secretion is initially suppressed but subsequently the dif-

ference in GH levels between ad libitum and DR animals becomes reversed as a

result of DR delaying or reducing age-related decline in GH secretion [44].

Plasma IGF-1 levels are suppressed in DR animals, although hepatic levels of

IGF-1 mRNA in DR mice may be elevated rather than reduced [38].

Studies in long-lived mutant and gene knockout mice provide very strong

evidence for the role of somatotropic and insulin signaling in the control of

mammalian aging and longevity and indirectly support the role of these signal-

ing pathways in mediating the effects of DR. Results obtained in these mutants

also emphasize very important differences between the consequences of partial

versus complete inhibition of the release or actions of IGF-1 and insulin. Mice

with the absence of IGF-1 signaling due to disruption of IGF-1 or IGF-1R

genes rarely survive to adulthood [36]. In contrast, IGF-1R �/� mice, with

approximately 50% reduction in the levels of IGF-1 receptors, and Ames, Snell

and GHRKO mice, with an organ-specific deficiency of GH-dependent IGF-1

Table 3. Effects of DR on the levels of proteins and phosphorylated proteins related

to insulin and IGF-1 signaling in the liver and skeletal muscle in GHRKO as compared to

normal mice

Affected in both Affected only in Affected only in

GHRKO and normal mice GHRKO mice

normal mice

pAkt ↓

Liver PPAR-� ↑ p38 MAPK ↑

pCREB ↑ PPAR-�/� ↓

Akt-2 ↑

Muscle pAMPK ↑ pJNK-1 ↓

pJNK-2 ↑ PGC-1� ↑ pAkt ↓

Arrows pointing up indicate increases; arrows pointing down indicate decreases.

pCREB � Phosphorylated cAMP response element-binding protein.


expression, live significantly longer than their normal siblings [4, 6, 8–10].

Similarly, absence of insulin or its actions in mice or humans leads to diabetes

and death, while reduced levels of insulin in dwarf and GHRKO mice and

adipocyte-specific absence of IRs in FIRKO mice are associated with extended

longevity [7–10].

Following this evidence, it appears that longevity benefits are related to a

modest or organ-limited deficiency of IGF-1 and insulin signaling, while severe

or complete suppression of the same signaling pathways may be detrimental or

lethal. This conclusion helps to address a conundrum of qualitatively similar

endocrine conditions that lead to serious disease in humans but delay aging and

prolong life in mice. A similar conclusion derived from multiple studies – that

reduced somatotropic and insulin signaling is involved in mediating the effects

of DR on aging – can now be sharpened by suggesting that DR produces a favor-

able combination of modest, likely organ-specific reduction of IGF-1 expres-

sion, reduced insulin release and enhanced sensitivity to insulin actions.

Examining the interaction of mutations affecting somatotropic and/or

insulin signaling with DR may facilitate the identification of mechanisms that

link altered endocrine function with delayed aging and extended longevity.

Considerable overlap of phenotypic characteristics of long-lived Ames

dwarf and GHRKO mice with the characteristics of normal mice subjected to

DR would seem to suggest that these mutants are unlikely to benefit from DR.

Indeed, one may assume that the additive effects of these mutations and DR on

growth and glucose homeostasis might be incompatible with survival of these

animals. Surprisingly, the mutants seem unimpaired; both Ames dwarf and

GHRKO mice readily tolerate 30% DR. Moreover, Ames dwarfs respond to

30% DR by an additional extension of their life span [31].

Interestingly, our recent results indicate that an identical regimen of DR has

very little effect on the longevity of GHRKO mice; the median life span was not

affected and the extension of the estimated maximal life span was small and lim-

ited by gender. These unexpected observations create some novel opportunities

for identifying those effects of DR that are likely to be causally related to

extended longevity. For example, reduction in hepatic levels of phosphorylated

Akt and PPAR-�/� protein, along with an increase in phosphorylated p38

MAPK and a reduction in the levels of phosphorylated pJNK-1 in the skeletal

muscle were produced by DR in normal but not in GHRKO mice. Although

association does not imply causality, it is noteworthy that these DR effects were

seen in animals in which DR extended life span but not those in which it did not.

Comparison of the effects of DR in normal and GHRKO mice also sug-

gests that some of the changes that would seem likely to be of functional impor-

tance may have no (or a limited) role in mediating the effects of DR on

life span. For example, body weight was reduced and hepatic expression of


PGC-1�, Sirt-1 and PPAR-� were increased by DR in both normal mice that

lived longer and in GHRKO animals that did not. Thus, it could be suggested

that the well-documented induction of PGC-1� and Sirt-1 under conditions of

reduced food availability is either not sufficient to increase longevity or is not

effective in animals in which GH action is blocked. Similar suggestions can be

made about the alterations in the expression of a number of insulin-related

genes in the skeletal muscle and the heart, identified earlier in this review.

Further study will be necessary to elucidate the interaction of DR with var-

ious mammalian longevity genes in the control of aging. An exploration of the

responses of long-lived mouse mutants to both milder and more severe regi-

mens of DR would also be of considerable interest, as suggested by the findings

of Clancy et al. [45] in long-lived chico mutant Drosophila.

The effects of genes related to an insulin/IGF-1-like signaling pathway on

the life spans of worms and flies were studied in considerable detail [reviewed

in 46–49]. These genes exhibit considerable homology to IGF-1- and insulin-

related genes in mammals [46, 48]. In the worm Caenorhabditis elegans, the

life-extending effects of gene mutations in this pathway are generally additive

to the effects of DR, while in a fly (Drosophila melanogaster), the additive

effects of DR and insulin-related longevity genes have been difficult to demon-

strate [48]. Making direct comparisons between the effects of DR in these

species and in the mouse is challenging, because there are profound differences

in life cycles, physiology (heterothermic vs. homothermic) and in the ways in

which DR is implemented. In worms and flies, DR usually consists of reducing

the caloric and/or nutritional value of food by dilution or changes in composi-

tion, while in laboratory rodents DR involves ingestion of reduced amounts of a

complete, nutritionally balanced diet or a similar diet supplemented with a mix-

ture of micronutrients.

In future studies we will utilize differential responses to DR in normal,

Ames dwarf and GHRKO mice to identify those organ-specific alterations in

IGF-1 and insulin signaling that are associated with an increased life span [50].

The downstream targets of these genetic pathways should offer important clues

to the mechanisms of delayed aging and extended longevity in both long-lived

mutants and in normal animals subjected to DR.

Acknowledgement

This work was supported by the National Institutes of Health (NIH/NIA 1U19

AG023122–01A and RO1 AG19899), the Ellison Medical Foundation and the Southern

Illinois University Geriatrics Research Initiative. We thank Steve Sandstrom for his help in

preparing this manuscript and our colleagues who contributed to the studies discussed in this

article, especially Dr. John Kopchick, who provided us with GHRKO breeder mice to start


our colony of these animals, and to Drs. Daniel Turyn and Fernando Dominici, who demon-

strated alterations in insulin signaling in Ames dwarf and GHRKO mice.

References

1 Weindruch R: The retardation of aging by dietary restriction: studies in rodents and primates.

Toxic Pathol 1996;24:742–745.

2 Masoro EJ: Dietary restriction: an experimental approach to the study of the biology of aging;

in Masoro EJ, Austad SN (eds): Handbook of the Biology of Aging. San Diego, Academic Press,

2001, pp 396–420.

3 Brown-Borg HM, Borg KE, Meliska CJ, Bartke A: Dwarf mice and the ageing process. Nature

1996;384:33.

4 Flurkey K, Papaconstantinou J, Miller RA, Harrison DE: Lifespan extension and delayed immune

and collagen aging in mutant mice with defects in growth hormone production. Proc Natl Acad

Sci USA 2001;98:6736–6741.

5 Coschigano KT, Clemmons D, Bellush LL, Kopchick JJ: Assessment of growth parameters and

life span of GHR/BP gene-disrupted mice. Endocrinology 2000;14:2608–2613.

6 Holzenberger M, Dupont J, Ducos B, Leneuve P, Geloen A, Evens P, Cervera P, LeBouc Y: IGF-1

receptor regulates lifespan and resistance to oxidative stress in mice. Nature 2003;421:182–187.

7 Miskin R, Masos T: Transgenic mice overexpressing urokinase-type plasminogen activator in

the brain exhibit reduced food consumption, body weight and size, and increased longevity.

J Gerontol 1997;52A:B118–B124.

8 Bartke A, Turyn D: Mechanisms of prolonged longevity: mutants, knock-outs, and dietary restric-

tion. J Antiaging Med 2001;4:197–203.

9 Bartke A, Heiman M, Turyn D, Dominici F, Kopchick J: The role of growth hormone signaling in

the control of ageing; in Straub RH, Mocchegiani E (eds): The Neuroendocrine Immune Network

in Aging. Berlin, Elsevier, 2004, pp 123–137.

10 Bartke A: Minireview: role of the growth hormone/insulin-like growth factor system in mam-

malian aging. Endocrinology 2005;146:3718–3723.

11 Bartke A: Histology of the anterior hypophysis, thyroid and gonads of two types of dwarf mice.

Anat Rec 1964;149:225–235.

12 Andersen B, Pearse RV II, Jenne K, Sornson M, Lin SC, Bartke A, Rosenfeld MG: The Ames

dwarf gene is required for Pit-1 gene activation. Dev Biol 1995;172:495–503.

13 Sornson MW, Wu W, Dasen JS, Flynn SE, Norman DJ, O’Connell SM, Gukovsky I, Carrière C,

Ryan AK, Miller AP, Zuo L, Gleiberman AS, Anderson B, Beamer WG, Rosenfeld MG: Pituitary

lineage determination by the prophet of pit-1 homeodomain factor defective in Ames dwarfism.

Nature 1996;384:327–333.

14 Bartke A: Influence of luteotrophin on fertility of dwarf mice. J Reprod Fertil 1965;10:93–103.

15 Bartke A: Prolactin-deficient mice; in Alexander NJ (ed): Animal Models for Research on

Contraception and Fertility. Hagerstown, Harper & Row, 1979, pp 360–365.

16 Mattison JA, Wright JC, Bronson RT, Roth GS, Ingram DK, Bartke A: Studies of aging in Ames

dwarf mice: effects of dietary restriction. J Am Aging Assoc 2000;23:9–16.

17 Bartke A: Delayed aging in Ames dwarf mice: relationships to endocrine function and body size;

in Hekimi Z (ed): The Molecular Genetics of Aging: Results and Problems in Cell Differentiation.

Berlin, Springer, 2000, pp 181–202.

18 Brown-Borg H, Rakoczy S: Catalase: expression in delayed and premature aging mouse models.

Exp Gerontol 2000;35:199–212.

19 Brown-Borg H, Johnson W, Rakoczky S, Romanick M: Mitochondrial oxidant generation

and oxidative damage in Ames dwarf and GH transgenic mice. Am Aging Assoc 2001;24:

85–96.

20 Borg KE, Brown-Borg HM, Bartke A: Assessment of the primary adrenal cortical and pancreatic

hormone basal levels in relation to plasma glucose and age in the unstressed Ames dwarf mouse.

Proc Soc Exp Biol Med 1995;210:126–133.


21 Hunter WS, Croson WB, Bartke A, Gentry MV, Meliska CJ: Low body temperature in long-lived

Ames dwarf mice at rest and during stress. Physiol Behav 1999;67:433–437.

22 Miller RA, Harper JM, Galecki A, Burke DT: Big mice die young: early life body weight predicts

longevity in genetically heterogeneous mice. Aging Cell 2002;1:22–29.

23 Zhou Y, Xu BC, Maheshwari HG, He L, Reed M, Lozykowski M, Okada S, Wagner TE, Cataldo

LA, Coschigano K, Baumann G, Kopchick JJ: A mammalian model for Laron syndrome produced

by targeted disruption of the mouse growth hormone receptor/binding protein gene (the Laron

mouse). Proc Natl Acad Sci USA 1997;94:13215–13220.

24 Chandrashekar V, Bartke A, Coschigano KT, Kopchick JJ: Pituitary and testicular function in

growth hormone receptor gene knockout mice. Endocrinology 1999;140:1082–1088.

25 Danilovich N, Wernsing D, Coschigano KT, Kopchick JJ, Bartke A: Deficits in female reproduc-

tive function in GH-R-KO mice: role of IGF-I. Endocrinology 1999;140:2637–2640.

26 Coschigano KT, Holland AN, Riders ME, List EO, Flyvbjerg A, Kopchick JJ: Deletion, but not

antagonism, of the mouse growth hormone receptor results in severely decreased body weights,

insulin, and insulin-like growth factor I levels and increased life span. Endocrinology 2003;144:

3799–3810.

27 Bartke A, Chandrashekar V, Bailey B, Zaczek D, Turyn D: Consequences of growth hormone (GH)

overexpression and GH resistance. Neuropeptides 2002;36:201–208.

28 Liu JL, Coschigano KT, Robertson K, Lipsett M, Guo Y, Kopchick JJ, Kumar U, Liu YL:

Disruption of growth hormone receptor gene causes diminished pancreatic islet size and increased

insulin sensitivity in mice. Am J Physiol Endocrinol Metab 2004;287:E405–E413.

29 Hauck SJ, Hunter WS, Danilovich N, Kopchick JJ, Bartke A: Reduced levels of thyroid hormones,

insulin, and glucose, and lower body core temperature in the growth hormone receptor/binding

protein knockout mouse. Exp Biol Med 2001;226:552–558.

30 Han ES, Lu DH, Nelson JF: Food restriction differentially affects on messenger RNAs encoding

the major anterior pituitary tropic hormones. J Gerontol Biol Sci 1998;54A:B322–B329.

31 Bartke A, Wright JC, Mattison J, Ingram DK, Miller RA, Roth GS: Extending the lifespan of long-

lived mice. Nature 2001;414:412.

32 Miller RA, Chang Y, Galecki AT, Al-Regaiey K, Kopchick JJ, Bartke A: Gene expression patterns

in calorically restricted mice: partial overlap with long-lived mutant mice. Mol Endocrinol

2002;16:2657–2666.

33 Tsuchiya T, Dhahbi JM, Cui X, Mote PL, Bartke A, Spindler SR: Additive regulation of hepatic

gene expression by dwarfism and dietary restriction. Physiol Genomics 2004;17:307–315.

34 Dominici FP, Hauck S, Argentino DP, Bartke A, Turyn D: Increased insulin sensitivity and upreg-

ulation of insulin receptor, insulin receptor substrate (IRS)-1 and IRS-2 in liver of Ames dwarf

mice. J Endocrinol 2002;173:81–94.

35 Argentino DP, Dominici FP, Munoz MC, Al-Regaiey K, Bartke A, Turyn D: Effects of long-term

dietary restriction on glucose homeostasis and on the first steps of the insulin signaling system in

skeletal muscle of normal and Ames dwarf (Prop1df/Prop1df) mice. Exp Gerontol 2005;40:27–35.

36 Lupu F, Terwilliger JD, Lee K, Segre GV, Efstratiadis A: Roles of growth hormone and insulin-like

growth factor I in mouse postnatal growth. Dev Biol 2001;229:141–162.

37 Sun LY, Al-Regaiey K, Masternak MM, Wang J, Bartke A: Local expression of GH and IGF-1 in

the hippocampus of GH-deficient long-lived mice. Neurobiol Aging 2005;26:929–937.

38 Masternak M, Al-Regaiey K, Bonkowski M, Panici J, Sun L, Wang J, Przybylski GK, Bartke A:

Divergent effects of dietary restriction on gene expression in normal and long-lived mice.

J Gerontol A Biol Sci Med Sci 2004;59:784–788.

39 Masternak MM, Al-Regaiey KA, Bonkowski MS, Panici JA, Bartke A: Effect of every other day

feeding diet on gene expression in normal and in long-lived Ames dwarf mice. Exp Gerontol

2005;40:491–497.

40 Masternak MM, Al-Regaiey KA, Del Rosario Lim MM, Jimenez-Ortega V, Panici JA,

Bonkowski MS, Bartke A: Effects of caloric restriction on insulin pathway gene expression in

the skeletal muscle and liver of normal and long-lived GHR-KO mice. Exp Gerontol 2005;40:

679–684.

41 Masternak MM, Al-Regaiey KA, Del Rosario Lim MM, Bonkowski MS, Panici JA, Przybylski

GK, Bartke A: Caloric restriction results in decreased expression of peroxisome proliferator-


activated receptor superfamily in muscle of normal and long-lived growth hormone receptor/

binding protein knockout mice. J Gerontol A Biol Sci Med Sci 2005;60:1238–1245.

42 Al-Regaiey KA, Masternak MM, Bonkowski M, Sun L, Bartke A: Long-lived growth hormone

receptor knockout mice: interaction of reduced insulin-like growth factor 1/insulin signaling and

dietary restriction. Endocrinology 2005;146:851–860.

43 Al-Regaiey KA: Long-Lived Growth Hormone Receptor Knockout Mice: Interaction of Reduced

IGF-1/Insulin Signaling and Dietary Restriction; PhD dissertation, Department of Physiology,

Southern Illinois University, Carbondale, 2005, p 103.

44 Sonntag WE, Cefalu WT, Ingram RL, Bennett SA, Lynch CD, Cooney PT, Thornton PL, Khan AS:

Pleiotropic effects of growth hormone and insulin-like growth factor (IGF) on biological aging:

inferences from moderate caloric restricted animals. J Gerontol 1999;54A:B521–B538.



2001;292:104–106.


2003;299:1346–1351.

47 Longo VD, Finch CE: Evolutionary medicine: from dwarf model systems to healthy centenarians?

Science 2003;299:1342–1346.

48 Kenyon C: The plasticity of aging: insights from long-lived mutants. Cell 2005;120:449–460.

49 Kimura KD, Tissenbaum HA, Liu Y, Ruvkun G: daf-2, an insulin receptor-like gene that regulates

longevity and diapause in Caenorhabditis elegans. Science 1997;277:942–946.

50 Blüher M, Kahn B, Kahn CR: Extended longevity in mice lacking the insulin receptor in adipose

tissue. Science 2003;299:572.

51 Bonkowski MS, Rocha JS, Masternak MM, Al-Regaiey KA, Bartke A: Targeted disruption of

growth hormone receptor interferes with the beneficial actions of calorie restriction. Proc Natl

Acad Sci USA 2006;103:7901–7905.

Andrzej Bartke, PhD

Geriatrics Research, Departments of Internal Medicine and Physiology

Southern Illinois University School of Medicine

PO Box 19628, Springfield, IL 62794–9628 (USA)




Anti-Inflammatory Mechanisms ofDietary Restriction in Slowing AgingProcesses

T.E. Morgan, A.M. Wong, C.E. Finch

Leonard Davis School of Gerontology and USC College,

University of Southern California, Los Angeles, Calif., USA

AbstractDietary restriction (DR) remains the most powerful and general environmental manipu-

lation of aging processes in laboratory animals with strong beneficial effects on most age-

related degenerative changes throughout the body. Underlying the beneficial effects of DR is

the attenuation of system-wide inflammatory processes including those occurring within the

central nervous system. During normal aging a progressive neuroinflammatory state builds in

the brain involving astrocytes and microglia, the primary cellular components of neuroinflam-

mation. DR attenuates the age-related activation of astrocytes and microglia with concomitant

beneficial effects on neurodegeneration and cognition. Increasing evidence suggests that com-

mon pathways are emerging that link many normal aging inflammatory processes with age-

related diseases such as Alzheimer, cancer, diabetes and cardiovascular disease.


Dietary restriction (DR) remains the most powerful and general manipula-

tion of aging processes in laboratory animals. Evidence is now overwhelming

that DR increases life span by slowing the Gompertz mortality rate accelera-

tion. The Gompertz analysis of DR was first made by Berg [1] in 1976. This

fundamental effect of DR has been amply verified [2, p. 508; 3].

Corresponding to slowed mortality rates, most spontaneous degenerative

changes in aging are attenuated. In rodents, the age-related increases in tumor

and organ-specific pathology are delayed by DR, according to the genotype [2,

4–7]. In the widely used F344 rats, for example, chronic renal disease, which

may be the major cause of morbidity, is strikingly reduced [8]. Cardiomyopathy

of F344 rats is also strongly associated with the severity of kidney degeneration,

but the mechanisms may be different [9]. Nonetheless, we must confront the

Morgan/Wong/Finch 84

puzzle in F344 rats that about 25% of old DR rats have no gross organ pathol-

ogy at death [8]. We suggest the possibility of metabolic instability during DR

below in lesion-free aging rodents.

Dietary Restriction Attenuates Neuroinflammatory

Aspects of Aging

The first indication that DR is neuroprotective for aging came from a 1985

report [10]. In some rodent colonies, hind limb paralysis becomes increasingly

common during aging in association with degeneration of spinal motor neurons

(radiculoneuropathy) [10, 11]. The degeneration of myelin sheaths in spinal

roots arises after sporadic axonal atrophy and is associated with segmental

demyelination and local ballooning [12–14]. Hind limb paralysis was markedly

attenuated by DR [10, 11].

Hind limb paralysis varies widely between colonies and is unfamiliar to

current researchers of aging. The greatest incidence reported, 100%, was

observed in colonies before the era of modern husbandry (specific-pathogen

free). In the NIA contract colony at Charles River Laboratory, in 1978–1983,

rats (9 genotypes, both sexes) had a 25% incidence, with a mean age at lesion of

31 months; the incidence in mice (12 genotypes) was �0.1% [15]. These major

differences are puzzling and not easily attributed to improved husbandry and

health. Early rodent colonies before 1970 often carried a much higher load of

infections than the present specific-pathogen-free colonies.

While most attention has been given to the biochemical, metabolic and

genomic effects of DR, evidence is growing for the importance of system-wide

anti-inflammatory effects of DR in attenuating aging [16–18]. Our laboratory is

focused on neuroinflammatory changes of aging in rodents, primates and

humans. These generalized aging changes arise in the absence of specific neu-

rodegeneration [16, 17]. In aging rodents, the main brain aging changes are

glial activation (microglia and astrocytes)1 [17] and synaptic atrophy [19–24].

These changes are progressive during middle age into old age and arise in the

absence of disease. The type and extent of change are selective and differ exten-

sively between even closely connected brain systems. The opposing glial and

synaptic changes span a range of about 50% but are much larger than changes

in cell number. In fact, several exacting studies have looked for but did not

detect age changes in the total numbers of neurons [25] or glia [26]. Thus, in

1Microglia are bone-marrow-derived monocytes which are constantly repopulated in adult

brains. Astrocytes are of neural crest origin and share the same stem cell precursors as neurons.

Anti-Inflammatory Mechanisms of Dietary Restriction 85

aging rodents and perhaps in humans, the main brain aging changes represent a

type of plasticity that remodels cell cytoarchitectonic relationships without

cell death. DR has a remarkable ability to attenuate these changes. White mat-

ter myelinated tracts are a robust example of the plasticity of neuroinflamma-

tory aging.

Age-Related Microglial Activation: White Matter Degeneration

Macroscopically, magnetic resonance imaging studies on aging humans

and monkeys show subtle structural changes in the corpus callosum, striatum

and other white-matter-rich tracts [27–29]. These changes may be caused by

the focal degeneration of myelin sheaths and differ by brain region. The later-

myelinated regions are more susceptible to demyelination during normal aging

and Alzheimer disease (AD) [30].

White matter aging is accompanied by increased microglial activation [31,

32], but cause and effect are unclear. Aging rodent models show robust increases

in markers of microglial activation, e.g. CR3 (complement receptor) and MHC

class II antigens (antigen presentation by macrophages) [33, 34]. These changes

are attenuated by DR [33]. Most recently, we found that the scavenger receptor

macrosialin (CD68), a member of the lysosomal/endosomal-associated mem-

brane glycoprotein family, shows the greatest age-related increase in the corpus

callosum of C57BL/6NNia mice; again, this is attenuated by DR (fig. 1) [35].

Because macrosialin is increased in peripheral macrophages by oxidized lipids

(low-density lipoproteins) [36] and because oxidized lipids generally promote

inflammation [37, 38], we hypothesize that the oxidation of white matter lipids is

a factor in microglial activation. In fact, we showed that oxidized low-density

Fig. 1. The age-related increase in macrosialin (CD68) expression is attenuated by

DR. Macrosialin immunoreactivity in the corpus callosum and corticostriatal bundles

(insets) of 4-month ad libitum (a), 24-month ad libitum (b) and 24-month calorie-restricted

(c) C57BL/6NNia mice. The arrow identifies macrosialin immunostaining at the periphery

of a corticostriatal bundle in 24-month ad libitum mice. Bars � 100 �m for micrographs,

30 �m for insets. Reprinted from Wong et al. [35], with permission from Elsevier.

a b c


lipoproteins induced CD68 in BV-2 microglial cells [35]. Moreover, CD68 is

induced by inflammatory stimuli (lipopolysaccharide plus �-interferon) in BV-2

cells [35]. Therefore, CD68 serves as an inflammatory marker as well as an indi-

cator of oxidative damage during normal brain aging.

Because DR clearly attenuates age-related increases in inflammatory

genes such as CD68, CR3 and MHC class II antigens, we hypothesize that DR

will protect against age-related demyelinating events. An ongoing study of DR

in rhesus monkeys has not given definitive information for technical reasons.

After 11–13 years of DR, middle-aged (�24 years old) and old monkeys (�24

years old) had smaller putamen volumes than ad libitum fed animals [39].

However, there were no initial magnetic resonance imaging data to establish the

baseline (before or at the beginning of DR). Thus, it is unresolved if the smaller

putamen volumes in DR animals resulted from DR, or if the volume differences

were present at the beginning of the study.

Age-Related Astrocytic Activation: Glial Fibrillary Acidic Protein

Astrocytes are an important source of neurotrophic factors, axonal guid-

ance molecules and extracellular matrix molecules crucial for neuron survival

and sprouting. In response to injury or disease, astrocytes take on an activated

phenotype that is characterized by cell hypertrophy and upregulation of the

intermediate filament proteins, glial fibrillary acidic protein (GFAP) and

vimentin, as well as inflammatory mediators and extracellular matrix molecules

[40, 41]. However, during normal aging astrocytes become activated with con-

comitant increases in GFAP and vimentin in the absence of overt pathology

[42–44]. This age-related astrocytic activation [45, 46] contributes to age-

related increased inflammatory and oxidative damage [44, 47], decreased neu-

rogenesis [48] and synaptic atrophy [19].

We are investigating the hypothesis that the increase in GFAP expression is

a primary cause in synaptic atrophy and impaired synaptogenesis during nor-

mal aging [46]. We have developed a heterochronic cell culture model to test

this hypothesis. In brief, test neurons (E18 cortex) are seeded on monolayers of

primary cultures of astrocytes from young adult or aging rat cerebral cortex.

The old-rat-derived astrocytes retain the high GFAP per cell [49] as observed in

vivo [33]. Moreover, the E18 neurites outgrow poorly on old-rat-derived astro-

cytes. These age impairments in neurotrophic support are rapidly reversed by

downregulating GFAP by small interfering RNA [46]. The mechanism involves

an inverse relationship between GFAP expression and secretion of laminin, a

critical component of the extracellular matrix that guides neurite outgrowth.

Additional support for the critical role of GFAP comes from studies on mice

lacking both GFAP and vimentin which have improved synaptic regeneration

and increased neurogenesis [50, 51].


Just as age-related microglial activation is reduced by DR (discussed

above), DR is also effective at attenuating many of the genotypic and pheno-

typic changes that astrocytes undergo during aging. The age-related increase in

GFAP is attenuated by DR [43, 44, 52] and this occurs at the transcriptional

level [33, 53]. Microarray profiling confirmed the effects of DR on GFAP [17,

44]. Although neuropathologists have long used GFAP as a marker of neurode-

generation, our work clearly shows that the age increase in GFAP arises in the

absence of neuron cell death and may be an upstream factor in synaptic atrophy

during aging. Because of the concurrent activation of microglial inflammatory

markers, we provisionally consider that GFAP is embedded in a neuroinflam-

matory network. The beneficial effect of DR on glial activation may underlie

DR’s ability to attenuate age-related declines in synaptic plasticity and neuroge-

nesis [54–58]. Ongoing studies are evaluating if DR improves the neurotrophic

support of aging glia.

Age-Related Neurodegenerative Disease: Experimental Rodent Models

DR also protects against neurodegenerative processes in experimental

rodent models. For example, DR protects neurons from many toxins, including

methylphenyltetrahydropyridine [59], kainic acid [60], 3-nitropropionic acid and

malonate [60, 61]. AD-like changes do not arise in aging rodents, possibly

because the rodent ß-peptide has several amino acid substitutions that decrease

its aggregation into fibrillar amyloids that are characteristic of AD [62].

However, mice carrying human transgenes for early-onset familial AD develop

fibrillar amyloids and various other specific AD-like neuropathological changes

during aging. We have recently shown that DR attenuated brain deposits of brain

amyloid by 50% within the short time of several months [63] (fig. 2a). These

changes were accompanied by a reduction in GFAP in astrocytes surrounding

the plaque (fig. 2b). We demonstrated these beneficial effects of DR in two

transgenic mouse models of AD, APPswe/ind and APP�PS1 [63]. These effects

of DR also extend to a third genotype, Tg2576 [64].

Low-energy diets in humans are being considered as an approach to lower-

ing AD risk, because in retrospective studies, AD victims tended to have higher

calorie intake [65, 66]. Of course, it is much harder to establish causality of diet

in humans, because individuals who adopt special diets also often pursue other

health-promoting activities, such as exercise which may protect against cogni-

tive declines in normal aging [67, 68].

Another example of DR providing age-related neuroprotective activities is

in the experimental model of retinal ischemia/reperfusion [69]. As observed in

cortical and hippocampal regions (see above), microglia and astrocytes become

progressively activated in the aged retina [69]. Further glial activation occurs

when the aged retina is subjected to ischemia/reperfusion with concomitant


neuronal damage. In this model of ischemia/reperfusion with individual eyes,

DR attenuated retinal glial activation and neuronal damage [69]. In fact, these

authors suggest that the beneficial effects of DR are directly related to its effect

on glial activation supporting the hypothesis that the anti-inflammatory actions

of DR on glia may mediate neuroprotection.

Dietary Restriction Attenuates Inflammatory Processes

Microarray Profiling Highlights Anti-Inflammatory Effects of

Dietary Restriction

The broad scope of inflammatory gene expression during brain aging has

become clear through the numerous publications utilizing microarray gene

expression profiling [44, 70, 71]. These studies showed that inflammation-related

genes increased during aging. Importantly, DR attenuated the age-related increase

in inflammatory genes [44]. In fact, DR prevented the age-related increased

expression of 65% of those genes involved in the inflammatory response in the

neocortex [44] suggesting that this is a primary mechanism underlying the ben-

eficial effect DR has on brain aging processes.

Suppression of Inflammation in Acute Dietary Restriction

Inflammatory responses are attenuated by DR throughout the body [17,

72]. We begin with examples from the skin. In the classic pharmacological

Fig. 2. DR reduces �-amyloid (A�) number and �-amyloid-associated astrocyte acti-

vation relative to ad libitum (AL) feeding. a Plaque size and total �-amyloid plaques were

reduced by DR in APPswe/ind (*p � 0.05, n � 7–8). b Sholl analysis of concentric rings

around �-amyloid plaques (inset) showed reduced GFAP immunoreactivity nearest to

plaques in DR versus ad libitum feeding (p � 0.05). Reprinted from Patel et al. [63], with

permission from Elsevier.

20

15

10

Nu

mb

er

of

A�

pla

qu

es

5

0

70

60

50

GF

AP

(%

are

a)

40

30

20

0

0 20 40 60 80 100

Distance from plaque (�m)

120

DR

140

10

�10

Plaque size

Total

*

*

AL

AL DR

a b


model of footpad edema induced by subcutaneous injection, DR shortened the

inflammatory responses in young mice on DR for 8 weeks [73]. In clinical stud-

ies, dermatitis was also improved by 8 weeks on a low-energy diet with

micronutrient supplements. All patients responded to some degree, with the

reductions of edema, oozing and skin sloughing (excoriation) being correlated

with weight loss [74]. Inhibition of keratinocyte proliferation, an observed

effect of DR in young mice [75], may contribute to the reduced excoriation.

In humans, serum C-reactive protein (CRP) was 80% lower in a self-

selected group that had maintained DR for 8 years [76]. CRP is an acute-phase

protein secreted by the liver in humans [77], which is an important host defense

molecule by binding to Gram-negative bacteria and enhancing their clearance

by phagocytosing macrophages. However, CRP also has major importance in

vascular disease as a risk indicator and for its potential direct role in lipid accu-

mulations by macrophages (foam cells) in atheromas. Serum CRP is elevated

during obesity and, not surprisingly, short-term weight reduction decreased

serum CRP by 30% [78, 79]. Here we confront the complexities of weight

reduction. DR could enhance the host defense by lowering blood glucose

[80–82], yet DR diminishes CRP and possibly other defenses.

Changes in gene expression in the liver during short-term DR (3–30

weeks) have been profiled by microarrays in several studies [83–87].

Agreement is emerging, despite differences in the choice of rodent genotypes,

duration of DR and microarray technologies. Short-term DR induces and

represses many mRNAs in the liver that mediate increased gluconeogenesis,

increased protein and fatty acid catabolism, and decreased synthesis of choles-

terol, fatty acids and triglycerides [84, 87]. The Krebs cycle (tricarboxylic acid

cycle) drives these changes, with increased shunting of pyruvate to oxaloacetate

in the liver by increased activity of pyruvate carboxylase [88]. The increased

oxaloacetate feeds into gluconeogenesis after conversion by malate dehydroge-

nase, which is also increased by DR. Besides transcriptional changes in these

genes, levels of activity in some enzymes are allosterically regulated, e.g. pyruvate

carboxylase is activated by acetyl-CoA, which is increased by the ß-oxidation

of fatty acids liberated during lipolysis. Acute-phase response mRNAs are also

decreased, including serum amyloid A4 and several complement system factors

(mannose-binding lectin, C4-binding protein, C9) [84]. DNA repair is upregu-

lated (Rad511), as are CYP450 family genes that mediate detoxification and

decrease DNA damage. Again, there is impressive overlap of genes associated

with lipid metabolism and vascular disease.

Overall, these 50–100 mRNA changes are a small subset (�1%) of all the

genes active in the liver. The race is on to find transcription factors that are

shared key regulators of these gene subsets. The effects of DR on many diseases

of aging with inflammatory components give a basis to look for transcription


factors that could modulate inflammatory gene subsets implicated in AD, can-

cer, diabetes and vascular disease [17, 72]. Corton et al. [84] have shown in the

liver that the transcription factors peroxisome proliferator-activated receptor

(PPAR), liver X receptor and retinoid X receptor, which regulate many genes

during DR, also have major roles in inflammation. Additional experimental

models include PPAR knockout mice and drug antagonists, which induce

mRNA changes that overlap with DR to some extent (see below).

Mechanisms Underlying Anti-Inflammatory

Actions of Dietary Restriction

Glucocorticoids

DR increases blood glucocorticoids by 20% or more [18, 73, 89]. The

increased glucocorticoids are a homeostatic response to increase the catabo-

lism of fatty acids for energy (gluconeogenesis), while decreasing the synthe-

sis of fatty acids and cholesterol. If the energy deficit is prolonged, protein

catabolism is also increased. DR also decreases the tissue content of oxida-

tively damaged proteins and lipids, which are always present with enough food

intake and which accumulate during aging. Importantly, glucocorticoids

have broad anti-inflammatory effects mediated by the direct interaction

between the glucocorticoid receptor and the transactivation domain of NF-B

which serves as a key transcription factor in the regulation of inflammation

[90, 91]. Because chronically elevated glucocorticoids are also broadly associ-

ated with neuronal damage and neuronal death, it is paradoxical that DR is

neuroprotective [18].

Glucose and Advanced Glycation End Products

DR lowers blood glucose by about 10–15%. Blood glucose levels directly

influence the formation of oxidation products, as was outlined two decades ago

in Cerami’s hypothesis of glucose as a mediator of aging [92]. Glucose and

other reducing sugars react spontaneously (nonenzymatically) with free amino

groups of proteins (e.g. –NH2 of lysine) to form an initial ‘glycation’ product by

the Amadori reaction, which is assayed as furosine. Then, Amadori glycation

products become oxidized to ‘glycoxidation products’, assayed as pentosidine,

which are also referred to as advanced glycation end products [93]. DR inhibits

glycoxidation during aging in rodent skin, whereas diabetes and end-stage renal

disease accelerate glycoxidation [93–95].

Advanced glycation end product adducts are recognized by a macrophage

scavenger receptor, the RAGE (receptor for advanced glycation end products)

of monocytes (macrophages, microglia) and other cells. RAGEs are also


activated by the �-amyloid peptide and other stress-associated proteins

(S100/calgranulins). A working hypothesis is that advanced glycation end prod-

ucts and RAGEs mediate feed-forward loops of oxidative stress and inflamma-

tion that increase bystander molecular damage in atherosclerosis, AD and other

chronic inflammatory diseases [96, 97]. In turn, RAGE activation enhances

proinflammatory pathways that release cytokines (e.g. interleukin 6) and leuko-

cyte adhesion factors (e.g. monocyte chemoattractant protein 1 and vascular

cell adhesion molecule 1), and that induce the enzymatic synthesis of reactive

oxygen species through NAD(P)H oxidases (e.g. gp91phox) and mitochondrial

electron transport. Lastly, RAGE activation may stimulate feed-forward vicious

cycles by autoinduction in the same cell [98–100]. RAGE downstream signal-

ing pathways include phosphatidylinositol triphosphate kinase, NF-B and

JAK/stat. Feedback loops include the induction of RAGE by tumor necrosis

factor through production of reactive oxygen species, mediated by NF-B

[101]. RAGE-dependent processes are also implicated in AD.

The lower glucose may also be a risk factor in sudden death. Recall the

puzzle that some DR rats died without evidence of gross pathology. We suggest

the precedent of the sudden ‘dead-in-bed syndrome’ of humans. Transient

hypoglycemia is implicated in sudden death from cardiac arrest in type 1 dia-

betics (insulin-deficient), who have 3-fold more unexpected death than healthy

young individuals [102].

Peroxisome Proliferator-Activated Receptors

As discussed earlier, the nuclear hormone superfamily of PPARs may play

a critical role in mediating many of the transcriptional effects of DR in periph-

eral systems. Indeed, in the rat kidney PPAR mRNA, protein and DNA binding

activities are decreased with age and these changes are attenuated with DR

[103]. While the PPARs show wide distribution among glia and neurons in the

brain [104], the effects of age or DR have not been documented. Although

PPARs are best known for their precise transcriptional control of metabolic

events, certain subtypes (in particular, PPAR-�) mediate inflammatory

processes [105–107]. Regarding the brain, PPAR stimulation reduces neuroin-

flammation, both in vivo [108, 109] and in vitro [105, 110, 111]. Thus, PPAR

mediation of the anti-inflammatory effects of DR in the brain seems likely.

Conclusion

DR attenuates many age-related inflammatory events in the CNS and periph-

ery of experimental animal models in concert with increasing life span. In the

aging brain, DR suppresses the activation of microglia and astrocytes which are


associated with demyelination, synaptic atrophy and neurodegeneration. These

events are believed to be the underlying causes of age-related cognitive decline.

Rodent models suggest that DR may also protect against age-related neurodegen-

erative diseases involving inflammation such as AD and ischemia/reperfusion.

Even short-term DR can attenuate inflammation and affect metabolic and

DNA repair pathways. Mechanisms by which DR suppresses peripheral inflamma-

tion include the elevation of glucocorticoids, lowering of glucose and activation of

PPARs. Although the effects of DR are less understood in the brain, common path-

ways are emerging that link many normal aging inflammatory processes with age-

related diseases such as AD, cancer, diabetes and cardiovascular disease.

Acknowledgement

Supported by grants to C.E.F. and T.E.M. (AG13499 and the Alzheimers Association

Temple Award) and A.M.W. (AG00093).

References

1 Berg BM: Pathology and aging; in Everitt AV, Burgess JA (eds): Hypothalamus, Pituitary, and

Aging. Springfield, Thomas, 1976, pp 43–67.

2 Finch CE: Longevity, Senescence, and the Genome. Chicago, University of Chicago Press, 1990,

p 922.

3 Merry BJ: Oxidative stress and mitochondrial function with aging – The effects of calorie restric-


4 Sacher G: Life table modification and life prolongation; in Finch CE, Hayflick L (eds): Handbook

of the Biology of Aging. New York, Van Nostrand Reinhold, 1977, pp 582–638.

5 Weindruch RH, Walford RL: The Retardation of Aging and Disease by Dietary Restriction.


6 Dirx MJ, Zeegers MP, Dagnelie PC, van den Bogaard T, van den Brandt PA: Energy restriction and

the risk of spontaneous mammary tumors in mice: a meta-analysis. Int J Cancer 2003;106:766–770.

7 Kritchevsky D: The effect of over- and undernutrition on cancer. Eur J Cancer Prev 1995;4: 445–451.

8 Shimokawa I, Higami Y, Hubbard GB, McMahan CA, Masoro EJ, Yu BP: Diet and the suitability

of the male Fischer 344 rat as a model for aging research. J Gerontol 1993;48:B27–B32.

9 Maeda H, Gleiser CA, Masoro EJ, Murata I, McMahan CA, Yu BP: Nutritional influences on

aging of Fischer 344 rats. 2. Pathology. J Gerontol 1985;40:671–688.

10 Everitt AV, Seedsman NJ, Jones F: The effects of hypophysectomy and continuous food restriction,

begun at ages 70 and 400 days, on collagen aging, proteinuria, incidence of pathology and

longevity in the male rat. Mech Ageing Dev 1980;12:161–172.

11 Berg BN, Wolf A, Simms HS: Nutrition and longevity in the rat. 4. Food restriction and the radicu-

loneuropathy of aging rats. J Nutr 1962;77:439–442.

12 Kazui H, Fujisawa K: Radiculoneuropathy of ageing rats: a quantitative study. Neuropathol Appl

Neurobiol 1988;14:137–156.

13 Mitsumori K, Maita K, Shirasu Y: An ultrastructural study of spinal nerve roots and dorsal root

ganglia in aging rats with spontaneous radiculoneuropathy. Vet Pathol 1981;18:714–726.

14 Krinke G: Spinal radiculoneuropathy in aging rats: demyelination secondary to neuronal dwin-

dling? Acta Neuropathol (Berl) 1983;59:63–69.


15 Bronson RT: Rate of occurrence of lesions in 20 inbred and hybrid genotypes of rats and mice sac-

rificed at 6 month intervals during the first years of life; in Harrison DE (ed): Genetic Effects on

Aging. Caldwell, Telford Press, 1990, pp 279–357.

16 Finch CE, Longo VD: The gero-inflammatory manifold; in Rogers J (ed): Neuroinflammatory

Mechanisms in Alzheimer Disease Basic and Clinical Research. Boston, Birkhäuser, 2001, pp 238–258.

17 Finch C, Morgan T, Rozovsky I, Xie Z, Weindruch R, Prolla T: Microglia and aging in the brain;

in Streit W (ed): Microglia in the Regenerating and Degenerating Central Nervous System. New

York, Springer, 2002, pp 275–305.

18 Patel NV, Finch CE: The glucocorticoid paradox of caloric restriction in slowing brain aging.


19 Masliah E, Mallory M, Hansen L, DeTeresa R, Terry RD: Quantitative synaptic alterations in the

human neocortex during normal aging. Neurology 1993;43:192–197.

20 Morgan DG, May PC, Finch CE: Dopamine and serotonin systems in human and rodent brain:

effects of age and neurodegenerative disease. J Am Geriatr Soc 1987;35:334–345.

21 Wang GJ, Volkow ND, Logan J, Fowler JS, Schlyer D, MacGregor RR, Hitzemann RJ, Gur RC,

Wolf AP: Evaluation of age-related changes in serotonin 5-HT2 and dopamine D2 receptor avail-

ability in healthy human subjects. Life Sci 1995;56:PL249–PL253.

22 Keck BJ, Lakowski JM: Neurochemistry of receptor dynamics in the aging brain; in Hof PR,

Mobbs CV (eds): Functional Neurobiology of Aging. New York, Academic Press, 2001, pp 21–29.

23 Teter B, Finch CE: Caliban’s heritance and the genetics of neuronal aging. Trends Neurosci

2004;27:627–632.

24 Moore TL, Schettler SP, Killiany RJ, Herndon JG, Luebke JI, Moss MB, Rosene DL: Cognitive

impairment in aged rhesus monkeys associated with monoamine receptors in the prefrontal cortex.

Behav Brain Res 2005;160:208–221.

25 Rasmussen T, Schliemann T, Sörensen JC, Zimmer J, West MJ: Memory impaired aged rats: no

loss of principal hippocampal and subicular neurons. Neurobiol Aging 1996;17:143–147.

26 Long JM, Kalehua AN, Muth NJ, Calhoun ME, Jucker M, Hengemihle JM, Ingram DK, Mouton

PR: Stereological analysis of astrocyte and microglia in aging mouse hippocampus. Neurobiol

Aging 1998;19:497–503.

27 Abe O, Aoki S, Hayashi N, Yamada H, Kunimatsu A, Mori H, Yoshikawa T, Okubo T, Ohtomo K:

Normal aging in the central nervous system: quantitative MR diffusion-tensor analysis. Neurobiol

Aging 2002;23:433–441.

28 Salat DH, Tuch DS, Greve DN, van der Kouwe AJ, Hevelone ND, Zaleta AK, Rosen BR, Fischl B,

Corkin S, Rosas HD, Dale AM: Age-related alterations in white matter microstructure measured

by diffusion tensor imaging. Neurobiol Aging 2005;26:1215–1227.

29 Matochik JA, Chefer SI, Lane MA, Woolf RI, Morris ED, Ingram DK, Roth GS, London ED: Age-

related decline in striatal volume in monkeys as measured by magnetic resonance imaging.


30 Bartzokis G: Age-related myelin breakdown: a developmental model of cognitive decline and

Alzheimer’s disease. Neurobiol Aging 2004;25:5–18.

31 Sloane JA, Hollander W, Moss MB, Rosene DL, Abraham CR: Increased microglial activation and

protein nitration in white matter of the aging monkey. Neurobiol Aging 1999;20:395–405.

32 Hinman JD, Duce JA, Siman RA, Hollander W, Abraham CR: Activation of calpain-1 in myelin

and microglia in the white matter of the aged rhesus monkey. J Neurochem 2004;89:430–441.

33 Morgan TE, Xie Z, Goldsmith S, Yoshida T, Lanzrein AS, Stone D, Rozovsky I, Perry G, Smith

MA, Finch CE: The mosaic of brain glial hyperactivity during normal ageing and its attenuation

by food restriction. Neuroscience 1999;89:687–699.

34 Ogura K, Ogawa M, Yoshida M: Effects of ageing on microglia in the normal rat brain: immuno-

histochemical observations. Neuroreport 1994;5:1224–1226.

35 Wong AM, Patel NV, Patel NK, Wei M, Morgan TE, de Beer MC, de Villiers WJS, Finch CE:

Macrosialin increases during normal brain aging are attenuated by caloric restriction. Neurosci

Lett 2005;390:76–80.

36 de Beer MC, Zhao Z, Webb NR, van der Westhuyzen DR, de Villiers WJ: Lack of a direct role for

macrosialin in oxidized LDL metabolism. J Lipid Res 2003;44:674–685.


37 Fischer B, von Knethen A, Brune B: Dualism of oxidized lipoproteins in provoking and attenuat-

ing the oxidative burst in macrophages: role of peroxisome proliferator-activated receptor gamma.

J Immunol 2002;168:2828–2834.

38 Khan M, Pelengaris S, Cooper M, Smith C, Evan G, Betteridge J: Oxidised lipoproteins may pro-

mote inflammation through the selective delay of engulfment but not binding of apoptotic cells by

macrophages. Atherosclerosis 2003;171:21–29.

39 Matochik JA, Chefer SI, Lane MA, Roth GS, Mattison JA, London ED, Ingram DK: Age-related

decline in striatal volume in rhesus monkeys: assessment of long-term calorie restriction.


40 Ridet JL, Malhotra SK, Privat A, Gage FH: Reactive astrocytes: cellular and molecular cues to

biological function. Trends Neurosci 1997;20:570–577.

41 Eddleston M, Mucke L: Molecular profile of reactive astrocytes – implications for their role in

neurologic disease. Neuroscience 1993;54:15–36.

42 Nichols NR, Day JR, Laping NJ, Johnson SA, Finch CE: GFAP mRNA increases with age in rat

and human brain. Neurobiol Aging 1993;14:421–429.

43 Major DE, Kesslak JP, Cotman CW, Finch CE, Day JR: Life-long dietary restriction attenuates

age-related increases in hippocampal glial fibrillary acidic protein mRNA. Neurobiol Aging

1997;18:523–526.

44 Lee CK, Weindruch R, Prolla TA: Gene-expression profile of the ageing brain in mice. Nat Genet

2000;25:294–297.

45 Shetty AK, Hattiangady B, Shetty GA: Stem/progenitor cell proliferation factors FGF-2, IGF-1,

and VEGF exhibit early decline during the course of aging in the hippocampus: role of astrocytes.

Glia 2005;51:173–186.

46 Rozovsky I, Wei M, Morgan T, Finch C: Reversible age impairments in neurite outgrowth by

manipulations of astrocytic GFAP. Neurobiol Aging 2005;26:705–715.

47 Mrak RE, Griffin ST, Graham DI: Aging-associated changes in human brain. J Neuropathol Exp

Neurol 1997;56:1269–1275.

48 Kuhn HG, Dickinson-Anson H, Gage FH: Neurogenesis in the dentate gyrus of the adult rat: age-

related decrease of neuronal progenitor proliferation. J Neurosci 1996;16:2027–2033.

49 Rozovsky I, Finch CE, Morgan TE: Age-related activation of microglia and astrocytes: in vitro

studies show persistent phenotypes of aging, increased proliferation, and resistance to down-

regulation. Neurobiol Aging 1998;19:97–103.

50 Wilhelmsson U, Li L, Pekna M, Berthold CH, Blom S, Eliasson C, Renner O, Bushong E,

Ellisman M, Morgan TE, Pekny M: Absence of glial fibrillary acidic protein and vimentin pre-

vents hypertrophy of astrocytic processes and improves post-traumatic regeneration. J Neurosci

2004;24:5016–5021.

51 Larsson A, Wilhelmsson U, Pekna M, Pekny M: Increased cell proliferation and neurogenesis in

the hippocampal dentate gyrus of old GFAP(�/�)Vim(�/�) mice. Neurochem Res 2004;29:

2069–2073.

52 Nichols NR, Finch CE, Nelson JF: Food restriction delays the age-related increase in GFAP

mRNA in rat hypothalamus. Neurobiol Aging 1995;16:105–110.

53 Morgan TE, Rozovsky I, Goldsmith SK, Stone DJ, Yoshida T, Finch CE: Increased transcription of

the astrocyte gene GFAP during middle-age is attenuated by food restriction: implications for the

role of oxidative stress. Free Radic Biol Med 1997;23:524–528.

54 Ingram DK, Weindruch R, Spangler EL, Freeman JR, Walford RL: Dietary restriction benefits

learning and motor performance of aged mice. J Gerontol 1987;42:78–81.

55 Moroi-Fetters SE, Mervis RF, London ED, Ingram DK: Dietary restriction suppresses age-related

changes in dendritic spines. Neurobiol Aging 1989;10:317–322.

56 Hori N, Hirotsu I, Davis PJ, Carpenter DO: Long-term potentiation is lost in aged rats but pre-

served by calorie restriction. Neuroreport 1992;3:1085–1088.

57 Eckles-Smith K, Clayton D, Bickford P, Browning MD: Caloric restriction prevents age-

related deficits in LTP and in NMDA receptor expression. Brain Res Mol Brain Res 2000;78:

154–162.

58 Mattson MP, Duan W, Guo Z: Meal size and frequency affect neuronal plasticity and vulnerability

to disease: cellular and molecular mechanisms. J Neurochem 2003;84:417–431.


59 Duan W, Mattson MP: Dietary restriction and 2-deoxyglucose administration improve behavioral

outcome and reduce degeneration of dopaminergic neurons in models of Parkinson’s disease. J

Neurosci Res 1999;57:195–206.

60 Bruce-Keller AJ, Umberger G, McFall R, Mattson MP: Food restriction reduces brain damage and

improves behavioral outcome following excitotoxic and metabolic insults. Ann Neurol 1999;45:

8–15.

61 Finch CE: The neurotoxicology of hard foraging and fat-melts. Proc Natl Acad Sci USA

2004;101:17887–17888.

62 Johnstone EM, Chaney MO, Norris FH, Pascual R, Little SP: Conservation of the sequence of the

Alzheimer’s disease amyloid peptide in dog, polar bear and five other mammals by cross-species

polymerase chain reaction analysis. Brain Res Mol Brain Res 1991;10:299–305.

63 Patel NV, Gordon MN, Connor KE, Good RA, Engelman RW, Mason J, Morgan DG, Morgan TE,

Finch CE: Caloric restriction attenuates A� deposition in Alzheimer transgenic models. Neurobiol

Aging 2005;26:995–1000.

64 Wang J, Ho L, Qin W, Rocher AB, Seror I, Humala N, Maniar K, Dolios G, Wang R, Hof PR,

Pasinetti GM: Caloric restriction attenuates �-amyloid neuropathology in a mouse model of

Alzheimer’s disease. FASEB J 2005;19:659–661.

65 Luchsinger JA, Mayeux R: Dietary factors and Alzheimer’s disease. Lancet Neurol 2004;3:

579–587.

66 Luchsinger JA, Tang MX, Shea S, Mayeux R: Caloric intake and the risk of Alzheimer disease.

Arch Neurol 2002;59:1258–1263.

67 Scarmeas N, Stern Y: Cognitive reserve and lifestyle. J Clin Exp Neuropsychol 2003;25:625–633.

68 Laurin D, Verreault R, Lindsay J, MacPherson K, Rockwood K: Physical activity and risk of cog-

nitive impairment and dementia in elderly persons. Arch Neurol 2001;58:498–504.

69 Kim KY, Ju WK, Neufeld AH: Neuronal susceptibility to damage: comparison of the retinas of

young, old and old/caloric restricted rats before and after transient ischemia. Neurobiol Aging

2004;25:491–500.

70 Blalock EM, Chen KC, Sharrow K, Herman JP, Porter NM, Foster TC, Landfield PW: Gene

microarrays in hippocampal aging: statistical profiling identifies novel processes correlated with

cognitive impairment. J Neurosci 2003;23:3807–3819.

71 Lu T, Pan Y, Kao S-Y, Li C, Kohane IS, Chan J, Yankner BA: Gene regulation and DNA damage in

the ageing human brain. Nature 2004;429:883–891.

72 Longo VD, Finch CE: Evolutionary medicine: from starvation and dwarf model systems to healthy

centenarians? Science 2003;299:1342–1346.

73 Klebanov S, Diais S, Stavinoha WB, Suh Y, Nelson JF: Hyperadrenocorticism, attenuated inflam-

mation, and the life-prolonging action of food restriction in mice. J Gerontol A Biol Sci Med Sci

1995;50:B79–B82.

74 Kouda K, Tanaka T, Kouda M, Takeuchi H, Takeuchi A, Nakamura H, Takigawa M: Low-energy

diet in atopic dermatitis patients: clinical findings and DNA damage. J Physiol Anthropol Appl

Human Sci 2000;19:225–228.

75 Hsieh EA, Chai CM, de Lumen BO, Neese RA, Hellerstein MK: Dynamics of keratinocytes in

vivo using HO labeling: a sensitive marker of epidermal proliferation state. J Invest Dermatol

2004;123:530–536.

76 Fontana L, Meyer TE, Klein S, Holloszy JO: Long-term calorie restriction is highly effective in

reducing the risk for atherosclerosis in humans. Proc Natl Acad Sci USA 2004;101:6659–6663.

77 Szalai AJ: The biological functions of C-reactive protein. Vasc Pharmacol 2002;39:105–107.

78 Tchernof A, Nolan A, Sites CK, Ades PA, Poehlman ET: Weight loss reduces C-reactive protein

levels in obese postmenopausal women. Circulation 2002;105:564–569.

79 Heilbronn LK, Noakes M, Clifton PM: Energy restriction and weight loss on very-low-fat diets

reduce C-reactive protein concentrations in obese, healthy women. Arterioscler Thromb Vasc Biol

2001;21:968–970.

80 Bochicchio GV, Salzano L, Joshi M, Bochicchio K, Scalea TM: Admission preoperative glucose is

predictive of morbidity and mortality in trauma patients who require immediate operative inter-

vention. Am Surg 2005;71:171–174.


81 Butler SO, Btaiche IF, Alaniz C: Relationship between hyperglycemia and infection in critically ill

patients. Pharmacotherapy 2005;25:963–976.

82 Swenne CL, Lindholm C, Borowiec J, Schnell AE, Carlsson M: Peri-operative glucose control and

development of surgical wound infections in patients undergoing coronary artery bypass graft.

J Hosp Infect 2005;61:201–212.

83 Miller RA, Chang Y, Galecki AT, Al-Regaiey K, Kopchick JJ, Bartke A: Gene expression patterns

in calorically restricted mice: partial overlap with long-lived mutant mice. Mol Endocrinol

2002;16:2657–2666.

84 Corton JC, Apte U, Anderson SP, Limaye P, Yoon L, Latendresse J, Dunn C, Everitt JI, Voss KA,

Swanson C, Kimbrough C, Wong JS, Gill SS, Chandraratna RA, Kwak MK, Kensler TW, Stulnig

TM, Steffensen KR, Gustafsson JA, Mehendale HM: Mimetics of caloric restriction include ago-

nists of lipid-activated nuclear receptors. J Biol Chem 2004;279:46204–46212.

85 Dhahbi JM, Mote PL, Wingo J, Tillman JB, Walford RL, Spindler SR: Calories and aging alter

gene expression for gluconeogenic, glycolytic, and nitrogen-metabolizing enzyme. Am J Physiol

1999;277:E352–E360.

86 Dhahbi JM, Cao SX, Mote PL, Rowley BC, Wingo JE, Spindler SR: Postprandial induction of

chaperone gene expression is rapid in mice. J Nutr 2002;132:31–37.

87 Dhahbi JM, Kim HJ, Mote PL, Beaver RJ, Spindler SR: Temporal linkage between the phenotypic

and genomic responses to caloric restriction. Proc Natl Acad Sci USA 2004;101:5524–5529.

88 Hagopian K, Ramsey JJ, Weindruch R: Krebs cycle enzymes from livers of old mice are differen-

tially regulated by caloric restriction. Exp Gerontol 2004;39:1145–1154.

89 Sabatino F, Masoro EJ, McMahan CA, Kuhn RW: Assessment of the role of the glucocorticoid

system in aging processes and in the action of food restriction. J Gerontol 1991;46:B171–B179.

90 McKay LI, Cidlowski JA: Molecular control of immune/inflammatory responses: interactions

between nuclear factor-B and steroid receptor-signaling pathways. Endocr Rev 1999;20:

435–459.

91 Smoak KA, Cidlowski JA: Mechanisms of glucocorticoid receptor signaling during inflammation.

Mech Ageing Dev 2004;125:697–706.

92 Cerami A: Hypothesis: glucose as a mediator of aging. J Am Geriatr Soc 1985;33:626–634.

93 Ulrich P, Cerami A: Protein glycation, diabetes, and aging. Recent Prog Horm Res 2001;56:1–21.

94 Monnier VM, Kohn RR, Cerami A: Accelerated age-related browning of human collagen in dia-

betes mellitus. Proc Natl Acad Sci USA 1984;81:583–587.

95 Sell DR, Carlson EC, Monnier VM: Differential effects of type 2 (non-insulin-dependent) diabetes

mellitus on pentosidine formation in skin and glomerular basement membrane. Diabetologia

1993;36:936–941.

96 Ramasamy R, Vannucci SJ, Yan SS, Herold K, Yan SF, Schmidt AM: Advanced glycation end

products and RAGE: a common thread in aging, diabetes, neurodegeneration, and inflammation.

Glycobiology 2005;15:16R–28R.

97 Lu C, He JC, Cai W, Liu H, Zhu L, Vlassara H: Advanced glycation endproduct (AGE) receptor 1

is a negative regulator of the inflammatory response to age in mesangial cells. Proc Natl Acad Sci

USA 2004;101:11767–11772.

98 Basta G, Lazzerini G, Del Turco S, Ratto GM, Schmidt AM, De Caterina R: At least 2 distinct

pathways generating reactive oxygen species mediate vascular cell adhesion molecule 1 induction

by advanced glycation end products. Arterioscler Thromb Vasc Biol 2005;25:1401–1407.

99 Wautier MP, Chappey O, Corda S, Stern DM, Schmidt AM, Wautier JL: Activation of NADPH

oxidase by AGE links oxidant stress to altered gene expression via RAGE. Am J Physiol


100 Feng L, Matsumoto C, Schwartz A, Schmidt AM, Stern DM, Pile-Spellman J: Chronic vascular

inflammation in patients with type 2 diabetes: endothelial biopsy and RT-PCR analysis. Diabetes

Care 2005;28:379–384.

101 Mukherjee TK, Mukhopadhyay S, Hoidal JR: The role of reactive oxygen species in TNF-dependent

expression of the receptor for advanced glycation end products in human umbilical vein endo-

thelial cells. Biochim Biophys Acta 2005;1744:213–223.

102 Heller SR: Abnormalities of the electrocardiogram during hypoglycaemia: the cause of the dead in

bed syndrome? Int J Clin Pract Suppl 2002;129:27–32.


103 Sung B, Park S, Yu BP, Chung HY: Modulation of PPAR in aging, inflammation, and calorie

restriction. J Gerontol A Biol Sci Med Sci 2004;59:997–1006.

104 Moreno S, Farioli-Vecchioli S, Ceru MP: Immunolocalization of peroxisome proliferator-activated

receptors and retinoid X receptors in the adult rat CNS. Neuroscience 2004;123:131–145.

105 Jiang C, Ting AT, Seed B: PPAR-�agonists inhibit production of monocyte inflammatory

cytokines. Nature 1998;391:82–86.

106 Ricote M, Welch JS, Glass CK: Regulation of macrophage gene expression by the peroxisome

proliferator-activated receptor �. Horm Res 2000;54:275–280.

107 Sundararajan S, Landreth GE: Anti-inflammatory properties of PPAR-� agonists following

ischemia. Drug News Perspect 2004;17:229–236.

108 Heneka MT, Klockgether T, Feinstein DL: Peroxisome proliferator-activated receptor � ligands

reduce neuronal inducible nitric oxide synthase expression and cell death in vivo. J Neurosci

2000;20:6862–6867.

109 Dehmer T, Heneka MT, Sastre M, Dichgans J, Schulz JB: Protection by pioglitazone in the MPTP

model of Parkinson’s disease correlates with IB induction and block of NF-B and iNOS acti-

vation. J Neurochem 2004;88:494–501.

110 Petrova TV, Akama KT, Van Eldik LJ: Cyclopentenone prostaglandins suppress activation of

microglia: down-regulation of inducible nitric-oxide synthase by 15-deoxy-�12,14-prostaglandin

J2. Proc Natl Acad Sci USA 1999;96:4668–4673.

111 Combs CK, Johnson DE, Karlo JC, Cannady SB, Landreth GE: Inflammatory mechanisms in

Alzheimer’s disease: inhibition of �-amyloid-stimulated proinflammatory responses and neuro-

toxicity by PPAR-� agonists. J Neurosci 2000;20:558–567.

T.E. Morgan, PhD

Leonard Davis School of Gerontology

University of Southern California, 3715 McClintock Avenue

Los Angeles, CA 90089–0191 (USA)




Dietary Restriction in the NematodeCaenorhabditis elegans

Koen Houthoofd a,b, David Gemsb, Thomas E. Johnsonc,

Jacques R. Vanfleterena

aDepartment of Biology, Ghent University, Ghent, Belgium; bDepartment of Biology,

UCL Centre for Research on Ageing, University College London, London, UK;cDepartment of Integrative Physiology, Institute for Behavioral Genetics, University of

Colorado at Boulder, Boulder, Colo., USA

AbstractThe nematode Caenorhabditis elegans has proved to be an excellent model organism

for the study of development and aging. Many aging mutants have been discovered in the

past two decades, and much has been discovered about the physiology of long-lived mutants.

It therefore seems surprising that dietary restriction (DR) has not been extensively studied

using C. elegans. The main reason for this is the lack of an ideal method to subject C. elegans

to DR. However, several authors have tried to study the effect of DR on the metabolism and

physiology of C. elegans, and epistasis-type interaction studies have been carried out in order

to detect genes that might be involved in DR effects. These studies show that DR life exten-

sion is not caused by a reduced metabolic rate, consistent with results in other species.

Moreover, the well-known insulin/IGF-1 pathway seems not to mediate life-extending

effects. One possibility is that target of rapamycin signaling mediates the effects of DR on

life span in C. elegans.


The beneficial effect of food restriction on life span was first described in

rodents 70 years ago and later shown to occur in a wide range of vertebrate and

invertebrate taxa [1–4]. Because of its wide occurrence it is believed that this

effect enhances fitness and represents an evolutionary adaptation. The underly-

ing reasoning is that there are trade-offs between longevity and reproduction.

Fitness is strongly determined by reproductive success. It will therefore be

advantageous to allocate energy resources to reproduction as long as investment

Dietary Restriction in C. elegans 99

in maintenance is sufficient to support a longevity that is only limited by envi-

ronmental hazards, e.g. predation. When food is scarce, reproductive success

will decrease to some point where it becomes advantageous to invest as much

as possible in somatic maintenance thereby delaying reproduction until food

supply improves. This disposable soma theory of aging is very plausible and

mathematical modeling suggests that it is applicable to rodents [5, 6]. The

notion that life span extension by dietary restriction (DR) is a direct evolution-

ary adaptation suggests the existence of regulatory (signaling) pathways that

sense nutrient availability and enhance investment in somatic maintenance

accordingly to maximize life time fitness. However, nonadaptive explanations

for the effect of DR remain possible. For example, DR might attenuate

oxidative damage accumulation inflicted by reactive oxygen species (ROS)

by lowering ROS production. Alternatively, DR could stimulate degradation of

macromolecules for recycling when basic units for synthesis (e.g. amino acids,

monosaccharides, purines, pyrimidines) are scarce. Enhanced recycling could

assure rapid clearance of damaged macromolecules to the benefit of somatic

maintenance.

Simple invertebrate species including the nematode Caenorhabditis ele-

gans and the fly Drososphila melanogaster are experimentally tractable model

organisms for studying the interplay between environmental conditions and the

genes and signaling pathways that mediate life extension. Experiments in

C. elegans suggest that the insulin (Ins)/IGF-1 pathway is not involved in

DR-induced life extension and point to a more important role of the target of

rapamycin (TOR) pathway.

Studying Dietary Restriction in C. elegans

C. elegans is a free-living, microbivorous soil-dwelling nematode. Food is

detected via olfactory and chemosensory perception by amphids, paired sense

organs in the head region of the worm that contain the ciliated endings of sen-

sory neurons allowing the worm to respond to changes in the environment.

Food is taken up via peristaltic contractions of muscles in the pharyngeal

region. The microbes (e.g. bacteria) are ground in the terminal bulb of the phar-

ynx, and the remaining debris is passed into the intestine, which runs most of

the body length. The intestine is a 1-cell-thick epithelial tube with microvilli on

the luminal side. The intestinal cells are thought to absorb nutrients via pinocy-

tosis while smaller molecules are probably taken up by specific receptors; how-

ever, the biology of nutrient uptake remains poorly characterized. Nutrients are

then most probably secreted through the basal surface into the pseudocoelomic

fluid, which contacts most tissues [7, 8].

Houthoofd/Gems/Johnson/Vanfleteren 100

In the laboratory, C. elegans is usually cultured on a lawn of Escherichia

coli bacteria on agar plates. Typically, the slow-growing OP50 strain, which is

auxotrophic for uracil, is used because the thin bacterial lawn that it forms

makes the microscopic study of C. elegans easier. A thin, live E. coli lawn is

thus considered as the normal, nonrestricted diet of the worm. C. elegans can

also be grown in liquid suspension cultures with E. coli as food source, but

shaking vigorously is needed if the depth of the medium exceeds a few milli-

meters, to prevent hypoxic stress to the worms [9].

The ideal method for studying the importance of calories in the DR effect

in C. elegans would be the use of a medium containing all essential nutrients

needed for a maximal life span and a reduced amount of calories (e.g. in the

form of E. coli). However, this is difficult to achieve for several reasons. The

first problem is that the normal food source of C. elegans in the laboratory,

E. coli, is slightly toxic to the worm. In old worms, E. coli cells frequently

accumulate in and block the pharynx, the intestine and the uterus of the worm

[10; own observations]. Feeding C. elegans with E. coli that was killed by UV

irradiation or by antibiotics resulted in a 16–40% increase in mean life span

[10, 11]. Treating the bacteria with a bacteriostatic agent also resulted in life

extension, suggesting that something associated with the proliferation of bacte-

ria reduces the life span of the worm [10]. This is consistent with previous

claims that toxins that are produced by proliferating bacteria might be the

causative agent of life span reduction [12, 13]. Reducing the E. coli intake thus

not only lengthens the worm’s life span by reduced caloric intake, but also by

reducing E. coli toxicity.

A second difficulty in studying DR in the worm is that the beneficial

effects of reduced caloric intake are possibly offset by malnutrition, since both

calories and essential nutrients are provided by the same food source (E. coli

cells). Thus, reducing the E. coli intake also reduces the availability of com-

pounds that are necessary for maximizing the life span of the worm.

Thirdly, wild-type C. elegans that are fed E. coli that has a deficiency in

the synthesis of ubiquinone, live substantially longer [14]. Therefore, reducing

the amount of bacterial uptake might lead to life span extension due to reduced

ubiquinone uptake. It is therefore necessary to keep in mind that the beneficial

effects of reduced bacterial food intake are not only due to reducing calories.

Given that no evidence has ever been presented that it is the reduction of calo-

ries that is critical to DR effects in C. elegans, we use the term ‘dietary restric-

tion’ rather than ‘caloric restriction’ (CR) throughout the text.

Many different methods have been used to investigate the effects of DR on

various parameters in C. elegans, but all suffer from at least one of the above-

mentioned potential problems. The methods used can be roughly grouped into

three classes: (1) reducing the available amount of bacteria, (2) using synthetic


media and (3) reducing levels of the receptors necessary for the uptake of mole-

cules into the intestinal cells. In a pioneering study, Klass [15] decreased the bac-

terial concentration in suspension culture to impose DR on C. elegans. He found

a mean life span extension of 60% when the bacterial density was decreased

from 109 to 108 bacterial cells/ml (higher concentrations lead to a decreased life

span and decreased reproductive capacity, probably because of hypoxic stress).

Under these conditions, progeny production was decreased more than fourfold.

Hosono et al. [16] reduced bacterial concentrations on agar plates by decreasing

the amounts of bactopeptone. They also observed life extension, but no reduction

of reproductive capacity or body volume was seen in this case. The advantage of

restricting worms by reducing bacterial concentration is that this treatment can

be applied in a quantitative manner, which is useful if one wants to test the inter-

action with other life-extending mechanisms [17].

Reduced bacterial uptake can also be obtained genetically by using

mutants with a reduced pumping rate. Such Eat mutants have a starved appear-

ance and were used by Lakowski and Hekimi [18] to study the genetics of DR.

[Note that in C. elegans nomenclature, Eat refers to the phenotype, eat-1

(ad427) to the gene (allele) and EAT-1 to the protein encoded by eat-1.] These

authors found that most Eat mutants were indeed long-lived, with a maximal

life extension of about 50%. However, smaller or no effects on life span were

found in some other laboratories [19]. A likely explanation is that these mutants

experience DR depending on the environmental conditions such as the thick-

ness of the bacterial lawn. For example, we saw life extension when the Eat

mutants were grown in liquid culture, but not on plates [Houthoofd, unpubl.

results]. Perhaps reduced bacterial intake of Eat mutants is not limiting when

they are grown on plates with plenty of E. coli. Reducing the amount of E. coli

is the method that shows the most similarity with DR studies in other species

but suffers from the above-mentioned problems.

C. elegans can also be grown in sterile axenic media (axenic: grown in the

absence of any other species). One example of such a medium is Caenorhabditis

briggsae maintenance medium, a defined medium containing 54 compounds

[20]. A more frequently used, semidefined, axenic medium is composed of

yeast extract and soy peptone [21, 22]. A sterol and heme source must be added

to axenic media since C. elegans is not able to synthesize these compounds. The

heme requirement was originally met by adding tissue extracts, e.g. liver extract

or chicken embryo extract. Later it was found that pure hemoglobin is a suitable

supplement [23]. Sufficient sterols are supplied as impurities in yeast extract,

soy peptone and the heme source. When grown in axenic media, the life span of

worms is about twice as long as in populations maintained on E. coli [22, 24].

Axenically cultured worms have a retarded development and severely reduced

fertility compared to monoxenic culture conditions, which is also observed


under other DR regimens. That axenic culture entails DR is also suggested by

the observation that worms grown in axenic medium show several metabolic

and stress defense alterations similar to those seen in worms that are restricted

by eat mutation or by lowering bacterial food supply [22, 25]. However, axenic

media are generally rich in nutrients and it is therefore puzzling that worms that

are grown axenically seem to experience DR. Possible explanations are that

axenic medium cannot be taken up by the worms efficiently, either because

worms are filter feeders, spitting out most of the liquid medium, or because

compounds are not taken up efficiently by the intestine. Another possibility is

that some nutrients in the medium cannot be metabolized by the worm. Finally,

this medium might simply not fully meet the nutritional needs of this worm,

though this seems unlikely. Since axenic media are sterile, life extension is par-

tially caused by the absence of pathogenic bacteria and possibly ubiquinone.

And, clearly, axenic medium cannot be used as a means to partially reduce

caloric intake: it is loaded with calories.

Knocking down the activity of several transporters has also led to life exten-

sion in C. elegans. For example, RNAi (RNA-mediated interference) of nac-2

(transporter of di- and tricarboxylates) or nac-3 (transporter of dicarboxylates)

leads to a life extension of 19 and 15%, respectively [26, 27]. Decreased NAC-2

activity also caused a reduction in body size and intestinal lipid content, pheno-

types not seen in worms with lower NAC-3 activity. nac-2 is an orthologue of the

Drosophila Indy (‘I’m not dead yet’) gene. As its name suggests, a mutation in

Indy also causes life extension, but these flies have no lower brood size (when

fed ad libitum) [28]. PEP-2 (formerly known as OPT-2) is a proton-dependent

carrier responsible for the uptake of di- and tripeptides. Mutation in pep-2 leads

to a smaller body size and reduced developmental rate and fertility but does not

increase life span. However, mutation in pep-2 extends the life span of long-lived

daf-2 mutants [29]. The reason for this is unclear, but it might be that the pep-2

mutation is too strong, causing malnutrition effects offsetting the positive effects

of DR in wild-type worms, but not in daf-2 worms, which have more fat stores,

possibly protecting them against starvation. The NHX-2 Na�/H� exchanger is

needed to prevent acidification of the cytoplasm [30]. RNAi of nhx-2 increases

life span, among other DR phenotypes, probably because uptake of di- and

tripeptides is inhibited when the cytoplasmic pH drops.

Possible Mechanisms of Dietary-Restriction-Mediated

Life Extension

The mechanism by which DR extends life span is still unknown. Stochastic

as well as regulated mechanisms have been proposed. Most early models


attribute the action of DR to a reduction of ROS production, consistent with the

free radical theory of aging [31]. Drawing on the discovery of several signaling

pathways that regulate the aging rate of C. elegans, there is a growing belief that

DR effects may be mediated by (one of) these pathways [19].

Reduced Metabolic Rate?

Early in the 20th century, an inverse relationship between life span and

mass-specific oxygen consumption was observed in a comparison of eight

mammals [32]. Others found a similar inverse relationship between life span

and environmental temperature in insects [33]. These observations were used

by Pearl [34] as a basis for his rate-of-living theory (‘live fast, die young’). This

theory revived strongly when Harman formulated the free radical theory of

aging in 1956 [31]: a lower metabolic rate would, it was thought, lead to

reduced mitochondrial ROS production. It seemed reasonable that DR could

lead to a reduced metabolic rate since less fuel is available for driving the mito-

chondrial electron transport chain, and it was thus not surprising that one of the

most commonly proposed mechanisms of DR-induced life extension was

reduced metabolic rate. Since then it was realized however that a higher aerobic

metabolism is not necessarily linked to increased free radical generation, since

ROS production is dependent on the inner mitochondrial membrane potential.

In fact, membrane potential and ROS production are inversely related with res-

piratory activity: high in resting mitochondria and low in actively respiring

mitochondria [35–37]. Uncoupling proteins can also lower the membrane

potential, again leading to a lowering of free radical generation. For instance,

Speakman et al. [38] found that individuals with the highest metabolism, in a

mouse population, had the highest mitochondrial uncoupling rate and the

longest life span. A reduced ROS production rate could be caused by shifting to

an anaerobic metabolism [39] or by increasing the efficiency of the electron

transport chain [40].

In an attempt to study the interaction between dietary restriction and meta-

bolic rate, Lakowski and Hekimi [18] constructed double mutants of eat-2 and

clk-1, a mutant with a slowed behavior and long life span [41–43]. They found

that mutation in the clk-1 gene could not further extend the life span of eat-2

mutants and hence concluded that both mutations lengthen the C. elegans life

span via the same mechanism. Since it was believed that clk mutants were long-

lived due to a reduction of metabolic rate, it was concluded that DR postpones

aging by lowering the metabolic rate. However, later studies showed that clk

mutants had no reduced respiration or heat production rate [44, 45], undermin-

ing the proposed hypothesis.

Houthoofd et al. [22, 25, 46] measured respiration and heat production rate

directly in worms that were restricted by E. coli dilution, eat mutation or growth


in axenic medium. Surprisingly, they found that DR leads to an increase in

metabolic rate, at least when expressed per unit of body mass. They next deter-

mined the ATP content in these worms and found that DR causes lower ATP

concentrations. These authors also found that DR led to a higher reductive

capacity, measured as the maximal potential to reduce XTT in the presence of

exogenous NAD(P)H. A possible reason for these observations is that the high

metabolic rate is needed for the synthesis of molecules that are freely available

in the diet of ad libitum fed worms, but absent or at a lower concentration in

restricted worms. These anabolic reactions would require ATP and reductive

reactions. Another possible energy-demanding process is increased protein

turnover, as observed in mammals and yeast subjected to DR [47, 48]. In prin-

ciple, this could retard aging by lowering levels of damaged proteins in the cell.

The protein turnover rate has not been tested in C. elegans subjected to DR yet.

An increased respiration rate as a response to DR is consistent with experiments

in yeast. Lin et al. [49] have shown that DR causes a shift from fermentative

towards respirative metabolism and that life extension caused by DR depends

on the activity of the tricarboxylic acid cycle.

Mitochondria from DR rodents show a lower membrane potential and pro-

duced less ROS [reviewed in 40, 50]. The ROS production rate in response to

the nutritional regime of C. elegans awaits testing.

Increased Stress Resistance?

Life span extension in C. elegans is often correlated with increased resis-

tance to various stresses [51–53], and an upregulation of the activity of ROS-

detoxifying enzymes is often observed in long-lived mutant strains [52, 54, 55].

Houthoofd et al. [22, 24] determined the resistance to oxidative and heat stress

in worms that were grown under axenic conditions. They found that restricted

worms had a superior resistance to both stresses. Moreover, this was accompa-

nied by higher activities of superoxide dismutase and catalase, two enzymes

that are involved in the breakdown of ROS. pep-2 mutants have a higher heat

tolerance and an increased resistance to oxidative stress [29], but are not long-

lived, as mentioned previously. An upregulation of stress resistance in response

to DR could be the consequence of a hormetic response (i.e. stress-induced

stress resistance) [56, 57]. By this view, DR is a low-intensity stressor, and ani-

mals subjected to DR react to this stressor by upregulating the stress defense

system which also protects them against aging.

Reduced Ins/IGF-1 Signaling?

If increased stress defense is necessary for DR-induced life extension, the

DR response is likely mediated by a signaling pathway that regulates the

expression of a life-extending program in response to the nutritional status of


the organism. Good candidates are the JNK [58] and the Ins/IGF-1-like signal-

ing pathways. Both converge on the transcription factor DAF-16. The Ins/IGF-

1-like signaling pathway is an evolutionary conserved pathway similar to the

Ins and IGF-1 mammalian pathways. This pathway regulates the aging rate in

worms, flies and mice [reviewed in 59]. Inactivation of this pathway by muta-

tion in the Ins/IGF-1 receptor daf-2 or in one of the downstream genes (e.g.

age-1) in the worm results in a substantial life span extension that is dependent

on the transcription factor DAF-16 [60]. DAF-16 accumulates in the nucleus of

worms with reduced signaling activity, resulting in the increased expression of

many genes that confer resistance to stress or detoxification, and enhance mean

and maximum life span [61–65] (fig. 1).

The similarity with the mammalian Ins pathway and several observations

in the worm suggested a role for the Ins/IGF-1 pathway in life span extension

caused by DR. First of all, the Ins/IGF-1 pathway plays an important role in the

formation of dauers (a long-lived and stress-resistant larval stage) in C. elegans,

and food availability is one of the regulating factors for dauer formation [67].

Secondly, several of the 39 Ins-like peptides are expressed in amphid neurons

[68] and the longevity phenotype of mutants with defective olfactory percep-

tion is dependent on the DAF-16 transcription factor, suggesting the involve-

ment of the Ins/IGF-1 pathway in the food-sensing process [69]. Also, it is

reported by one group that DAF-16 activity in the intestinal cells, which are

responsible for the uptake of nutrients and for the transport of nutrients to the

pseudocoelomic fluid, is necessary and sufficient to regulate the worm’s life

span [70]. The germline also influences Ins/IGF-1 signaling: worms lacking

germline proliferation are long-lived, and this phenotype is also dependent on

DAF-16 [71]. Since DR causes lower brood size, it is possible that germline

proliferation is reduced. Finally, mutants with reduced Ins/IGF-1 signaling

activity share the increased stress resistance phenotype with individuals sub-

jected to DR. These results appear to show that Ins/IGF-1 signaling controls the

allocation of energy usage from reproductive growth to somatic maintenance

and longevity, depending on nutrient availability.

C. elegans is a convenient model organism to test the role of Ins/IGF-1 sig-

naling in the DR response. Many longevity mutants with impaired activity of

the Ins/IGF-1 pathway are available and can be used to see whether Ins/IGF-1

signaling and DR act via common or different mechanisms. Several authors

have used epistatic-like experiments to investigate the role of Ins/IGF-1 signal-

ing in DR responses. For example, Johnson et al. [72] cultured the longevity

mutant age-1 in liquid culture with different E. coli concentrations and found

that the life span of age-1 could also be extended by DR, suggesting that the

Ins/IGF-1 pathway is not needed for mediating DR life span effects. Lakowski

and Hekimi [18] made double mutants of eat-2 and daf-2, and found additive


Fig. 1. Model describing the potential involvement of TOR and Ins/IGF-1 signaling in

the response to food and regulation of life span in C. elegans. Food is detected via amphids,

sensory organs in the head region of the worm. Specific neurons in this region produce

insulin-like peptides (ILP), possibly in response to food. These can regulate the activity of

the phosphorylation cascade Ins/IGF-1 pathway, a main regulator of longevity. Signals from

the reproductive system also influence life span via this pathway. Nutrients are taken up by

intestinal cells through specific transporters or via pinocytosis and transported to the pseudo-

coelomic fluid. Cells can then take up these nutrients via unknown mechanisms. In mam-

mals, the TOR/Raptor pathway activity is regulated by nutrients, and by the AMP/ATP ratio

(via AMP kinase). It seems therefore plausible that the life span extension observed in C. elegans

worms grown under DR conditions is regulated via a similar mechanism. There is evidence

for cross talk between Ins/IGF-1 and TOR signaling (not shown in the figure): pep-2 and

daf-15 expression is under the control of DAF-16 [29, 63, 66]. In mammals, TOR activity is

also regulated by phosphatidylinositol 3-kinase and AKT proteins.

Food

Food sensing

system

ILP secretion

(in response to food and

signals from grem cells?)

DAF-2

Ins/IGF-1

receptor

Ins/IGF-1

signaling

Life span extension

Target cell

(Any cell type?)

TOR

signaling

Nutrients

TOR

Raptor

NucleusExpression of life

maintenance program

Forkhead

trasncription

factor

AMPK

PI3-kinase

PIP3

PIP2

Germ cells

?

?

?

?

Digestive system

NutrientsGut lumen

Intestinal cell

Pseudocoelomic fluid Nutrients

PEP-2 NAC-2 NAC-3 NHX-2

di-and tri-peptides

di-and tri-carboxylates

Na�

H�

Pinocytosis

HighAMP/ATP

JNK-1JNK-1

PDK-1 AKT-1

AAK-2

LET-363

DAF-15

DAF-16

AGE-1 AAP-1

AKT-2SGK-1


life-extending effects, again an indication of different mechanisms of life span

extension. Consistent with these results, Houthoofd et al. [24, 73, 74] cultured

daf-2 mutants in axenic medium and found that daf-2 mutants live substantially

longer in axenic medium. Axenic culture and reduced Ins/IGF-1 signaling had

additive effects on the upregulation of stress defenses and altered metabolism

[24, 46]. Meissner et al. [29] found that the life span of daf-2 mutants can be

further extended by an additive mutation in pep-2 and that additive effects were

also seen on heat tolerance of the double mutant. However, one difficulty with

interpreting results of this trend is that the long-lived Ins/IGF-1 mutants are

‘reduction of function’ mutants and thus still have residual activity. One could

therefore argue that a further life extension of Ins/IGF-1 mutants is simply the

result of a further reduction of Ins/IGF-1 activity [17].

More definitive conclusions may been drawn from studies of daf-16

mutants. A variety of mutant alleles are available for this gene, including null

and near-null mutants, and these mutations completely suppress the long life

span of the long-lived Ins/IGF-1 mutants. If the life extension caused by DR is

mediated by Ins/IGF-1 signaling, then mutation in daf-16 should suppress the

long life span of DR worms. DR-mediated life span extension was not sup-

pressed by daf-16 mutations when DR was imposed by using eat-2 mutants

[18]. The elevated stress defense of pep-2 mutants was also unaffected by muta-

tion in the daf-16 gene [29]. Finally, daf-16 failed to suppress the life extension,

metabolism and stress resistance of worms that were cultured in axenic medium

[24, 46]. These results are consistent with the cytosolic localization of DAF-16

in eat-2 mutants and in wild-type worms grown in axenic medium [24, 61]. The

predicted role of Ins/IGF-1 signaling is therefore not supported by experimental

testing. The dependence of JNK signaling on DAF-16 [58] similarly argues

against its potential role in mediating the effect of DR on life span. However, it

should be stressed that in the above-mentioned experiments, no null alleles of

daf-16 were used. Although the used alleles completely suppress the daf-2 Age

phenotype, it cannot be excluded that some residual activity causes the

increased life span of DR-treated worms. Since the life extension of mutants

with reduced food sensing is dependent on DAF-16, while DR is not,

chemosensory perception of food and absorption of nutrients might differen-

tially affect life span in C. elegans. Following similar reasoning, it can be con-

cluded that the starvation response, which depends on DAF-16 [61], is different

from responses to milder forms of DR.

Flies carrying a mutation in the insulin receptor substrate gene chico

respond to DR less efficiently; wild-type flies under DR show a maximal life

span at a lower food concentration than mutant flies do [75]. The authors

explain this by saying that Ins/IGF-1 signaling is involved in the DR response in

flies. However, other reasons could underlie this effect. chico flies could feed


less for example or could use other metabolic pathways for their energy supply,

making them more vulnerable to nutritional deprivation. In rodents, reduced

caloric intake leads to lower Ins, IGF-1 and growth hormone concentrations in

the blood stream [4]. CR feeding also results in a decreased proton-motive force

and ROS production, and these effects are reversed by subjecting CR animals to

a short period of Ins treatment [50]. However, Bartke et al. [76] reported that

CR further extends the life span of Ames dwarf mice, which produce less

growth hormone and IGF-1. These authors therefore concluded that mutants

with a reduced activity of the growth hormone/IGF-1 axis slow down the aging

process by a mechanism that is different from CR. However, this conclusion

was criticized by Clancy et al. [75] arguing that Ames dwarf mice have residual

IGF-1 activity that could be further decreased by DR, resulting in a further life

extension. Studying the effect of reduced FOXO activity on DR-treated mice

and flies might give a more definitive proof of the potential role of Ins/IGF-1

signaling in the DR response in these species.

Altered DNA Silencing?

The life span of C. elegans can also be extended by extra copies of sir-2.1

[77] or (slightly) by resveratrol [78], once thought to be a stimulator of SIR pro-

teins [79, 80]. sir-2.1 is a homologue of the yeast SIR-2 gene that encodes a his-

tone deacetylase, overexpression of which increases the replicative life span of

yeast mother cells [81, 82]. It was thought that CR increases the life span of

yeast mother cells by regulating SIR-2 activity, because the life span of Sir-2

mutants does not respond to variations in caloric uptake [83]. However, this

effect appears to be very strain dependent since other researchers, using differ-

ent yeast strains, did not observe such an effect [84, 85]. This issue has recently

been resolved by Lamming et al. [86] who detected that Hst-2, a Sir-2 homolog,

mediates Sir-2-independent life span extension by CR. Longevity induced by

increased SIR-2.1 activity in C. elegans is dependent on DAF-16 [77], and since

DR promotes longevity independently from DAF-16 in the worm, SIR-2.1

activity seems unlikely to mediate the DR-mediated life span extension in

C. elegans. However, the C. elegans genome contains 4 different sir-2 homo-

logues, and it cannot be excluded that other sir-2 genes might regulate the DR

response, independently of DAF-16. A direct test of the DR life span effect in

C. elegans mutants with altered SIR-2 activity, either by mutation or induction

by resveratrol, has not yet been reported.

Reduced TOR Signaling?

Another likely candidate for mediating a DR response is the TOR pathway.

In mammals and fruit flies, TOR senses the cellular amino acid pool and regu-

lates cell growth by a coordinated action on transcription, translation and protein


degradation [reviewed in 87]. When amino acids are abundant, mammalian TOR

(mTOR) phosphorylates, and thereby inactivates, the repressor of mRNA trans-

lation, initiation factor 4E binding protein, resulting in enhanced translation.

Activated mTOR also phosphorylates and activates ribosomal S6 kinase, also

favoring protein synthesis. Conversely, amino acid deprivation reduces mTOR

signaling and leads to increased autophagy and protein degradation [87].

A mutation in let-363, the C. elegans TOR homologue, or in daf-15, the

C. elegans homologue of Raptor (regulatory associated protein of TOR) causes

arrest and death as dauer-like larvae [66]. Heterozygous daf-15 mutants are

long-lived [66]. Knocking down let-363 by RNAi from the first day of adult-

hood also leads to increased life span, and this effect does not require DAF-16

[88]. Evidence for the role of TOR signaling in the DR response comes from

Meissner et al. [29]. These authors found that RNAi against let-363 did not

extend the life span of pep-2 mutants. This is consistent with pep-2 acting

upstream of TOR. In mammals, TOR activity is stimulated by Ins (and other

growth factors) via phosphatidylinositol 3-kinase and Akt [89] whereas in

C. elegans, DAF-16 controls the expression of daf-15 [66] and pep-2 [29, 63].

Thus, Ins/IGF-1 and TOR signaling might cooperate in a complex metabolic

control circuit that optimizes metabolism and life span as a function of nutrient

availability (fig. 1).

AMPK (AMP-activated protein kinase) activation leads to a decrease in

mammalian TOR activity as measured by S6K phosphorylation [90]. AMPK

proteins are potential candidates for the regulation of life span under DR condi-

tions. The C. elegans genome contains two homologues of AMPK, aak-1 and

aak-2. Overexpression of aak-2 extends life span after being activated by a high

AMP/ATP ratio. Since reduced caloric intake is likely to increase this ratio, it

seems plausible that aak-2 mediates DR-induced life extension [91]. This

hypothesis has not been tested directly however. AAK-2 functions independently

from DAF-16 to regulate life span. However, aak-2 is somehow regulated by

daf-2 [91]. Thus, it seems plausible that TOR regulates the aging rate via stimu-

lation by nutrients and by sensing the AMP/ATP ratio in the cell (fig. 1).

Conclusions

C. elegans has been proven to be a very useful model system to elucidate

the role of specific genes on the aging process. Several methods have been used

to study the effects of DR on the life span, stress resistance, metabolism and

activity of signaling pathways in C. elegans. It seems that DR increases rather

than decreases the metabolic rate of the worm, but the effect of DR on the ROS

production is yet unknown. The increased life span that is seen in restricted


worms is however accompanied by increased resistance to environmental stres-

sors and elevated activity of stress defense enzymes, possibly leading to less

molecular damage to macromolecules, and hence retarded aging effects. It

would be interesting to know if DR influences repair capacity, protein turnover

or both, but this issue has not been explored yet.

Several signaling pathways have been proposed to be involved in the DR

effects. Work has mostly focused on Ins/IGF-1 signaling as a potential regulator

of DR-induced life extension, but experimental verification has refuted this

hypothesis. TOR and AAK signaling seem to be likely candidates, but experi-

ments to prove or disprove their role have not been performed yet. Performing

epistasis-like experiments by combining nutritional restriction and reducing

TOR expression via RNAi is not simple. RNAi is most commonly achieved by

feeding worms with E. coli expressing double-stranded RNA for the target

gene. Thus, implementing DR by reducing the E. coli concentration would

inevitably reduce the amount of double-stranded RNA taken up by the worms

resulting in a reduction of functional gene knockdown. Possibly this problem

can be avoided by growing the worms under replete conditions up to the fourth

juvenile or young adult stage harvest and clean the worms and continue growth

on a restricted diet of the standard E. coli strain. Alternative approaches could

be considered e.g. by adding double-stranded RNA to the axenic culture

medium, but this approach is labor intensive, and as discussed previously it has

not been proven that axenic culture extends life span uniquely by imposing DR.

Nevertheless such experiments are urgently needed.

Interestingly, the TOR pathway has been shown to link nutrient sensing with

protein synthesis and degradation in mammals, and there is evidence of cross talk

between TOR and Ins/IGF-1 signaling [89]. The possibility that TOR plays a role in

controlling longevity in invertebrates and mammals is therefore plausible (fig. 1).

Acknowledgements

K.H. is a postdoctoral fellow with the Fund for Scientific Research-Flanders, Belgium.

D.G. acknowledges the financial support of the Wellcome Trust. J.R.V. is supported by the

Fund for Scientific Research-Flanders (Project G.0002.02) and the European Union

(Contract LSHM-CT-2004-512020).

References

1 McCay C, Crowell M, Maynard L: The effect of retarded growth upon the length of life and upon

ultimate size. J Nutr 1935;10:63–79.

2 Comfort A: The Biology of Senescence, ed 3. Edinburgh, Churchill Livingstone, 1979.


3 Weindruch R, Walford RL: The Retardation of Aging and Disease by Dietary Restriction.


4 Masoro E: Caloric Restriction: A Key to Understanding and Modulating Aging. Amsterdam,

Elsevier, 2002.

5 Shanley DP, Kirkwood TB: Calorie restriction and aging: a life-history analysis. Evolution Int J

Org Evolution 2000;54:740–750.

6 Kirkwood TB, Shanley DP: Food restriction, evolution and ageing. Mech Ageing Dev 2005;

126:1011–1016.

7 Avery L, Thomas J: Feeding and defecation; in Riddle D, et al (eds): C. elegans II. Plainview, Cold

Spring Harbor Laboratory Press, 1997, pp 679–716.

8 www.wormatlas.be

9 Fabian TJ, Johnson TE: Production of age-synchronous mass cultures of Caenorhabditis elegans.

J Gerontol A Biol Sci Med Sci 1994;49:B145–B156.

10 Garigan D, Hsu AL, Fraser AG, Kamath RS, Ahringer J, Kenyon C: Genetic analysis of tissue

aging in Caenorhabditis elegans: a role for heat-shock factor and bacterial proliferation. Genetics

2002;161:1101–1112.

11 Gems D, Riddle DL: Genetic, behavioral and environmental determinants of male longevity in

Caenorhabditis elegans. Genetics 2000;154:1597–1610.

12 Hansen E, Buecher EJ, Yarwood EA: Development and maturation of Caenorhabditis briggsae in

response to growth factor. Nematologica 1964;10:623–630.

13 Croll NA, Smith JM, Zuckerman BM: The aging process of the nematode Caenorhabditis elegans

in bacterial and axenic culture. Exp Aging Res 1977;3:175–189.

14 Larsen PL, Clarke CF: Extension of life-span in Caenorhabditis elegans by a diet lacking coen-

zyme Q. Science 2002;295:120–123.

15 Klass MR: Aging in the nematode Caenorhabditis elegans: major biological and environmental

factors influencing life span. Mech Ageing Dev 1977;6:413–429.

16 Hosono R, Nishimoto S, Kuno S: Alterations of life-span in the nematode Caenorhabditis elegans

under monoxenic culture conditions. Exp Gerontol 1998;24:251–264.

17 Gems D, Pletcher S, Partridge L: Interpreting interactions between treatments that slow aging.

Aging Cell 2002;1:1–9.


Acad Sci USA 1998;95:13091–13096.

19 Walker G, Houthoofd K, Vanfleteren JR, Gems D: Dietary restriction in C. elegans: from rate-of-

living effects to nutrient sensing pathways. Mech Ageing Dev 2005;126:929–937.

20 Buecher EJ, Hansen EL, Yarwood EA: Ficoll activation of a protein essential for maturation of the

free-living nematode Caenorhabditis briggsae. Proc Soc Exp Biol Med 1966;121:390–393.

21 Vanfleteren JR: Axenic culture of free-living, plant-parasitic, and insect-parasitic nematodes.

Annu Rev Phytopathol 1978;16:131–157.

22 Houthoofd K, Braeckman BP, Lenaerts I, Brys K, De Vreese A, Van Eygen S, Vanfleteren JR:

Axenic growth up-regulates mass-specific metabolic rate, stress resistance, and extends life span

in Caenorhabditis elegans. Exp Gerontol 2002;37:1371–1378.

23 Vanfleteren JR: Nematodes as nutritional models; in Zuckerman BM (ed): Nematodes as Biological

Models. New York, Academia Press, 1980, vol 2: Aging and other model systems, pp 47–79.


independent of the Ins/Igf-1 signalling pathway in Caenorhabditis elegans. Exp Gerontol

2003;38:947–954.



1359–1369.

26 Fei YJ, Inoue K, Ganapathy V: Structural and functional characteristics of two sodium-coupled

dicarboxylate transporters (ceNaDC1 and ceNaDC2) from Caenorhabditis elegans and their

relevance to life span. J Biol Chem 2003;278:6136–6144.

27 Fei YJ, Liu JC, Inoue K, Zhuang L, Miyake L, Miyauchi S, Ganapathy V: Relevance of Nac-2, an

Na�-coupled citrate transporter, to life span, body size and fat content in Caenorhabditis elegans.

Biochem J 2004;379:191–198.




29 Meissner B, Boll M, Daniel H, Baumeister R: Deletion of the intestinal peptide transporter affects

insulin and Tor signaling in Caenorhabditis elegans. J Biol Chem 2004;279:36739–36745.

30 Nehrke K: A reduction in intestinal cell Ph[I] due to loss of the Caenorhabditis elegans Na�/H�

exchanger Nhx-2 increases life span. J Biol Chem 2003;278:44657–44666.

31 Harman D: Aging: a theory based on free radical and radiation chemistry. J Gerontol 1956;11:

298–300.

32 Rubner M: Das Problem der Lebensdauer und seine Beziehungen zum Wachstum und Ernährung.

Munich, Oldenbourg, 1908.

33 Loeb J., Northrop JH: On the influence of food and temperature upon the duration of life. J Biol

Chem 1917;32:102–121.

34 Pearl R: The Rate of Living. New York, Knopf, 1928.

35 Korshunov SS, Skulachev VP, Starkov AA: High protonic potential actuates a mechanism of pro-

duction of reactive oxygen species in mitochondria. FEBS Lett 1997;416:15–18.

36 Brand MD: Uncoupling to survive? The role of mitochondrial inefficiency in ageing. Exp

Gerontol 2000;35:811–820.

37 Nicholls DG: Mitochondrial membrane potential and aging. Aging Cell 2004;3:35–40.

38 Speakman JR, Talbot DA, Selman C, Snart S, Mclaren JS, Redman P, Krol E, Jackson DM,

Johnson MS, Brand MD: Uncoupled and surviving: individual mice with high metabolism have

greater mitochondrial uncoupling and live longer. Aging Cell 2004;3:87–95.

39 Rea S, Johnson TE: A metabolic model for life span determination in Caenorhabditis elegans.

Dev Cell 2003;5:197–203.

40 Merry BJ: Molecular Mechanisms Linking Calorie Restriction and Longevity. Int J Biochem Cell

Biol 2002;34:1340–1354.

41 Sacher GA: Life table modifications and life prolongation; in Finch CE, Hayflick L (eds):

Handbook of the Biology of Aging. New York, Van Nostrand Reinhold, 1977, pp 582–638.

42 Lakowski B, Hekimi S: Determination of life-span in Caenorhabditis elegans by four clock genes.

Science 1996;17:1010–1013.

43 Wong A, Boutis P, Hekimi S: Mutations in the clk-1 gene of Caenorhabditis elegans affect devel-

opmental and behavioral timing. Genetics 1995;139:1247–1259.

44 Braeckman BP, Houthoofd K, Brys K, Lenaerts I, De Vreese A, Van Eygen S, Raes H, Vanfleteren JR:

No reduction of energy metabolism in Clk mutants. Mech Ageing Dev 2002;123:1447–1456.

45 Braeckman BP, Houthoofd K, De Vreese A, Vanfleteren JR: Apparent uncoupling of energy pro-

duction and consumption in long-lived Clk mutants of Caenorhabditis elegans. Curr Biol

1999;9:493–496.

46 Houthoofd K, Braeckman BP, Lenaerts I, Brys K, Matthijssens F, De Vreese A, Van Eygen S,

Vanfleteren JR: DAF-2 pathway mutations and food restriction in aging Caenorhabditis elegans

differentially affect metabolism. Neurobiol Aging 2005;26:689–696.

47 Lewis SE, Goldspink DF, Phillips JG, Merry BJ, Holehan AM: The effects of aging and chronic

dietary restriction on whole body growth and protein turnover in the rat. Exp Gerontol

1985;20:253–263.

48 Jazwinski SM: Metabolic mechanisms of yeast ageing. Exp Gerontol 2000;35:671–676.



2002;418:344–348.

50 Lambert AJ, Merry BJ: Effect of caloric restriction on mitochondrial reactive oxygen species pro-

duction and bioenergetics: reversal by insulin. Am J Physiol Regul Integr Comp Physiol

2004;286:R71–R79.

51 Larsen PL: Aging and resistance to oxidative damage in Caenorhabditis elegans. Proc Natl Acad

Sci USA 1993;90:8905–8909.

52 Vanfleteren JR: Oxidative stress and ageing in Caenorhabditis elegans. Biochem J 1993;292:605–608.

53 Johnson TE, de Castro E, Hegi de Castro S, Cypser J, Henderson S, Tedesco P: Relationship

between increased longevity and stress resistance as assessed through gerontogene mutations in

Caenorhabditis elegans. Exp Gerontol 2001;36:1609–1617.


54 Houthoofd K, Fidalgo MA, Hoogewijs D, Braeckman BP, Lenaerts I, Brys K, Matthijssens F, De

Vreese A, Van Eygen S, Munoz MJ, Vanfleteren JR: Metabolism, physiology and stress defense in

three aging Ins/IGF-1 mutants of the nematode Caenorhabditis elegans. Aging Cell 2005;4:

87–95.

55 Vanfleteren JR, De Vreese A: The gerontogenes age-1 and daf-2 determine metabolic rate poten-

tial in aging Caenorhabditis elegans. FASEB J 1995;9:1355–1361.

56 Turturro A, Hass B, Hart RW: Hormesis – Implications for risk assessment caloric intake (body

weight) as an example. Hum Exp Toxicol 1998;17:454–459.

57 Masoro EJ: Hormesis and the antiaging action of dietary restriction. Exp Gerontol 1998;33:

61–66.

58 Oh SW, Mukhopadhyay A, Svrzikapa N, Jiang F, Davis RJ, Tissenbaum HA: JNK regulates life-

span in Caenorhabditis elegans by modulating nuclear translocation of forkhead transcription

factor DAF-16. Proc Natl Acad Sci USA 2005;102:4494–4499.

59 Partridge L, Gems D: Mechanisms of ageing: public or private? Nat Rev Genet 2002;3:165–175.

60 Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R: A C. elegans mutant that lives twice as long

as wild type. Nature 1993;366:461–464.

61 Henderson ST, Johnson TE: daf-16 integrates developmental and environmental inputs to mediate

aging in the nematode Caenorhabditis elegans. Curr Biol 2001;11:1975–1980.

62 McElwee J, Bubb K, Thomas JH: Transcriptional outputs of the Caenorhabditis elegans forkhead

protein DAF-16. Aging Cell 2003;2:111–121.

63 Murphy CT, Mccarroll SA, Bargmann CI, Fraser A, Kamath RS, Ahringer J, Li H, Kenyon C:

Genes that act downstream of Daf-16 to influence the lifespan of Caenorhabditis elegans. Nature

2003;424:277–284.

64 McElwee JJ, Schuster E, Blanc E, Thomas JH, Gems D: Shared transcriptional signature in

Caenorhabditis elegans dauer larvae and long-lived daf-2 mutants implicates detoxification sys-

tem in longevity assurance. J Biol Chem 2004;279:44533–44543.

65 Gems D, McElwee JJ: Broad spectrum detoxification: the major longevity assurance process

regulated by insulin/IGF-1 signaling? Mech Ageing Dev 2005;126:381–387.

66 Jia K, Chen D, Riddle D: The TOR pathway interacts with the insulin signaling pathway to regu-

late C. elegans larval development, metabolism and life span. Development 2004;131:3897–3906.

67 Riddle DL, Albert PS: Genetic and environmental regulation of dauer larva development; in

Riddle DL, Blumenthal T, Meyer BM, Priess JR (eds): C. elegans II. Plainview, Cold Spring

Harbor Laboratory Press, 1997, pp 739–768.

68 Pierce SB, Costa M, Wisotzkey R, Devadhar S, Homburger SA, Buchman AR, Ferguson KC,

Heller J, Platt DM, Pasquinelli AA, Liu LX, Doberstein SK, Ruvkun G: Regulation of DAF-2

receptor signaling by human insulin and ins-1, a member of the unusually large and diverse C. elegans

insulin gene family. Genes Dev 2001;15:672–686.

69 Apfeld J, Kenyon C: Regulation of lifespan by sensory perception in Caenorhabditis elegans.

Nature 1999;402:804–809.

70 Libina N, Berman JR, Kenyon C: Tissue-specific activities of C. elegans DAF-16 in the regulation

of lifespan. Cell 2003;115:489–502.

71 Hsin H, Kenyon C: Signals from the reproductive system regulate the lifespan of C. elegans.

Nature 1999;399:362–366.

72 Johnson T, Friedman DB, Foltz N, Fitzpatrick PA, Shoemaker JE: Genetic variants and mutations

of Caenorhabditis elegans provide tools for dissecting the aging process; in Harrison D (ed):

Genetic Effects of Aging. Caldwell, Telford, 1990, vol II, pp 101–126.

73 Houthoofd K, Braeckman BP, Vanfleteren JR: The hunt for the record life span in Caenorhabditis

elegans. J Gerontol A Biol Sci Med Sci 2004;59:408–410.

74 Houthoofd K, Braeckman BP, Johnson TE, Vanfleteren JR: Extending life-span in C. elegans.

Science 2004;305:1238–1239.


Science 2002;296:319.




77 Tissenbaum HA, Guarente L: Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis

elegans. Nature 2001;410:227–230.

78 Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D: Sirtuin activators mimic

caloric restriction and delay ageing in metazoans. Nature 2004;430:686–689.

79 Kaeberlein M, McDonagh T, Heltweg B, Hixon J, Westman EA, Caldwell SD, Napper A, Curtis R,

DiStefano PS, Fields S, Bedalov A, Kennedy BK: Substrate-specific activation of sirtuins by

resveratrol. J Biol Chem 2005;280:17038–17045.

80 Borra MT, Smith BC, Denu JM: Mechanism of human SIRT1 activation by resveratrol. J Biol

Chem 2005;280:17187–17195.

81 Kaeberlein M, McVey M, Guarente L: The SIR2/3/4 complex and SIR2 alone promote longevity

in Saccharomyces cerevisiae by two different mechanisms. Genes Dev 1990;13:2570–2580.

82 Chang KT, Min KT: Regulation of lifespan by histone deacetylase. Ageing Res Rev

2002;1:313–326.

83 Lin SJ, Defossez PA, Guarente L: Requirement of NAD and SIR2 for life-span extension by calo-

rie restriction in Saccharomyces cerevisiae. Science 2000;289:2126–2128.

84 Jiang JC, Wawryn J, Shantha Kumara HM, Jazwinski SM: Distinct roles of processes modulated

by histone deacetylases Rpd3p, Hda1p, and Sir2p in life extension by caloric restriction in yeast.

Exp Gerontol 2002;37:1023–1030.

85 Kaeberlein M, Kirkland KT, Fields S, Kennedy BK: Sir2-independent life span extension by calo-

rie restriction in yeast. PLos Biol 2004;2:E296.

86 Lamming DW, Latorre-Esteves M, Medvedik O, Wong SN, Tsang FA, Wang C, Lin SJ, Sinclair

DA: HST2 mediates SIR2-independent life-span extension by calorie restriction. Science

2005;309:1861–1864.

87 Oldham S, Hafen E: Insulin/IGF and target of rapamycin signaling: a TOR de force in growth con-

trol. Trends Cell Biol 2003;13:79–85.

88 Vellai T, Takacs-Vellai K, Zhang Y, Kovacs A, Orosz L, Muller F: Influence of TOR kinase on

lifespan in C. elegans. Nature 2003;426:620.

89 Hay N, Sonenberg N: Upstream and downstream of mTOR. Genes Dev 2003;18:1926–1945.

90 Kimura N, Tokunaga C, Dalal S, Richardson C, Yoshino K, Hara K, Kemp BE, Witters LA,

Mimura O, Yonezawa K: A possible linkage between AMP-activated protein kinase (AMPK) and

mammalian target of rapamycin (mTOR) signalling pathway. Genes Cells 2003;8:65–79.

91 Apfeld J, O’Connor G, McDonagh T, DiStefano PS, Curtis R: The AMP-activated protein kinase

AAK-2 links energy levels and insulin-like signals to lifespan in C. elegans. Genes Dev

2004;18:3004–3009.

Prof. Jacques R. Vanfleteren

Department of Biology

Ghent University, K.L. Ledeganckstraat 35

BE–9000 Ghent (Belgium)

Tel. �32 9 264 52 12, Fax �32 9 264 87 93, E-Mail [email protected]



Diet Restriction in Drosophila melanogaster

Design and Analysis

Marc Tatar

Division of Biology and Medicine, Brown University, Providence, R.I., USA

AbstractDiet restriction (DR) was first shown to extend adult survival in Drosophila only a bit

longer than a dozen years ago. Limiting the amount of dietary yeast was sufficient to

increase life span. In the short time since this initial observation, work with Drosophila has

revealed several insights into the mechanisms of DR. It has also uncovered many unantici-

pated technical issues. This paper describes how resolving the way we study DR in

Drosophila is a prerequisite to discover the way nutrition modulates aging. Key empirical

problems include the necessity of measuring the impact of DR upon life span with multiple

levels of diet, analysis of the demographic response to diet with mortality data and, in the

context of reaction norms, methods of diet modification, and uncertainty as to how diet dilu-

tion translates to changes in actual nutrient uptake. We review the accumulated literature of

DR in Drosophila from this methodological lens to distill four important results: yeast

restriction alone is sufficient to increase survival; diet affects survival through two distinct

physiological responses, starvation and longevity assurance; mortality has no memory of its

past with respect to nutrition; the molecular operation of DR may involve processes of

deacetylation via Sir-2 and Rpd-3. Finally, it remains unknown whether or not DR functions

through insulin-related signaling.


Drosophila was introduced as a model organism at the turn of the last cen-

tury. In the following decades, biologists extensively studied its diet to perfect

methods of culture and to explore emerging concepts of animal nutrition. By

1930 Alpatov [1] could summarize this progress and contribute a novel obser-

vation: males lived longer when they were transferred to fresh food every 2

days rather than daily. If the action of transfer itself did not kill flies, this may

be the first evidence of extended longevity upon diet restriction (DR) in

Tatar 116

Drosophila melanogaster. Remarkably, 60 years passed before a report unam-

biguously documented DR in D. melanogaster [2]. Adults maintained on sugar

medium with yeast lived longer and laid fewer eggs when yeast was scarce

rather than abundant. Since attempts to document DR with Drosophila prior to

this time found limited diets to reduce survival, Chapman and Partridge [3]

resolved this contradiction by testing flies across a range of nutrient concentra-

tions, from 1.25% w/v to 16% w/v of sugar and yeast (SY diet, see table 1 for a

summary of diet compositions). The median life span was greatest upon a diet

with 5% SY, more dilute diets reduced both survival and fecundity, and richer

diets reduced survival but increased fecundity. To extend D. melanogaster life

span by DR, nutrients must be reduced within a physiological range where

trade-offs occur between reproduction and somatic survival. At lower levels of

diet, starvation due to malnutrition impairs fitness by reducing both survival

and fecundity.

That DR can extend D. melanogaster survival is no longer in doubt. But

whether DR does so because it retards senescence requires further analysis of

the life table. Demographic studies with Drosophila measure the age at death of

every individual in synchronous experimental and control cohorts. From the

distribution of deaths we calculate median life span, survivorship (Lx, the pro-

portion remaining alive at each age x), and, in some cases, a measure of ‘maxi-

mum life span’. These common life table statistics are useful summaries of the

age at death distribution, yet they need not describe senescence. Life table data

reveal senescence when the mortality rate progressively increases with age [4].

Mortality rates increase exponentially with age in D. melanogaster although

this pattern is obscure at early ages when sample size is small and absent at late

ages where mortality levels off [5, 6]. We judge that senescence is retarded

(slowed, postponed, reduced) when the trajectory of age-dependent mortality is

consistently lower in the treatment group relative to its concurrent control.

Median life span and survivorship are increased under this condition, but the

converse need not be true. Life span can differ among groups for reasons unre-

lated to aging, as when the proportion alive is diminished by accidental deaths

that occur in one time period among young adults of one cohort [7, 8].

To accurately describe the impact of DR on senescence from life table data

we must study the mortality pattern. Mortality analysis can be straightforward:

plot mortality as a function of age. A useful approximation of the mortality rate

mx is �ln(1 � dx/Nx), where dx is the number of deaths in the census interval x

to x � 1 and Nx is the number of individuals alive at age x [9]. Plot ln(mx) for

each cohort (but do not interpolate across ages where dx � 0 since these mx are

undefined, see Promislow et al. [5]). Across ages where ln(mx) increases pro-

gressively, evaluate if the trajectory for the DR group is consistently reduced

relative to its control.

Diet Restriction in D. melanogaster 117

The life table data of Chippindale et al. [2] are replotted as mortality rates to

illustrate this approach (fig. 1a, b). DR consistently reduces mortality and we

conclude that yeast restriction retards senescence – this increases the median life

span and survivorship. Figure 1c and d illustrates a contrasting outcome for

females maintained with and without yeast [10]. While survivorship is increased

among females maintained without yeast, this difference arose from a temporary

spike in mortality among young yeast-fed females (at approx. day 38); these data

do not demonstrate that DR retards senescence [11]. Naturally, statistical tests are

required to make strong inferences about mortality patterns. The nonparametric

log rank test is simple but its interpretation requires some care since this evaluates

mortality irrespective of consistency in age-dependent patterns. Proportional haz-

ard and parametric (e.g. Gompertz function) methods can explicitly evaluate the

temporal consistency of mortality differences and estimate their magnitude.

Guidance on these tools can be found in standard references [9, 12, 13].

The Practice of Drosophila Diet Restriction

As with humans, there are many ways to put a fly on a diet. A common

approach reduces the concentration of all nutrients within a fixed volume of

agar-based medium. The base medium may or may not also contain cornmeal.

An alternative design reduces only the concentration of nutrient yeast while

holding the sugar concentration constant. Since both methods are able to extend

life span (table 1), we might conclude that it is sufficient to limit a specific

nutrient component from yeast rather than caloric intake per se to retard fly

senescence. This simple idea proves difficult to verify. Because all these meth-

ods dilute nutrients rather than limit their absolute availability we do not know

whether flies on DR media actually eat less yeast, calories or both. This basic

problem has been addressed in several recent reports without reaching a com-

mon conclusion.

Early work on feeding behavior explored how food intake changed in

response to the concentration of specific dietary nutrients. Driver et al. [14]

estimated food consumption by the rate of fecal deposition and concluded that

feeding varied inversely with nutrient concentration. Edgecomb et al. [15]

assessed mated, yeast-fed flies after they had been maintained for 3 days on

sugar-only diets where sucrose varied from 0.5 to 17% w/v. Feeding was meas-

ured by the frequency of proboscis prints upon the food, and by the uptake and

excretion of a soluble, indigestible dye. Especially among females, adults com-

pensated for reduced sugar by feeding at higher rates.

The diets of these behavioral inquiries were not designed to study aging.

Therefore, to investigate how food intake varies under conditions where DR

Tatar 118

Table 1. Diet compositions in the literature

Report Diet components Relative longevity (DR/AL) Fecundity(DR/AL)

varied constant female male

Chippindale et al. [2] Y C, S 1.24 (a) 1.28 (a) reduced

Y C, S 1.30 (a) 1.14 (a) reduced

Y C, S 1.14 (a) 1.06 (a) reduced

Y C, S 0.99 (a) n.s. 1.11 (a) reducedChapman and Partridge [3] Y�S 1.56 reduced

Y�S 1.17

Clancy et al. [62] Y�S 1.33

Rogina et al. [46] Y�S 1.41

Pletcher et al. [72] Y�S 1.82 (b)

Mair et al. [42] Y�S 1.50 (c)

Wood et al. [26] Y�S 1.89 1.29Y C, S 1.19 (a) 1.07 (a)

Mair et al. [48] Y�S 1.36

Y�S 1.53

Magwere et al. [73] Y�S 1.64 1.32

Rogina and Helfand [64] Y�S 1.23 1.26

Mair et al. [18] Y�S 1.70 (a)

Y S 1.53 (a)

S Y 1.13 (a)

Min and Tatar [16] Y C, S 1.67 1.50

Carvalho et al. [17] Y�S C 1.57 (b)

van Herrewege [74] casein S 1.29 (b) 1.54 (b)

S casein 1.16 (b)

Min [75] casein S 1.58 1.69 reduced

Diets consisted of agar, water, antimicrobials and yeast (Y), sugars (S) and sometimes cornmeal (C), exceptwhen casein replaces yeast.

Relative longevity calculated from median adult life expectancy as DR/ad libitum (AL) unless noted: a � ratio ofreplicate averages, b � ratio of mean life-span, c � ratio of maximum life-span, defined by reporting author. Allcases significantly increased survival of DR adults, as reported by author, except where noted as not significant (n.s.).


Yeast type Mating conditions Wild-type Diet notesbackground

live yeast (paste) male/female pairs Ives B 1.5 mg/vial Y or 0.15 mg/vial Y oncharcoal medium

live yeast (paste) male/female pairs Ives O 1.5 mg/vial Y or 0.15 mg/vial Y oncharcoal medium

live yeast (paste) male/female pairs Ives D 1.5 mg/vial Y or 0.15 mg/vial Y oncharcoal medium

live yeast (paste) male/female pairs Ives C 1.5 mg/vial Y or 0.15 mg/vial Y oncharcoal medium

autolyzed yeast males constant Dahomey SY at 1.25, 2.5, 5, 10, 15% with agar constant;flakes longevity maximal at 5% SY

autolyzed yeast males intermittent Dahomey SY at 1.25, 2.5, 5, 10, 15% with agar constant;flakes longevity maximal at 5% SY

autolyzed yeast virgin females Dahomey SY at 1.25, 3.0, 5, 6.5, 8, 10, 15% with agarflakes constant; longevity maximal at 6.5% SY

autolyzed yeast Canton S SY at 5 or 15%; agar-only baseautolyzed yeast females once mated Dahomey dilute medium; AL � 15% SY, DR � 5% SY

flakesautolyzed yeast females once mated Dahomey dilute medium; AL � 15% SY, DR � 6.5% SYflakes

autolyzed yeast mixed-sex vial Canton S AL 15% SY vs. DR 5% SY in agar-only baseautolyzed yeast mixed-sex cage yw AL � 3% Y vs. DR � 2% Y in 10.5% S

(SAF™) with cornmeal baseautolyzed yeast males constant Dahomey dilute medium; AL � 15% SY, DR � 6.5% SYflakes

autolyzed yeast females once mated Dahomey dilute medium; AL � 15% SY, DR � 6.5% SYflakes

autolyzed yeast mated, then single-sex Dahomey SY at 2, 4, 6, 8, 10, 12, 14, 16%; agarflakes bottles from 12.5 to 20 g/l

autolyzed yeast mixed-sex vials Canton S SY at 5 or 15%; agar-only base

autolyzed yeast females once mated Dahomey DR � 6.5% Y, 6.5% S vs. AL � 15% Y, 15% Sflakes

autolyzed yeast females once mated Dahomey DR � 6.5% Y, 15% S vs. AL � 15% Y, 15% Sflakes

autolyzed yeast females once mated Dahomey DR � 15% Y, 6.5% S vs. AL � 15% Y, 15% Sflakes

autolyzed yeast mixed-sex cage Canton S AL � 16% Y, DR � 2% Y; cornmeal 5.2%, (SAF™) sugar 11%

yeast extract (Bacto™) virgin females SY at 1, 5, 10, 15%; cornmeal 8% (larvae rearedon Lewis medium)

mixed-sex vials Champétières S � 0.75%, essential mix, casein at 0, 1, 2, 3, wild � vestigial 4, 5%; max. at 2% (F) and 1% (M) casein

mixed-sex vials Champétières casein � 5%, essential media, S at 0, 1, 2, 3, wild � vestigial 4, 5%; max. at 3%

casein mixed-sex cage Canton S 11% S, 1.1% agar and casein at 0.5, 1, 2 or 4%

Tatar 120

Fig. 1. Assessing the influence of DR on aging from survivorship and mortality.

Evidence of retarded aging with DR [2]: reduced yeast increased survivorship (a) because it

consistently decreased mortality rate (b). Evidence inconsistent with retarded aging with DR

[10]: flies without dietary yeast increased survivorship (c) because many control flies (fed

yeast) died in one census interval (d).

1.0

0.8

0.6

0

0 20 40

Age (days)

60 80

�1

�2

�3

�4

�5

�6

0.4

0.2

0

0 20

Su

rviv

ors

hip

Mo

rtalit

y r

ate

(In

)

40

Age (days)

60 80

1.0

0.8

0.6

0.4

0.2

0

0 20

Surv

ivo

rship

40

Age (days)

60 80

�1.5

0 20 40

Age (days)

60 80

�2.0

�2.5

�3.0

�3.5

�4.0

Mo

rtalit

y r

ate

(In

)

a b

c d

Low yeast

High yeast

No yeast

Fed yeast

Low yeast

High yeast

No yeast

Fed yeast

extends life span, Min and Tatar [16] assessed a range of diet that varied in yeast

from 1 to 16% with a constant concentration of dietary sugar. Age-dependent

mortality was minimized by a 2% yeast diet; this consistently reduced mortality

relative to a 16% yeast diet and in turn increased the median life span by 16 days.

Accordingly, the feeding rate was measured by dye intake and fecal deposition

on medium with 2 and 16% inactive yeast (in agar-cornmeal-sucrose base) in

mated females. In contrast to the compensatory feeding that occurs at low levels

of dietary sugar, females on 16% yeast consumed more diet than those on 2%

yeast. Since fecundity is elevated on yeast-rich diets, the high feeding rate of

these females may correspond to the metabolic demands of egg production.


Carvalho et al. [17] took a somewhat different approach. They varied the

concentration of both sugar and yeast extract in agar-cornmeal base (SYE diet)

and labeled diets with a soluble radioactive tracer in the form of the nucleotide

[�-32P]dCTP. Tracer uptake and survivorship were measured in virgin females.

Survival was greatest upon a diet of 1% SYE. At diets between 2 and 15% SYE

there were no clear differences in age-dependent mortality despite some varia-

tion in mean life span. Across the full range of diets, tracer uptake increased

as nutrient concentration declined. These data show compensatory feeding and

its consequences: tracer intake on the 1% diet was only 40% of its consumption

on the 15-fold richer diet. Flies on diluted diet acquired fewer nutrients as

expected, but the quantity of consumed marker was not proportional to the

nutrient concentration of the diet. It is also notable that although Carvalho et al.

[17] and Min and Tatar [16] both found less total consumption on diluted diets,

these studies reported that feeding was stimulated by opposite conditions. The

reproductive status of females may explain this difference. Carvalho et al. [17]

studied virgin females, which have little metabolic demands from egg produc-

tion. Compensatory feeding may be the default behavior in the absence of

reproduction.

Mair et al. [18] provide our final perspective. They independently varied the

concentration of both sugar and yeast in agar base. Newly hatched females were

mated and subsequently studied without males. Survivorship was markedly

increased on diets that restricted yeast while holding sugar constant. On the other

hand, when holding the amount of yeast constant, survivorship was only mod-

estly increased by diets with less sugar. These data confirm the importance of

dietary yeast in DR as reported in Chippindale et al. [2] and provide a potential

way to assess the relative importance of specific nutrients and calories. Mair

et al. [18] noted that solitary females extended their proboscides for the same

amount of time on each type of diet and suggest from this behavior that females

will acquire the same amount of calories if the diets have similar energetic con-

tents. Since the low-yeast/high-sugar and the high-yeast/low-sugar diets were

energetically equivalent, Mair et al. argued that DR mediates life span because it

limits specific nutrients rather than calories. This inference, however, requires

that nutrient acquisition is proportional to nutrient concentration. The actual

relationship between proboscis extension and food consumption is unknown.

Furthermore, different experimental conditions were used to measure proboscis

extension (undisturbed, solitary females) than was used to measure longevity

because females in the demographic trials had complex patterns of feeding

behavior. If females in fact increase their food intake on high yeast diet under the

demographic conditions, as they may to support egg production, their net caloric

intake will be greater on high-yeast/low-sugar than on low-yeast/high-sugar

media. On the other hand, if these females feed more when yeast or sugar is

Tatar 122

reduced, differences in net caloric intake will be less than anticipated from the

energetic value of the diet. In either case, caloric intake and nutrient quality will

be correlated and we cannot determine which factor modulates aging.

To move forward we must directly control food intake or explicitly meas-

ure assimilated nutrients. Markers placed in the medium are useful but indirect;

dyes measure gut content and radio-tagged nucleotides will correlate with gut

content and assimilation. An additional complexity is that not all components of

a diet are equally soluble, and this applies to markers as well as to sugar and

yeast. Adult Drosophila feed by sponging material from the media surface;

solutes of sugar (and of marker) will be readily acquired but insoluble meta-

bolites from yeast that are embedded below the agar surface will be relatively

inaccessible.

One strategy to assess whether calories mediate Drosophila DR is to meas-

ure energy flux (caloric value of eggs, soma, excreta and heat). A specific role

of calories can be ruled out if a restricted diet increases survival but these flies

process the same amount of energy as ad libitum controls. An alternative

approach would be to experimentally regulate consumption. This was feasible

with the Mediterranean fruit fly [19] and the housefly [20] where adults were

provided a defined volume of liquid diet that was fully consumed during a nor-

mal feeding cycle. In both cases and contrary to precedence, reduced intake of

nutrients had no positive effect on life span. A fixed food intake study of DR

should be a high priority with Drosophila, although the small scale of this fly

makes for many technical challenges. A third option would be to label carbon

and nitrogen of dietary sugar and yeast with stable isotopes and then track their

acquisition and metabolic flux by elemental specific mass spectrometry [21].

Through such methods O’Brien et al. [22] showed how the butterfly Heliconius

charitonius acquired essential amino acids from dietary pollen to produce eggs

and perhaps to support its long-lived soma. Stable isotope labeling of fly nutri-

ents might help identify specific metabolites that limit egg production but

increase in somatic tissue when DR extends life span.

Although we cannot yet resolve the relative importance of caloric intake

and specific nutrients, it is clear that reduced dietary yeast is sufficient to

extend life span. Yeast is a rich source of carbohydrates, sterols, fatty acids,

vitamins, minerals and amino acids. Amino acids deserve attention because

reduced methionine extends life span in rats and in mice [23, 24]. In mice this

also retards the age-dependent decline in immunity, metabolism and stress

resistance. Whether D. melanogaster survival can be improved by limiting

dietary amino acids has proved difficult to address since defined diets opti-

mized for larvae are not suitable for adults [25]. As an alternative we have measured

the survival of flies maintained on sugar agar alone or when supplemented with

casein at 0.5, 1, 2 and 4% [75]. In both males and females, survival was greatest


at intermediate and low levels of casein, and this arose demographically

because the age-dependent trajectory of mortality was consistently reduced.

Importantly, females on 4% casein produced most eggs, although net reproduc-

tion was much less than when females feed on yeast. While preliminary, these

observations meet the criteria for DR and suggest that amino acid restriction

may specifically influence Drosophila aging. The low level of absolute fecun-

dity indicates that other nutrients are essential for egg production and these may

as well affect aging.

Regardless of the metabolic currency, models to explain how restricted diet

modulates life span largely focus on the use of nutrients for reproduction rela-

tive to somatic repair [27, 28]. Before discussing this allocation concept, we

note that nutrients may mediate aging through a very different route – via sen-

sory cues. Environmental cues that predict future conditions regulate major life

history phenotypes of invertebrates. Dauer in Caenorhabditis elegans is

induced by pheromones and the perception of low food concentration, while

mutations within specific sensory neurons extend adult life span [29]. Among

insects, each stage of the life cycle may be controlled by cues that predict envi-

ronmental suitability for growth relative to quiescence [30]. Adult reproductive

diapause is no exception [31]. The black blowfly Phormia regina can detect

potential food supplies through taste hairs on its tarsi [32], and adults of this fly

will not initiate egg production until it feeds on protein [33]. In a similar way

Drosophila may sense dietary yeast to modulate neuroendocrine signals that

control commitment to reproductive physiology. It should be fascinating to

determine whether the perception of different concentrations of dietary yeast is

sufficient to modulate Drosophila aging.

Mechanisms of Diet Restriction in Drosophila

Models of resource acquisition and allocation provide the classic explana-

tion for how DR modulates aging [34]. Available resources are allocated from

reproduction to somatic maintenance when nutrient acquisition is limited

(fig. 2a). However, while many data are consistent with the correlations

predicted by the allocation model, there are few data to test its assumptions. It

will be important to discuss the type of evidence needed to evaluate resource

allocation and to consider a mechanistic alternative – direct constraints of

reproduction [7, 35].

Manipulative studies of life history traits often find negative correlations

between reproduction and survival. For instance, the female Drosophila life

span is increased when their reproduction is experimentally repressed [10].

Likewise, females on a restricted diet have increased survival but produce

Tatar 124

fewer eggs [3]. Each of these outcomes is anticipated if there is a limiting

resource essential for both somatic and reproductive function but the data are

equally consistent with an alternative explanation (fig. 2b). Nutrients mediate

the level of reproduction, and reproduction imposes direct constraints on

somatic functions that affect survival [7, 35]. Reproduction for instance may

activate metabolism and thereby incur damage through exposure to reactive

oxygen species. Reproduction may also inhibit the expression of somatic pro-

tection systems. For instance, misexpression of hsp-70 reduces age-specific

mortality in young females but this simultaneously diminishes egg hatchabil-

ity without reducing egg production [36, 37]. Selection will favor systems to

negatively regulate hsp-70 because the fitness benefit of early reproduction is

greater than the value of survival to old age. By such direct constraints from

reproduction, if DR reduces fecundity it can increase survival without resource

allocation.

Fig. 2. Conceptual models for how nutrients mediate life span. a The ‘Y model’ of

resource acquisition and allocation. When nutrients become limited, acquired resources are

preferentially allocated to somatic maintenance at the expense of current reproduction. b The

‘direct constraints’ model. The activity of reproduction directly constrains somatic mainte-

nance and persistence by imposing direct damage or by inhibiting the process of somatic

repair. When nutrients become limited, reproduction is reduced and constraints on somatic

persistence are attenuated.

Resource

acquisition

Soma

Reproduction

Inhib

itory

Neg

ativ

e

Resource

acquisition

Soma

Reproduction

a

b


How can we experimentally distinguish between these alternative mech-

anisms since allocation and direct constraints both predict negative correlations

between reproduction and survival? One approach is to jointly manipulate

nutrient acquisition and reproduction. If resources are competitively allocated

to somatic function at the expense of reproduction, experimentally reduced

reproduction should have a larger effect on survival when resources are limited

than when they are abundant [38, 39]. When testing this idea with the bean bee-

tle Callosobruchus maculatus we found that resource allocation could explain

only a portion of the mortality differential associated with egg production [35].

From this observation we proposed that direct reproductive constraints account

for the remaining effects of reproduction on mortality.

As with C. maculatus, resource allocation alone may not be sufficient to

explain DR in D. melanogaster. Recent studies have physiologically decou-

pled age-specific fecundity and mortality. For instance, third instar larvae that

complete development without dietary yeast produce females with low fecun-

dity despite feeding on yeast as adults [40]. Yet, these females have the same

age-dependent mortality trajectory as highly fecund controls. Diet manipula-

tion during adulthood appears to have the same capacity to decouple repro-

duction and mortality [41]. Females maintained on a sugar-only diet survive

for many weeks, produce no eggs and have relatively high age-specific

mortality (fig. 3a). When these females are switched to a diet with yeast at

late ages they rapidly acquire the mortality trajectory of control females

heretofore maintained on full-yeast media (fig. 3b). Control females at this

age lay few eggs but the switched females now produce many eggs; at these

ages differences in fecundity are not accompanied by differences in mortality.

An important extension of this design confirmed that age-specific mortality

trajectories are similarly plastic when diet is varied in conditions where DR

extends life span [42]. Adults were switched between DR and ad libitum con-

ditions, and mortality rapidly adjusted to the level of the continuous diet

treatment (fig. 3c). Although reproduction was not measured across these

diets, Chippindale et al. [2] have previously documented that such a switch

rapidly adjusts age-specific fecundity. It would be useful to simultaneously

measure age-specific fecundity and mortality in a trial of ad-libitum-to-DR

switch to determine if reproduction is independent of current mortality.

Together these studies provide important insights into the nature of DR.

Current mortality has no mechanistic connection to past nutrition and repro-

duction, contrary to expectation based on resource allocation. Rather, nutri-

tion modulates the current level of susceptibility to fail in response to

underlying systems that degenerate with age.

Genetic analyses provide further challenges to the traditional view of

resource allocation. We now recognize many genes that extend adult survival

Tatar 126

when mis-expressed in D. melanogaster [43]. Some concomitantly reduce

fecundity (e.g. InR), but many (e.g. INDY, foxo, rpd-3) do not reduce reproduc-

tion within the benign conditions of the laboratory [44–46]. While a trade-off

might occur under more natural conditions [47], these cases demonstrate that

somatic survival can be increased without diminishing the allocation of

resources to reproduction. Sterility mutants demonstrate the same principle.

The ovoD genotype blocks oogenesis at an early stage but DR remains an effec-

tive way to extend life span in these sterile females [48].

While these data challenge concepts about resource allocation, they also

present a broader paradox: reproduction can be decoupled from mortality,

either with or without nutrient interactions. In this case, is it still possible for

DR to mediate aging through direct costs of reproduction? It may, providing

Fig. 3. Age-specific mortality and reproduction decoupled by diet switch. a, b Females

maintained on sugar-only diet have a relatively high mortality and produce no eggs [41].

When switched to an SY diet (day 28, arrow), females rapidly assume the mortality trajec-

tory of control females that were continuously fed the SY diet. This illustrates the amnesia of

mortality with respect to diet. At ages after 35 days, females of the switched group produce

many more eggs than the fully fed females, yet the groups have similar mortality rates. c The

amnesia of mortality with respect to diet illustrated when females are switched from

restricted to full diet [42].

0 10 20 30 40 50

Age (days)

�9

�8

�7

�6

�5

�4

�3

�2

�1

0

Mo

rtalit

y r

ate

(In

)

0 10 20 30 40 50 60

Age (days)

�7

�6

�5

�4

�3

�2

�1

0

Mo

rtalit

y r

ate

(In

)

0 10

Eg

gs

20 30 40 50 60

Age (days)

0

20

40

60

80

Full diet

No yeast

Switch week 4 Full diet

Restricted diet

Switch week 2

a

b

c


that reproduction modulates somatic damage and maintenance through germ-

line-correlated signals [49, 50]. Mortality was decoupled from reproduction in

C. elegans in worms where the gonad was intact and or laser-ablated without

affecting life span [51]. However, life span was extended when the germ line

alone was eliminated [52]. To account for these observations, Kenyon et al. [51]

proposed that there is a balance of longevity modulating signals between the

germ line and the somatic gonad. These gonad signals could regulate direct

constraints of reproduction on somatic function. Although we have yet to define

roles for germ line and somatic gonad control of D. melanogaster aging, recent

data make a strong case for nutrient regulation of germ line stem cells in the

adult. Insulin is secreted by neurons within the adult brain in well-fed flies [40].

Germ line proliferation within the ovary is autonomously stimulated by these

insulin-like ligands; reduced diet and reduced insulin-producing cell secretion

is sufficient to slow germ line stem cell divisions [53, 54]. In parallel to these

reproductive responses, aging is retarded by systemic reduction of the insulin-

like receptor as well as by ablation of the neuronal insulin-producing cells

[55–57]. It shall be important in the future with D. melanogaster to determine

whether nutrients or insulin mediate demographic aging via their effects on the

activity of germ line stem cells.

The Interaction of Genes and Diet Restriction

As we discover the nutrient conditions whereby reduced diet extends fly

life span, we can apply Drosophila genetics to uncover the mechanistic basis for

DR. It should be possible through mutant screens to identify genes that are

essential for a restricted diet to extend life span. The focus of current work is on

candidate genes, which asks if misexpression reduces the longevity gain

induced by a restricted diet in matched wild-type controls. When they do, func-

tions associated with the gene are considered to overlap with mechanisms by

which diet modulates aging. To date this approach has been applied to genes

that retard aging when misexpressed. Prominent among these is chico, which

encodes the insulin-like receptor substrates 1–4 homolog. Ligand-activated

insulin receptor phosphorylates insulin-like receptor substrate, which in turn

signals through AKT to inactivate the dFOXO transcription factor [58] and to

permit activity of the translation regulator target of rapamycin [59]. Here we

discuss chico to understand the practice of gene-by-diet analysis and to assess

whether DR modulates fly aging via insulin signaling.

The mutant allele chico1 reduces age-dependent mortality in males and

females [60, 61]. Homozygote chico1/chico1 are dwarf and infertile. Clancy

et al. [62] investigated how survival of homozygote and wild-type females

Tatar 128

changed as diet ranged from 1.25 to 15% of both sugar and yeast in agar base

(SY diet). In general, the phenotypic response within a defined genotype across

environmental conditions is called a reaction norm or a gene-by-environment

interaction [63]. The reaction norm for control females, as expected, was con-

cave; median survival was greatest at an intermediate diet concentration, 6.5%

SY (fig. 4a). Females of chico1/chico1 produced a similar pattern except that the

optimal diet was 8.0% SY. The reaction norm of mutant homozygote flies was

shifted to the right such that DR produced the same maximum median survival

as observed for wild-type females.

In the analysis of reaction norms, we have evidence that some function of a

gene interacts with the manipulated environmental parameter (diet in our case)

Fig. 4. Gene-by-diet interaction plots. a Median life span of wild-type and chico dwarf

adults on SY diet of 7 concentrations [62]. b Median life span of adults with wild type or

sir-2 overexpression on SY diet at 2 concentrations [64]. c Median life span of adults with

wild type or mutant rdp-3 on SY diet at 2 concentrations [46].

60

50

Mean

life s

pan

(d

ays)

40

30

65

55

45

35

5

b c

a

Additional sir-2

Wild type

Wild type

Mutant rpd-3

15

SY concentration (%)

Med

ian life s

pan (d

ays)

1.5 3.0 5.0 6.5 8.0 10.0

Diet concentration (SY%)

15.0

65

55

45

35

5 10

SY concentration (%)

Med

ian life s

pan (d

ays)

chico1/chico1

Wild type


when the gene-by-environment interaction plots are not parallel (fig. 5). Data

sufficient to reject the null hypothesis of parallel reaction norms indicate that

there is some overlap between the function of the gene and how the environ-

ment affects the phenotype. Because Clancy et al. [62] measured survivorship

across many levels of nutrition they were able to define the diet of maximum

Fig. 5. How reaction norms are interpreted to determine when a gene participates in

the process whereby DR extends longevity. Schematic reaction norms are presented as con-

tinuous linear functions and are limited to the range of diet concentrations corresponding to

DR physiology: with reduced diet, survival increases and fecundity declines. The diet con-

centration axis is continuous from most restricted to full ad libitum feeding. This scheme can

represent many permutations. Our cases provide examples to understand when there is over-

lap of gene function and mechanisms of DR. The median life span of the wild genotype

(‘wild type’; dashed line) strongly responds to DR; it is represented with the same reaction

norm in each panel. The variant allele (‘mutant’; solid line) can be any well-defined genetic

variation of the candidate gene, for instance a loss of function mutant, hypomorphic mutant,

overexpression transgene or isolated natural polymorphism. For these examples, the variant

allele increases life span in standard laboratory conditions. Significant gene-by-DR interac-

tion (gene � DR) is evidence that the function of the candidate gene is involved in the

mechanism by which DR extends life span (cases 2, 3, 4). Reaction norms with nonzero

slope have ‘phenotypic plasticity’ – these show that the phenotype (median life span) varies

across environments (diet). The allelic variant may abolish phenotypic plasticity (case 2) or

change its extent (cases 3 and 4) – these equally imply DR functionality of the candidate

gene. Lack of gene � DR evidence occurs only in case 1, where DR induces proportional

reaction norms; the genotypes have equivalent phenotypic plasticity.

Case 1

No : gene�DR

Yes : DR plasticity in mutant

Case 2

Yes : gene�DR

No : DR plasticity in mutant

Case 3

Yes : gene�DR


Case 4

Yes : gene�DR


DR

Med

ian

life s

pan

ad libitum

Diet continuum

Tatar 130

longevity for both chico1/chico1 and wild-type flies, and their data recorded sur-

vivorship on either side of this maximum, that is under conditions of malnutri-

tion and when DR affects aging. As a consequence, the wild-type and

chico1/chico1 reaction norms intersect. From this evidence and based on the

apparent similarity among genotypes of their longevity maxima, Clancy et al.

[62] concluded that ‘chico1 induces a stage equivalent to submaximal, DR-

induced slowing of aging. These two pervasive interventions that slow aging

therefore act through overlapping mechanisms.’

To assess this conclusion we must consider the different ways nutrition can

affect survival and then how to test inferences from reaction norms of survival

data. The chico1 mutation appears to affect the interaction with nutrition in two

ways. First, chico1 is more prone to starvation due to malnutrition; in the range

of diets where restriction has negative effects on life span, chico1 has a shorter

life span than the wild type. Second, chico1 retards aging when nutrients are

physiologically sufficient; in the range of diets where restriction increases life

span, chico1 has superior survival. As originally noted by Chapman and

Partridge [3], the mechanisms that affect life span during starvation level diets

will differ from those that affect life span when DR slows aging [11]. When

malnourished, both fecundity and survival decline as diet is progressively

restricted, and physiology activated to ensure survival must be directed at cop-

ing with the effects of starvation. In contrast, in the range of nutrients that main-

tain sustenance, restricted diet increases survival as it decreases fecundity.

While we cannot yet describe mechanisms of DR that regulate survival and

fecundity, most likely they are distinct from processes that kill flies when

starved [65]. Do the data of Clancy et al. [62], when viewed in this way, indicate

that chico1 and DR slow aging through overlapping mechanisms?

If there are distinct biological processes that modulate survival under con-

ditions of malnutrition versus DR, we should separately evaluate how the can-

didate gene interacts with each process. In particular, to determine if functions

of chico overlap with the mechanisms of slow aging induced by restricted diet,

we must evaluate the reaction norms specifically in the diet range where nutri-

ents were sufficient, not in the range when flies are starving. The relevant

range of diets occurs at or above 8% SY for chico1 and 6.5% SY for wild-type

flies, and here the reaction norms appear remarkably parallel. If so, these data

provide no evidence that functions of chico overlap with the mechanism

of DR.

What is the likelihood that the reaction norms in the DR diet range of figure 4a

are in fact not parallel, that the data implicate chico to function within the mech-

anisms of DR? Presently we cannot evaluate this question because point esti-

mates of median survivorship do not describe the error variance of the mortality

differences, and the log rank test does not estimate mortality differentials.


Inferences on gene-by-environment (diet) interaction require survival regression

analysis where the difference in mortality is statistically evaluated with a model

where genotype (G) and diet level (D) are main effects and the interaction para-

meter (G � D) describes how genotype modifies the effect of diet on mortality.

Survival analysis can be conducted with proportional hazard or accelerated fail-

ure (parametric) methods; both use every death observation to simultaneously

estimate the main and interaction parameters and their associated standard

errors. These statistics describe the extent mortality is modified by genotype and

by diet, and whether these effects are significant. Central to the problem at hand,

when the interaction parameter (G � D) is statistically significant, we can infer

that the reaction norms are not parallel. Only then can we say that the way diet

modifies aging depends on the function of the tested gene (fig. 5).

In practice we test data to reject the null hypothesis of parallel reaction

norms. However, failure to detect a significant interaction parameter must be

interpreted with care. There may be a meaningful deviation from parallel reac-

tion norms but the data may be too few or too variable to make a statistically

powerful test. Furthermore, DR reaction norms may be nonlinear, or the inter-

action may be apparent only at diets beyond the studied range. These cautions

apply to work beyond Drosophila. Notably, eat mutants are used to simulate

restricted versus full diet in C. elegans. The apparent absence of interaction of

median life span between these proxies for nutrient intake and the longevity

gene daf-16 [66] is frequently interpreted to imply that DR is affected through

functions independent of insulin/IGF signaling [67]. To support this conclusion

we need data from multiple diet levels and survival analysis with enough power

to detect meaningful differences in mortality.

The framework of reaction norm analysis recommends that we apply two

operations to assess when a gene participates in mechanisms of DR. First, use

data within the range of nutrients where survival is modified by mechanisms of

DR and not by starvation. We can identify this range from survival measured

across a broad and fine-grained series of diets, as with chico [62, 68]. When a

study uses only two diets there is a chance that the diet concentration used for

the restricted condition is in the range of malnutrition. Reproduction provides

an additional criterion to define the region of a physiologically sufficient diet

since fecundity should vary inversely with survival in the nutrient range of DR.

Second, evaluate whether the reaction norms in the DR range are not parallel.

Evidence to reject the null hypothesis (parallel norms) suggests that there is a

function associated with the candidate gene that contributes to the mechanism

by which DR retards senescence.

Short of such formal analysis for chico we can be relatively confident that

there is no support to reject the hypothesis of parallel reaction norms within the

nutrient regions of DR reported in Clancy et al. [62]. Yet, overall we see the

Tatar 132

gene-by-environment plots intersect. How then might we interpret these results?

Since the molecular functions associated with chico1 affect both the response to

malnutrition and to aging, the mutation must influence a common and upstream

process, for instance by modulating nutrient perception or by changing the rate

of food intake. In these interpretations, chico1 does not elucidate a specific

pathway through which DR extends life span. Rather, chico functions at a broad

level of nutrient interactions to affect many aspects of physiology and life

history.

Besides chico, data to assess gene-by-DR interaction are available for the

histone deacetylases encoded by rpd-3 and sir-2. Misexpression of histone

deacetylases retards aging in yeast, C. elegans and Drosophila [69]. In C. elegans,

longevity extension induced by SIR-2 requires functional daf-16 [70]. In mam-

malian cells, SIRT deacetylates regulatory proteins such as p53, FOXO and

Ku70 and thereby mediates the balance between stress resistance and apopto-

sis [71]. To understand whether such aging-associated outcomes work through

pathways of DR, Rogina et al. [46] and Rogina and Helfand [64] assessed how

D. melanogaster rpd-3 and sir-2 affect longevity on media of low and high SY

diets. Figure 4b and c compares the median life span of rpd-3 mutants and of

sir-2 overexpression relative to their respective wild types. In both cases, the

reaction norms converge, and although the data were not tested by survival

regression analysis it is clear from pairwise tests that the lines cannot be paral-

lel. Unfortunately, with only two diet levels we cannot rule out that the diet

used at the lower concentration is within the region of malnutrition. The risk

here is for the apparent interaction of the reaction norms to involve both the

consequences of starvation and of aging. It would be useful to make life tables

for additional levels of diet and to measure fecundity. Some clues are presently

provided by available data on mRNA abundance. The message of sir-2 is

reduced 2-fold in long-lived rpd-3 mutants as well as in studies where diet is

restricted [46, 72]. Since rpd-3 and restricted diet affect the sir-2 message in

similar ways, the restricted diet used by Rogina and Helfand may indeed affect

survival through processes of DR rather than through starvation. Taken

together, these data provisionally demonstrate gene-by-DR interaction and

present the first genetic evidence on a mechanism of Drosophila DR. Reduced

diet retards aging through a pathway that involves processes of deacetylation.

What We Know of Diet Restriction in Drosophila

This paper focused on how we study DR in Drosophila and said little about

the way DR might work to slow aging in this animal. The literature is replete

with many attractive mechanistic hypotheses but these remain speculations


until we establish meaningful experimental designs and data interpretation.

Still, strong progress has been made with the fly [73, 74]. The first experimen-

tal treatment to successfully implement DR with the fly was published little

more than a decade ago and the first genes to modulate fly aging have only

recently been reported. In this short time we have collected a small number of

important insights.

(1) Yeast restriction is sufficient to increase survival. Whether this works

because flies acquire less of some yeast-specific nutrient or because they eat

fewer calories remains unknown. (2) Adult survival is influenced by two dis-

tinct processes: starvation due to malnutrition and longevity assurance physiol-

ogy due to DR. (3) Mortality has no memory of its past with respect to

nutrition. Nutrition modulates how susceptible individuals are to failure caused

by systems that degenerate with age. (4) Age-specific mortality and reproduc-

tion can be decoupled; DR does not retard aging simply by reducing the alloca-

tion of resources to eggs. (5) The molecular operation of DR may involve

processes of deacetylation; whether the mechanisms of DR function through

insulin-related signaling remains unknown. Finally, we can also see the poten-

tial that lies ahead: with new experimental, demographic and genetic tools we

expect to make rapid progress to understand the mechanistic basis for how DR

retards Drosophila aging.

References

1 Alpatov WW: Experimental studies on the duration of life. 13. The influence of different feeding

during the larval and imaginal stages on the duration of life of the imago of Drosophila

melanogaster.Am Nat 1930;64:37–55.

2 Chippindale AK, Leroi AM, Kim SB, Rose MR: Phenotypic plasticity and selection in Drosophila

life-history evolution. I. Nutrition and the cost of reproduction. J Evol Biol 1993;6:171–193.

3 Chapman T, Partridge L: Female fitness in Drosophila melanogaster: an interaction between the

effect of nutrition and of encounter rate with males. Proc R Soc Lond Biol Sci 1996;263:755–759.

4 Sacher GA: Life table modification and life prolongation; in Finch CE, Hayflick L (eds):

Handbook of the Biology of Aging. New York, Van Nostrand Reinhold, 1977, pp 582–638.

5 Promislow DEL, Tatar M, Pletcher S, Carey JR: Below threshold mortality: implications for stud-

ies in evolution, ecology and demography. J Evol Biol 1999;12:314–328.

6 Curtsinger JW, Fukui H, Townsend D, Vaupel J: Failure of the limited-lifespan paradigm in genet-

ically homogeneous populations of Drosophila melanogaster. Science 1992;258:461–463.

7 Tatar M: Senescence; in Fox CW, Roff DA, Fairbain DJ (eds): Evolutionary Ecology: Case

Studies. Oxford, Oxford University Press, 2001.

8 Tatar M: Comment on ‘long-lived Drosophila with overexpressed dfoxo in adult fat body’.

Science 2005;307:675a.

9 Lee ET: Statistical Methods for Survival Data Analysis, ed 2. New York, Wiley, 1992.

10 Partridge L, Green A, Fowler K: Effects of egg-production and of exposure to males on female

survival in Drosophila melanogaster. J Insect Physiol 1987;33:745–749.

11 Partridge L, Piper MDW, Mair W: Dietary restriction in Drosophila. Mech Ageing Dev 2005;126:

938–950.

12 Parmer MKB, Machin D: Survival Analysis: A Practical Approach. Chichester, Wiley, 1995.

Tatar 134

13 Pletcher SD, Khazaeli AA, Curtsinger JW: Why do lifespans differ? Partitioning mean longevity dif-

ferences in terms of age-specific mortality parameters. J Gerontol Biol Sci 2000;55A:B381–B389.

14 Driver CJI, Wallis R, Cosopodiots G: Is a fat metabolite the major diet dependent accelerator of

aging? Exp Gerontol 1986;21:497–507.

15 Edgecomb RS, Harth CA, Schneiderman AM: Regulation of feeding behavior in adult Drosophila

melanogaster varies with feeding regime and nutritional state. J Exp Biol 1994;197:215–235.

16 Min KJ, Tatar M: Drosophila diet restriction in practice: do flies consume fewer nutrients? Mech

Ageing Dev 2006;127:93–96.

17 Carvalho GB, Kapahi P, Benzer S: Direct quantification of food intake reveals compensatory

ingestion upon dietary restriction in Drosophila. Nat Methods 2005;2:813–815.

18 Mair W, Piper MDW, Partridge L: Calories do not explain extension of life span by dietary restric-

tion in Drosophila. PLoS Biol 2005;3:e223.

19 Carey JR, Liedo P, Harshman L, Zhang U, Muller H-G, Partridge L, Wang J-L: Life history

response of Mediterranean fruit flies to dietary restriction. Aging Cell 2002;1:140–148.

20 Cooper TM, Mockett RJ, Sohal BH, Sohal RS, Orr WC: Effect of caloric restriction on life span of

the housefly, Musca domestica. FASEB J 2004:03–1464fje.

21 Krijgsveld J, Ketting RF, Mahmoudi T, Johansen J, Artal-Sanz M, Verrijzer CP, Plasterk RH, Heck AJ:

Metabolic labeling of C. elegans and D. melanogaster for quantitative proteomics. Nat Biotechnol

2003;21:927–931.

22 O’Brien DM, Boggs CL, Fogel ML: Pollen feeding in the butterfly Heliconius charitonia: isotopic

evidence for essential amino acid transfer from pollen to eggs. Proc R Soc Lond B Biol Sci

2003;270:2631–2636.

23 Zimmerman JA, Malloya V, Krajcika R, Orentreicha N: Nutritional control of aging. Exp Gerontol

2003;38:47–52.

24 Miller RA, Buehner G, Chang Y, Harper JM, Sigler R, Smith-Wheelock M: Methionine-deficient

diet extends mouse lifespan, slows immune and lens aging, alters glucose, T4, IGF-I and insulin lev-

els, and increases hepatocyte MIF levels and stress resistance. Aging Cell 2005;4:119–125.

25 Lamb MJ: Ageing; in Ashburner m, Wright AR (eds): Genetics and Biology of Drosophila.

New York, Academic Press, 1978, pp 43–105.

26 Wood JG, Rogina B, Lavu S, Howitz K, Helfand SL, Tatar M, Sinclair D: Sirtuin activators mimic

caloric restriction and delay ageing in metazoans. Nature 2004;430:686–689.

27 Simmons FH, Bradley TJ: An analysis of resource allocation in response to dietary yeast in

Drosophila melanogaster. J Insect Physiol 1997;43:779–788.

28 Zera AJ, Harshman LG: The physiology of life history trade-offs in animals. Annu Rev Ecol

Systematics 2001;32:95–126.

29 Alcedo J, Kenyon C: Regulation of C. elegans longevity by specific gustatory and olfactory neu-

rons. Neuron 2004;41:45–55.

30 Dingle H: Evolution of Insect Migration and Diapause. Proceedings in Life Sciences. New York,

Springer, 1978.

31 Tatar M, Yin C-M: Slow aging during insect reproductive diapause: why butterflies, grasshoppers

and flies are like worms. Exp Gerontol 2001;336:723–738.

32 Dethier VG, Solomon RL, Turner LH: Sensory input and central excitation and inhibition in the

blowfly. J Comp Physiol Psychol 1965;60:303–313.

33 Stoffolano JG: Influence of diapause and diet on the development of the gonads and accessory

reproductive glands of the black blowfly, Phormia regina (Meigen). Can J Zool 1974;52:981–988.

34 Bell G, Koufopanou V: The cost of reproduction, in Dawkins R, Ridley M (Eds): Oxford Surveys

in Evolutionary Biology. Oxford, Oxford University Press, 1986, pp 83–131.

35 Tatar M, Carey JR: Nutrition mediates reproductive trade-offs with age-specific mortality in the

beetle Callosobruchus maculatus. Ecology 1995;76:2066–2073.

36 Tatar M, Khazaeli AA, Curtsinger JW: Chaperoning extended life. Nature 1997;390:30.

37 Silbermann R, Tatar M: Reproductive costs of heat shock protein in transgenic Drosophila

melanogaster. Evolution 2000;54:2038–2045.

38 van Noordwijk AJ, de Jong G: Acquisition and allocation of resources: their influence on variation

in life history tactics. Am Nat 1986;128:137–142.


39 de Jong G, van Noordwijk AJ: Acquisition and allocation of resources: genetic (co)variances,

selection, and life histories. Am Nat 1992;139:749–770.

40 Tu MP, Tatar M: Juvenile diet restriction and the aging and reproduction of adult Drosophila

melanogaster. Aging Cell 2003;2:327–333.

41 Good T, Tatar M: Age-specific mortality and reproduction respond to adult dietary restriction in

Drosophila melanogaster. J Insect Physiol 2001;47:1467–1473.

42 Mair W, Goymer P, Pletcher SD, Partridge L: Demography of dietary restriction and death in

Drosophila. Science 2003;19:1731–1733.

43 Helfand SL, Rogina B: Genetics of aging in the fruit fly, Drosophila melanogaster. Annu Rev

Genet 2003;37:329–348.



45 Hwangbo DS, Gershman B, Tu MP, Palmer MR, Tatar M: Drosophila dFOXO controls lifespan

and regulates insulin signaling in brain and fat body. Nature 2004;429:562–566.

46 Rogina B, Helfand SL, Frankel S: Longevity regulation by Drosophila Rpd3 deacetylase and

caloric restriction. Science 2002;29:298.

47 Walker DW, McColl G, Jenkins NL, Harris J, Lithgow GJ: Evolution of lifespan in C. elegans.

Nature 2000;18:296–297.

48 Mair W, Sgro CM, Johnson AP, Chapman T, Partridge L: Lifespan extension by dietary restriction

in female Drosophila melanogaster is not caused by a reduction in vitellogenesis or ovarian activ-

ity. Exp Gerontol 2004;39:1011–1019.

49 Leroi AM: Molecular signals versus the Loi de Balancement. Trends Ecol Evol 2001;16:24–29.

50 Tatar M: Germ-line stem cells call the shots. Trends Ecol Evol 2002;17:297–298.

51 Kenyon C, Chang J, Gensch E, Rudner A, Tabtiang R: A C. elegans mutant that lives twice as long

as wild type. Nature 1993;366:461–464.

52 Hsin H, Kenyon C: Signals from the reproductive system regulate the lifespan of C. elegans.

Nature 1999;399:362–366.

53 Drummond-Barbosa D, Spradling AC: Stem cells and their progeny respond to nutritional changes

during Drosophila oogenesis. Dev Biol 2001;231:265–278.

54 LaFever L, Drummond-Barbosa D: Direct control of germline stem cell division and cyst growth

by neural insulin in Drosophila. Science 2005;309:1071–1073.

55 Tatar M, Kopelman A, Epstein D, Tu M-P, Yin C-M, Garofalo RS: A mutant Drosophila insulin

receptor homolog that extends life-span and impairs neuroendocrine function. Science

2001;292:107–110.

56 Wessells RJ, Fitzgerald E, Cypser JR, Tatar M, Bodmer R: Insulin regulation of heart function in

aging Drosophila. Nat Genet 2004;36:1275–1281.

57 Broughton SJ, Piper MDW, Ikeya T, Bass TM, Jacobson J, Driege Y, Martinez P, Hafen E, Withers DJ,

Leevers SJ, Partridge L: Longer lifespan, altered metabolism, and stress resistance in Drosophila

from ablation of cells making insulin-like ligands. Proc Natl Acad Sci USA 2005;102:3105–3110.

58 Barthel A, Schmoll D, Unterman TG: FoxO proteins in insulin action and metabolism. Trends

Endocrinol Metab 2005;16:183–189.

59 Oldham S, Hafen E: Insulin/IGF and target of rapamycin signaling: a TOR de force in growth con-

trol. Trends Cell Biol 2003;13:79–85.



2001;292:104–106.

61 Tu M-P, Epstein D, Tatar M: The demography of slow aging in male and female Drosophila mutant

for the insulin-receptor substrate homolog chico. Aging Cell 2002;1:75–80.


Science 2002;296:319.

63 Pigliucci M: Phenotypic Plasticity: Beyond Nature and Nurture. Baltimore, Johns Hopkins

University Press, 2001.

64 Rogina B, Helfand SL: Sir2 mediates longevity in the fly through a pathway related to calorie

restriction. Proc Natl Acad Sci USA 2004;101:15998–16003.

Tatar 136

65 Zinke I, Schutz CS, Katzenberger JD, Bauer M, Pankratz MJ: Nutrient control of gene expression

in Drosophila: microarray analysis of starvation and sugar-dependent response. EMBO J

2002;21:6162–6173.


Acad Sci USA 1998;95:13091–13096.

67 Walker G, Houthoofd K, Vanfleteren JR, Gems D: Dietary restriction in C. elegans: From rate-of-

living effects to nutrient sensing pathways. Mech Ageing Dev 2005;126:929–937.

68 Gems D, Pletcher S, Partridge L: Interpreting interactions between treatments that slow aging.

Aging Cell 2002;1:1–9.

69 Guarente L, Picard F: Calorie restriction – the SIR2 connection. Cell 2005;120:473–482.

70 Lombard DB, Chua KF, Mostoslavsky R, Franco S, Gostissa M, Alt FW: DNA repair, genome sta-

bility, and aging. Cell 2005;120:497–512.

71 Tissenbaum HA, Guarente L: Increased dosage of a sir-2 gene extends lifespan in Caenorhabditis

elegans. Nature 2001;410:227–230.

72 Pletcher S, Macdonald SJ, Marguerie R, Certa U, Stearns SC, Goldstein DB, Partridge L:

Genome-wide transcript profiles in aging and calorically restricted Drosophila melanogaster.

Curr Biol 2002;12:712–723.

73 Magwere T, Chapman T, Partridge L: Sex differences in the effect of dietary restriction on life span

and mortality rates in female and male Drosophila melanogaster. J Gerontol A Biol Sci 2004;59:

B3–B9.

74 Van Herrewege J: Nutritional requirements of adult Drosophila melanogaster: the influence of the

casein concentration on the duration of life. Exp Gerontol 1974;9:191–198.

75 Min KJ, Tatar M: Restriction of amino acids extends lifespan in Drosophila melanogaster. Mech

Ageing Dev 2006;127:643–646.

Marc Tatar

Associate Professor

Division of Biology and Medicine

Box G–W, Brown University

Providence, RI 02912 (USA)




Dietary Restriction in Aging Nonhuman Primates

Julie A. Mattison, George S. Roth, Mark A. Lane, Donald K. Ingram

Intramural Research Program, Laboratory of Experimental Gerontology,

National Institute on Aging, Baltimore, Md., USA

AbstractDietary restriction (DR) has been shown to benefit health and longevity in a wide vari-

ety of species, although most have maximal life spans of only a few years. In 1987, the

National Institute on Aging began the first well-controlled long-term study in a species with

a considerably longer life span and a closer physiology to humans. Using rhesus monkeys

(Macaca mulatta), an extensive array of physiological measures have been conducted in both

males and females to evaluate the effects of DR. Monkeys benefit from DR with a lower

body weight, body fat, blood glucose and thus are at lower risk for developing diabetes.

Changes in several endocrine measures indicate an altered hormonal axis; however, circadian

patterns and timing relative to the onset of DR can obscure the differences. Despite the

caloric deficit, female monkeys are not reproductively compromised, and both males and

females may benefit immunologically. There remains much to be learned from this model of

DR including whether long-term DR will increase life span in a primate species.


Although dietary restriction (DR) as a prolongevity intervention has been

investigated since 1935, its application to a primate model is relatively new and

still unproven. Moreover, despite the considerable existing literature on the

topic, it will be several more years before any degree of certainty will emerge

for evaluating whether nonhuman primates (NHPs) on DR will exhibit

increased life span and health span. A considerable amount of data has been

generated to suggest many positive effects of DR in both humans and NHPs,

but it is uncertain whether or not this will translate to a longer life expectancy.

The first experimental study of DR in an NHP model was initiated in 1987

by intramural investigators at the National Institute on Aging (NIA) [1].

Subsequently, a similar study was undertaken at the University of Wisconsin

Mattison/Roth/Lane/Ingram 138

(UW) [2]. Male and female rhesus monkeys (Macaca mulatta) in both of these

longitudinal studies were grouped randomly into approximately equal numbers

of control and DR subjects. Monkeys at the NIA ranged in age from 2 to 23

years when they were initiated into the study; thus, some monkeys were prepu-

bertal while others were considered old. Monkeys in the UW study were all

adults (8–14 years of age) at initiation. All information gleaned about DR in

rhesus monkeys is derived from these two studies but with a median life span of

25 years and a maximum of 40 years, it will be several more years before

longevity data are conclusive.

A related investigation at the University of Maryland developed from stud-

ies of obesity and diabetes in rhesus monkeys in which weight titration imple-

mented to prevent obesity has produced many physiological effects similar to

DR [3]. However, the small sample of DR monkeys (n � 8), the lack of concur-

rent controls and random diet group assignment complicate the interpretation

of data emerging from this study. While it would be tempting to conclude from

their early evidence that DR increases survival in rhesus monkeys as Bodkin et

al. [4] made claim, the interesting relationships of nutrition, aging and disease

do not provide conclusive results at this time [5].

With such a long life span, studies of longevity in rhesus monkeys are

challenging and costly to conduct. Additionally, although a demonstration of

increased mean and maximum life span will be required for drawing definitive

conclusions, evidence for preserved health and function is also required. Since

conclusive survival data are still years away, the evidence for DR as an inter-

vention for decreasing the incidence and delaying the onset of age-related dis-

eases and pathology will be presented in the current review. The NIA study has

been designed to examine the effects of DR on cellular, organ, physiological

and behavioral function.

Body Composition and Dietary Intake

It would be expected that DR would produce a decline in body weight and

fat mass. Data from both the NIA and UW confirmed this expected result [2, 6].

Maturing monkeys, both control and DR, continued to gain weight into adult-

hood, but over the course of these studies, both male and female DR monkeys

weigh less than age-matched controls [6]. However, male monkeys have

responded to DR to a greater extent than females. NIA female monkeys have

maintained higher body weights compared to their respective controls at similar

levels of DR relative to males (fig. 1) [7]. A moderate sex difference was also

apparent in UW monkeys [8]. Factors contributing to this apparent sex differ-

ence including body composition and hormones are being explored, but UW

DR in Aging Nonhuman Primates 139

attributed some of this sex discrepancy to the low body fat of the females at the

start of the study [9].

In addition to lower body weight, examination by dual energy X-ray

absorptiometry indicated that DR monkeys have relatively less fat and lean

mass than controls [10]. DR monkeys also have less trunk fat and a reduced

trunk-to-leg fat ratio (fig. 2) [6]. DR monkeys in the UW study also had less

abdominal fat than controls [11]. Considering the health risks associated with

abdominal adiposity, this reduction indicates a favorable shift for cardiovascu-

lar risk factors.

After 15 years of study, DR monkeys in the NIA study continue to have

lower body weights compared to controls [7]. Additionally, consistent with

studies in humans, rhesus monkeys show a clear age-related decline in caloric

intake (fig. 3). Aging is commonly associated with decreased food intake in

humans [12]. This decline may be related to health issues, decreased energy

expenditure, decreased motivation (detailed in behavior section), changes in

body composition or hormonal status. There are several blood parameters that

may correlate with changes in feeding behavior and the NIA identified a nega-

tive relationship between globulin and caloric intake independent of age [12].

The significance of this relationship is being explored further.

Although consistent with humans, this observation of decreased intake

complicates the logistics of a DR study and maintaining a 30% reduction in

caloric intake compared to age-matched controls. The anorexia of aging and

associated weight loss predisposes older humans to malnutrition and disease

[13]. Thus, the NIA study has held food allotments constant for both the control

Fig. 1. Mixed effect model of body weight (BW) as a function of age in male and

female rhesus monkeys on a control diet (CON) or DR. Reprinted from Mattison et al. [7].

0 10 20 30 40

0

2

4

6

8

10

12

Age (years)

BW

(kg

)

CON males

DR males

CON females

DR females


and DR monkeys at their adult level for the duration of the study. Although the

intake for the control monkeys has been decreasing as they age (they have been

leaving behind more food), the DR monkeys have not been subject to a further

reduction.

Glucose, Insulin and Metabolic Rate

Considering the lower body weight and fat, it would be expected that DR

monkeys would be better able to regulate glucose than controls. A reduction in

fasting glucose was not evident immediately in the NIA monkeys [14], but levels

Fig. 2. DR reduces abdominal (trunk) fat. Each bar represents the mean (� SEM)

amount of trunk fat determined by dual energy X-ray absorptiometry after 6 (females; a) or

11 (males; b) years on DR. Ages at the time data were collected for females: juvenile 7–9,

adult 6–13 and old 22–27 years; for males: juvenile 12–13, adult 14–16 and old 28–34 years.

The effect of DR on reducing trunk fat was significant for both genders (p � 0.05).

Reprinted from Lane et al. [6].

Juvenile Adult

Age group

Age group

Old0

500

1,000

1,500

2,000

2,500

Juvenile Adult Old0

1,000

2,000

Tru

nk f

at

(g)

Tru

nk f

at

(g)

Control

DR

a

b


were reduced significantly after 3 years on DR [15]. This delayed glucose

response was also evident in the UW monkeys [16]. The NIA DR monkeys also

had a lower acute insulin response to a glucose challenge compared to controls

[15]. The UW study assessed glucose kinetics during an intravenous glucose tol-

erance test. Compared to controls, DR monkeys had lower basal �-cell sensitiv-

ity to glucose, greater insulin sensitivity and a lower first-phase plasma insulin

response [17]. Body fatness was highly predictive of glucose response in these

monkeys, and these factors may contribute to an overall reduced disease risk.

Both NHP studies have reported a reduction in metabolic rate in DR mon-

keys. In the NIA study, the reduction occurred early but equilibrated to levels

similar to the controls as body composition changed [18]. Blanc et al. [19]

reported that the reduction in total energy expenditure of DR monkeys in the

UW study was attributable to resting energy expenditure and was significant

even when adjusted for fat-free mass.

Endocrine Function

The endocrine system is important for regulating and maintaining complex

systems of metabolism, thermoregulation, reproduction, stress response, behav-

ior, homeostasis and immunity. Most of these processes are disturbed with

aging, and these changes may be attributed to a decline in endocrine activation

or signaling. Although the basal activity of some endocrine functions may not

be altered with age, the response to stimuli may be drastically compromized.

Fig. 3. Mixed effect model of daily caloric intake as a function of age in male and

female rhesus monkeys on a control diet (CON) or DR. Reprinted from Mattison et al. [7].

0 4 8 12 16 20 24 28 32 36

0

250

500

750

1,000

Age (years)

Calo

ries/d

ay

CON males

DR males

CON females

DR females


Thus, it was of interest to investigate how DR could affect various endocrine

parameters.

Melatonin

Melatonin is a hormone secreted by the pineal gland in a diurnal pattern

peaking during the night. It has been reported to improve sleep, lower blood

pressure, strengthen the immune system, act as an antioxidant and even

increase longevity [reviewed in 20]. Peak nocturnal concentrations are reached

at the age of 1–3 years in humans with a gradual decline thereafter [21]. The

pineal gland shrinks, melatonin secretion decreases and is often accompanied

by a phase shift of the pulsatile release. A similar trend with age is seen in

rodents [22] and monkeys [23].

It was of interest to determine if long-term DR would alter the age-related

changes in melatonin secretion in the NIA monkeys. Early studies in Fischer

344 rats showed that 40% DR attenuated the age-related decline in pineal func-

tion and melatonin secretion [24]. However, the immune response in rats fol-

lowing melatonin supplementation was not improved as it was with DR [25].

Cross-sectional data from 52 control-fed male and female monkeys in the

NIA study confirmed an age-related decline in peak melatonin levels [26].

However, unlike findings in a cross-sectional study in humans in which females

accounted for the age-related difference [27], there was no gender difference in

this cohort of monkeys. Additionally, monkeys that had been maintained on

DR for 12 years did not show an age-related decline; in fact, there was an age-

associated increase (fig. 4) [26]. Moreover, the old DR monkeys had significantly

higher serum melatonin levels than age-matched controls. This diet difference

was not apparent in the younger adult monkeys but they may not have experi-

enced a significant age-related decline yet. Similarly, urinary excretion of mela-

tonin in the adult monkeys at the UW was not different between diet groups [8].

Additional diet differences may emerge as both the NIA and UW adult mon-

keys reach older ages.

Dehydroepiandrosterone

The androgenic steroids, dehydroepiandrosterone (DHEA) and its sulfated

form DHEAS, are among the most abundant steroids in the body. In both humans

and NHPs, circulating levels of DHEAS are very high during early adult life and

then decrease markedly with aging. Elevated serum levels of DHEAS have been

related to a protective function against age-associated diseases, such as diabetes

[28], heart disease [29] and cancer [30]. As a result, this hormone has received

considerable attention as a possible intervention against the decrements of aging.

A large-scale survey of 792 laboratory-housed male and female rhesus

monkeys at the UW reported an approximately 90% reduction in DHEAS from


infancy through 36 years of age [31]. The magnitude of the decrease was great-

est during the first few years of life followed by a more gradual decline, averag-

ing 4.2% per year [31]. In humans, DHEAS peaks at around 20 years of age and

then decreases continuously thereafter in both men and women [32, 33].

Although the slope of the decline clearly differs in these two species, the rate is

about 2–2.5 times higher in rhesus monkeys compared to humans, a rate that is

consistent with the humans’ approximately 3-fold greater life span [35].

Blood samples from NIA monkeys have been tested on several occasions

to identify changes in DHEAS with age and DR. Roth et al. [34] reported an

age-related decrease in DHEA in a cross-sectional sample of males that was not

affected by 2–3 years of DR. Subsequently, Lane et al. [35] showed that the

postmaturational decline in DHEAS was attenuated by DR in a 3-year longitu-

dinal study. However, this report focused on the young adults and it remained

Fig. 4. The effect of aging and DR on plasma melatonin in rhesus monkeys.

a Relationship between age and melatonin levels (log transformed) for control (CON) and

DR rhesus monkeys. b Mean (� SEM) melatonin levels in adult and old monkeys. The

age � diet interaction is significant (p � 0.04) by two-way analysis of variance. ap � 0.04:

analysis of the simple main effect of age indicated a significant decrease in the control group

but not in the DR group; bp � 0.01: analysis of the simple main effect of diet indicated a sig-

nificant effect in the old group but not in the adult group. Reprinted from Roth et al. [26].

Adult Old0

1

2

3

Mela

tonin

(lo

g p

mo

l/l )

0 10 20 30 40

0

1

2

3

Age (years)

Mela

ton

in (lo

g p

mo

l/l)

CON

r2�0.07, p�0.06

0 10 20 30 40

DR

r2�0.14, p�0.01

Age (years)

a

b

Male

Female

CON DR

a

b


unclear if the effect of DR would be maintained into older age. Additionally,

measurements were based on blood samples collected at one time point in

the day, although at a consistent time point, they could not detect circadian

fluctuations.

In the UW DR study, an effect of age on DHEAS levels was not detected in

a cross-sectional analysis, although it was reported in the larger cohort cited

above. However, this finding may be explained by the narrow age range of the

monkeys, as all were considered middle-aged and thus subtle changes would

not be evident. The effect of DR in the UW cohorts was somewhat inconsistent.

One group of DR males had lower DHEAS levels than controls, but the DR

females had slightly higher levels. Overall, the results suggested that DR did

not alter DHEAS concentrations [8].

Using an advanced methodology to collect hormonal data, a cohort of NIA

rhesus monkeys housed at the Oregon National Primate Research Center

(ONPRC) was fitted with an indwelling catheter system. Serial blood samples

were collected at hourly intervals for a 24-hour period in young (approx. 10

years) and old (approx. 26 years) male monkeys following 4 years of 30%

restriction [36]. A clear diurnal pattern in DHEAS was evident in the young

monkeys with a peak occurring in the morning when the lights came on. There

was a dramatic drop in DHEAS concentration from young levels in both control

and DR old males with little evidence of a diurnal pattern. In this cross-

sectional cohort of male monkeys, DR did not attenuate the age-related decline

seen in DHEAS; in fact, DHEAS levels were slightly lower in the DR monkeys

of both age groups [36]. It is possible that DR was initiated too late in the old

monkeys, they were already 22 years of age, and a significant age-related

decline may have already occurred that could not be reversed.

Cortisol

Glucocorticoids are of interest in the context of aging since most studies

report that levels increase in rodents during late middle age and in humans during

old age, a change that could indicate hippocampal dysfunction [37]. In the con-

text of DR studies, glucocorticoids are of interest because elevated cortisol (the

primary glucocorticoid in primates; corticosterone in rodents) may be the result

of this low-intensity nutritional stress and actually mediating some of the positive

effects of DR. In a cross-sectional study of rhesus monkeys, the UW group did

not detect an age-related increase in cortisol during a 30-month assessment.

Additionally, there was no consistent difference between DR and control animals

[8]. In the monkeys housed at the ONPRC, both young (10 years) and old (26

years) males showed a diurnal pattern of release using an indwelling vascular

catheter collecting 1-hour interval samples. There was no significant age-related

change. Although mean and maximal levels were similar, an additional peak was


apparent in the young DR monkeys that was not evident in the age-matched con-

trol or older monkeys. The peak was consistent with their increased locomotor

activity. The old DR monkeys subjected to the same 4-year period of DR had

slightly lower 24-hour cortisol levels possibly indicating that their stress response

pathway was not activated comparably to the young monkeys [36].

Thyroid Hormones

Thyroid hormones regulate metabolism and thereby may play a role in the

mechanism for the metabolic adaptations that occur with DR. Additionally, nor-

mal aging has been associated with a slight decrease in thyroid-stimulating

hormone (TSH) release [38] and decreased peripheral degradation of thyroxine

(T4), resulting in lower serum triiodothyronine (T3) [39]. DR has been shown to

have immediate effects on thyroid hormones in Sprague-Dawley rats.

Decreases in T3 and T4 were sustained for 1 year but returned to normal levels

within 7 days of refeeding [40].

In a cross-sectional analysis of the NIA monkeys ranging in age from 8 to

32 years, T3 did not change with age, while T4 and TSH decreased [41]. T3 was

lower in monkeys within 1 month of initiating a DR diet when phased in gradu-

ally. However, the effect may be transient as the difference disappeared after

6 months on the diet. A diet effect was also evident in the old cohort of monkeys

in which TSH levels were increased by long-term DR [41]. UW monkeys,

which had been on DR for a briefer time, did not demonstrate changes in T3 [8].

This inconsistency between studies may relate to differences in body composi-

tion or macronutrients in the diet. Overall, results suggest that DR may con-

tribute to an altered thyroid hormone axis.

Reproductive Function

Mechanisms that control energy balance are linked to those that control

maturation and reproduction; therefore, it is plausible that when calories, thus

energy, are decreased, growth and reproduction would also be affected. It was of

interest to determine if it would also retard sexual maturation in rhesus mon-

keys as has been shown in rodents [40] and undernourished humans [42]. Male

rhesus monkeys subjected to 30% DR at 1–2 years of age experienced an

approximately 1-year delay in sexual maturation as evidenced by lower circulat-

ing testosterone levels compared to control monkeys [43].

Reproductive development was not monitored in the NIA female monkeys;

however, currently there is an active program investigating ovarian aging and

the transition to menopause. The pattern of reproductive senescence in rhesus

females is similar to that in humans; however, relative to life span, menopausal


Fig. 5. Total number (a) and percent normal (24–31 days; b) menstrual cycles in con-

trol (CON; n � 21) and 30% DR (n � 19) female rhesus monkeys over a 2-year period. Each

point represents data for individual monkeys. Linear regression analysis revealed that both

total number and percent normal menstrual cycles declined with age. DR did not affect men-

strual cycling (p � 0.05). Reprinted from Lane et al. [44].

0 10 20 30

0

10

20

30

40

CON DRp�0.03, r2�0.11

Nu

mb

er

of

cycle

s

0 10 20 30

0

50

100

150p�0.05, r2�0.08

Age (years)

Age (years)

Cycle

s (%

no

rmal)

a

b

changes in monkeys occur slightly later in life [44, 45]. The similar hormonal

changes leading to menopause offer rhesus monkeys as a valuable model for

this area of study. Previous NIA data from a single time point blood sample of

40 monkeys aged 7–27 years indicated a significant age-related decrease in

serum estradiol, increase in follicle-stimulating hormone and decrease in both

number and length of menstrual cycles (fig. 5) [44]. Progesterone and luteiniz-

ing hormone did not change with age (fig. 6). None of these parameters were

altered by 6 years of a 30% DR diet. More recently, the NIA has collected blood

samples from these same female monkeys during 3 consecutive menstrual

cycles, daily during the follicular phase and every third day during the luteal


phase. Data from this more rigorous sampling strategy are consistent with

previous findings in that all young monkeys cycled regularly, regardless of diet

group. Additionally, elevated follicle-stimulating hormone levels were apparent

in the older monkeys, and estradiol was not different between age groups [46].

Studies are under way to determine if DR delays the onset of menopause.

Long-term DR data contrast with those from a recent report in young rhe-

sus females (6–10 years) restricted to lose 20% of their body weight. The 4 ini-

tially lean monkeys became anovulatory in about 2 months while it took up to

10 months and a 46% reduction in body weight for the obese monkey [47].

Ovulation returned with refeeding but at a 28% greater caloric intake. This

apparent discrepancy was likely caused by the immediate stress of caloric

deficit which compensatory mechanisms can reverse over time.

Fig. 6. Serum estradiol (a), follicle-stimulating hormone (FSH; b), progesterone (c)

and luteinizing hormone (LH; d) concentrations in control (CON, n � 21) and 30% DR

(n � 19) female rhesus monkeys. Each point represents biochemical data for individual

monkeys at the corresponding age. Data were collected after 6 years of DR. Linear regres-

sion revealed significant effects of age on estradiol and follicle-stimulating hormone con-

centrations (p � 0.02). DR did not affect reproductive hormone concentrations. Reprinted

from Lane et al. [44].

0 10 20 30

0

50

100

150p�0.02, r2�0.24

Estr

ad

iol (p

g/m

l)

0 10 20 30

0

2.5

5.0

7.5p�0.02, r2�0.24

FS

H (ng

/ml)

0 10 20 30

0

0.25

0.50

0.75p�0.20, r2�0.07

Pro

geste

ron

e (n

g/m

l)

0 10 20 30

0

1

2

3

4 p�0.18, r2�0.09

Age (years)Age (years)

Age (years)Age (years)

LH

(ng

/ml)

CON DR

a c

b d


Immune Function

The role of the immune response in contributing to the prolongevity effects

of DR has not been explored extensively in NHPs. DR retards several disease

processes which have an immune component as has been shown by a decreased

incidence of lymphomas, hepatomas and lung tumors in mice and rats [48].

Jolly [49] also reported that DR was effective in modulating the severity of

symptoms in several autoimmune disease rodent models. Many studies of

immune function have focused on T lymphocytes, which regulate both the type

and the magnitude of the immune response. In rodents, the age-dependent loss

in T lymphocyte interleukin 2 (IL-2) production is slowed by DR, while the

population of naïve lymphocytes is maintained [50]. The mechanisms for these

effects in rodents are still under investigation and have limited applicability to

NHPs because many species differences exist in immune system biology.

Information on aging of the NHP immune system is limited, and data on

the effect of DR in NHPs are more scarce. Due to the nature of the studies, inva-

sive procedures and immune challenges in a long-term study are problematic.

Thus, much of the data are obtained from in vitro studies of lymphocytes. Old

control monkeys from the NIA study were reported to have a reduced intracel-

lular free Ca2� response from CD4� lymphocytes following stimulation com-

pared to their young counterparts [51]. In this early study, 4 years of DR in the

males and only 4 months in the female did not alter the response.

In the same monkeys, lymphopenia was reported following 7 years on a

DR diet, a finding similar to data in rodents. Additionally, those monkeys initi-

ated to DR at 1 year of age tended to have a reduced proliferative capacity of

mitogen-induced peripheral blood mononuclear cells (PBMCs), which was not

apparent in monkeys initiated at a slightly older age [52]. Following 4 years on

DR, monkeys at the UW showed no reduction in peripheral blood lymphocytes,

but mitogen responses were reduced in DR monkeys compared to controls [53].

However, when NIA monkeys were examined after 14 years on DR, there was

no indication of lymphopenia and only a slight trend toward decreased white

blood cell count in those monkeys that had been initiated on the DR diet at 1

year of age [54]. Additionally, the difference in PBMC proliferative response to

several mitogens in DR animals was not different when the animals had been

maintained on the diet for 9 years or longer [54].

Although age-related changes in immune function are often contradictory,

cytokine responses from male NIA monkeys are in general agreement with sev-

eral mouse and human studies. Despite variability, the monkeys demonstrated a

likely switch from a Th1 (�-interferon) to a Th2-associated (IL-10, IL-6) cytokine

profile during aging as evidenced by an increase in IL-10 and IL-6 response and

a decreased �-interferon response following stimulation of the PBMCs [55].


These same measures were conducted in a group of young (6–7 years) and old

(22–25 years) monkeys following 2 years on a DR diet and showed that DR might

be able to prevent this immune response switch. The old DR monkeys had signif-

icantly higher levels of the Th1-like cytokine �-interferon compared with old con-

trols [56]. This improved responsiveness could be a key factor in decreasing the

incidence of cancer and other age-associated diseases.

To date, the only data available on the effect of DR on the composition of

the peripheral T cell compartment comes from NIA monkeys housed at the

ONPRC. Nikolich-Z¤ ugich and Messaoudi [54] reported that both males (3–5

years old) and females (5–7 years old) on long-term DR initiated during early

adulthood have elevated percentages of naïve CD4 and CD8 cells and a decline

in effector memory and senescent T cells compared to controls. This same

preservation of naïve T cells was also evident in the PBMCs of adult animals on

DR for only 2 years and the lymph nodes of older animals started on DR at a

minimum of 17 years old [54]. Harper et al. [57] reported that the percentage of

naïve T cells was a strong biomarker of aging that correlated with longevity in

mice.

Gene Array/Oxidative Stress

UW investigators have used gene array technology to analyze age-related

changes in the pattern of gene expression from skeletal muscle and the effects

of DR in rhesus monkeys. In a comparison of young (mean age 8 years) and old

monkeys (mean age 26 years), an upregulation of some transcripts involved in

inflammation and oxidative stress and a downregulation in those associated

with mitochondrial electron transport and oxidative phosphorylation were

observed [58]. A downregulation of genes involved in energy metabolism and

an upregulation of some structural genes involved in cellular growth were

observed after 10 years of DR in middle-aged monkeys (mean age 20 years).

DR did not affect the progression of age-related changes in gene expression

which contrasts findings in mice [58].

The UW study has also focused on molecular markers of oxidative stress

using electron microscopy. In a cohort of 2- to 34-year-old rhesus monkeys,

there was an age-associated 2-fold increase in carbonyl levels from the vastus

lateralis [59]. However, in the adult monkeys (17–23 years old) following 10

years on a 30% DR diet, carbonyls and nitrotyrosine levels were significantly

lower than age-matched control-fed monkeys.

The NIA has measured serum isoprostane as an indicator of oxidative

stress. However, due to changes in assay techniques and sensitivity, results have

been inconsistent. Additional analyses are under way.


Behavioral Assessments

Both humans and NHPs experience declines in several cognitive domains

including the ability to recall recent events, acquire new information and shift

from one problem-solving strategy to another, as well as reduced motor abilities.

Tests of delayed responses taxing short-term memory are the best characterized in

aging monkeys. The NIA is actively developing and implementing a behavioral

program to evaluate age-related changes in memory and function to include tasks

such as: object discrimination as an association memory task; delayed match-to-

sample as a recognition memory task; object reversal for set shifting; motor move-

ment assessment panel to detect coarse and fine motor movement, and automated

locomotor activity detection. Many of these tasks are currently under study.

Fig. 7. Retrieval time (square root transformation) in a food motivation task

(means � SEM) for female (a) and male (b) rhesus monkeys in different age groups on a

control diet (CON) or DR for over 8 years. Reprinted from Mattison et al. [7].

Juvenile Adult Old0

1

2

3

4

5

6

7

(6)

(7)

(8)

(7)

(2) (4)

Age group

Retr

ieval tim

e (s)

Juvenile Adult Old0

1

2

3

4

5

6

7

(8)

(5) (5)

(5)

(3)(5)

Age group

Retr

ieval tim

e (s)

CON DR

a

b


Behavior tasks used in nutritional studies can be confounded by the com-

mon practice of using a food reward; thus, it was necessary to establish that DR

monkeys did not perform better in food-motivated tasks merely because they

were more hungry than controls. Monkeys were observed in a novel task that

measured the time they would spend working to get to a food reward [7].

Performance in this task was considered a measure of the activational aspect of

motivation as the monkey initiated and maintained behavior required to retrieve

the food [60]. Retrieval times between control and DR groups for the three age

cohorts juvenile, adult, and old for males and females were not different

(fig. 7). This phenomenon was observed in monkeys that had been on DR for

7–11 years and also in a cohort following only 3–5 years of DR [7]. Because

motivational aspects appeared similar between diet groups, the NIA has devel-

oped additional behavioral experiments which use food rewards.

Additionally, from this initial task, a clear age-related decline in motivation

was observed when data from control monkeys only were analyzed (fig. 8) [7].

This finding paralleled the age-related decline in caloric intake. Changes in

energy expenditure, body composition and hormonal influences may all con-

tribute to behavior and feeding changes.

Considering the metabolic changes associated with DR, it was of interest to

determine age-associated changes in locomotor activity. Following 6 years on

the study, NIA monkeys were monitored using ultrasonic motion detectors and

videotape. Compared to their respective control group, adult DR males were the

only cohort to display more pacing, gross movement, stereotypies and were less

passive [61]. A subsequent study in the female cohort showed no generalized

Fig. 8. Regression of age onto retrieval time (seconds) as a measure of motivation for

food in rhesus monkeys fed both a long- and short-term control diet. Reprinted from

Mattison et al. [7].

0 50 100 150 200 250 300 350 400

0

10

20

30

40

50

60

70

r�0.35, p�0.03

Age (months)

Retr

ieval tim

e (s)

Males

Females


diet effect on activity although DR juveniles (6–8 years) were slightly less active

than age-matched controls [62]. DR monkeys at the UW initially declined in

activity while the controls increased [2], but these differences were not sustained

[63, 64]. Duffy et al. [65] reported increased motor activity in DR rats which was

associated with feeding time. The NIA is again monitoring the locomotor activ-

ity level to evaluate age-related changes and the influence of long-term restric-

tion. Preliminary data indicate an age-related decline in home cage activity for

control-fed males and females, and that old DR monkeys appear slightly less

active than their control counterparts [unpubl. observation].

Brain

Analyses are ongoing to correlate behavior function with in vivo brain

imaging. Thus far, age-related declines in the volume of the striatum and

reduced binding potential of striatal dopamine D2 receptors have been identi-

fied [66]. DR did not attenuate this age-related decline in the striatum; in fact,

volumes of the putamen were larger in the middle-aged and old control mon-

keys compared to DR animals. The difference remained even when corrected

for body size [67]. The functional significance of this anatomical measure is

uncertain, and follow-up studies are planned.

A recent ancillary study conducted at the NIA has shown that a short-term

30% DR can lessen the severity of disease in a model of Parkinson’s disease

[68]. Adult male rhesus monkeys were injected unilaterally in the right carotid

artery with the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)

following 6 months on a DR or control diet. Following MPTP treatment, loco-

motor activity was markedly decreased in all monkeys; however, DR monkeys

showed higher levels of activity compared to controls. Additionally, the MPTP-

induced reduction in striatal levels of dopamine and dopamine metabolites was

attenuated in DR monkeys. Another important finding was that levels of glial-

derived neutrophic factor, which promotes survival of dopamine neurons, were

also higher in the caudate nucleus of DR monkeys. These findings suggest

that DR or other nutritional interventions may beneficially alter the course of

Parkinson’s disease.

Sensory Function

Several components of sensory function are affected with advanced age in

primates. The gradual losses to both the auditory and the visual systems com-

promise late-life abilities and independence. Rhesus monkeys are an excellent


model of human auditory capacity [69] and experience an age-related decline in

cochlear and neural function [70]. Similarly, the pattern and relative timing of

the age-dependent loss in accommodative function parallels that of humans [71,

72]. It was of interest to determine whether or not DR would influence age-

related changes in these sensory abilities.

Both the NIA and the UW studies have reported a clear age-related decline

in some measures of auditory function, but only the UW monkeys showed an

indication of diet differences. The NIA tested only the male monkeys and

showed evidence of an age-related decline in cochlear function as measured by a

decrease in distortion product otoacoustic emissions [73]. These are associated

with outer hair cell function within the cochlea [74], and a decline is indicative

of cochlear degeneration. Additionally, the NIA monkeys had some age-associated

decline in neural function as measured by a decline in some auditory brainstem

responses [73]. Their waveforms are recorded by electroencephalography, and

measures of wave amplitude, response latency and threshold indicate neural

function. There were no significant effects of DR on any auditory parameters.

UW monkeys also showed an auditory decline related to aging [75].

Additionally, sex and diet differences were apparent. Females were generally

younger than the males tested; thus, sex differences were likely confounded by

the age difference. There were poorer auditory brainstem response thresholds

with age; however, older DR males were better than the control counterparts

suggesting that DR may be beneficial in delaying presbycusis (high-frequency

progressive hearing loss). Actual sex differences in the measures will become

apparent in future studies when the females reach a similar old age.

NIA monkeys were measured on several parameters of visual function.

Consistent with previous studies in rhesus monkeys, lens thickness increased

with age and accommodative amplitude decreased; however, neither were

affected by diet (fig. 9) [76]. Lens thickening may combine with other lenticular

factors to decrease the lens’ ability to change shape during accommodation. This

study was the first to suggest that the effect of DR may not extend to the ocular

accommodative mechanism or lens clarity. However, it was possible that the

older animals may have already experienced age-related decline before initiation

of the DR diet that could not be reversed. Future studies when the younger mon-

keys have reached an advanced age will clarify potential diet differences.

Conclusion

Although DR continues to show great promise for its health benefits and

potential to extend life span, conclusive results from a long-living primate model

are still years away. Rhesus monkeys exhibit many beneficial effects from DR


that have been similarly documented in DR rodents, such as decreased fat,

improved glucoregulatory function, decreased risk factors for cardiovascular dis-

ease and diabetes. Additionally, factors that might be compromised by nutritional

stress, such as reproduction and immune function, have not been affected detri-

mentally and, in fact, appear predictive of enhanced survival. Both the NIA and

UW studies will continue to provide opportunities to assess aging parameters and

the effectiveness of DR in maintaining better health and function into old age.

Acknowledgements

The authors wish to acknowledge the valuable work of the staff at Poolesville, Md., includ-

ing Edward Tilmont, Jennifer Young, April Hobbs, Sue Pazzi, and the excellent veterinary care

provided by Drs. Doug Powell and Rick Herbert. Additionally, we thank our many collaborators

at other research institutions for their contributions. The work is supported by funds from the

NIA provided to the Veterinary Research Program through an Inter-Agency Agreement.

References

1 Ingram DK, Cutler RG, Weindruch R, Renquist DM, Knapka JJ, April M, Belcher CT, Clark MA,

Hatcherson CD, Marriott BM, Roth GS: Dietary restriction and aging: the initiation of a primate

study. J Gerontol 1990;45:B148–B163.

Fig. 9. Carbachol-induced accommodative amplitude as a function of age in rhesus

monkeys. Accommodative ability declined in both control (CON) and DR monkeys by

1.03 � 0.12 (p � 0.001) and 1.18 � 0.12 dpt/year (p � 0.001), respectively. There was no

difference between diet groups (p � 0.374). Reprinted from Mattison et al. [76].

Acco

mm

od

atio

n (d

pt)

Age (years)

10 15 20 25 30 35

10

0

10

20

30

CON

DR

Regression CON

Regression DR


2 Kemnitz JW, Weindruch R, Roecker EB, Crawford K, Kaufman PL, Ershler WB: Dietary restric-

tion of adult male rhesus monkeys: design, methodology and preliminary findings from the first

year of the study. J Gerontol 1993;48:B17–B26.

3 Bodkin NL, Ortmeyer HK, Hansen BC: Long-term dietary restriction in older-aged rhesus mon-

keys: effects on insulin resistance. J Gerontol 1995;50:B142–B147.

4 Bodkin NL, Alexander TM, Ortmeyer HK, Johnson E, Hansen BC: Mortality and morbidity in

laboratory-maintained rhesus monkeys and effects of long-term dietary restriction. J Gerontol

2003;58A:212–219.

5 Lane MA, Mattison JA, Roth GS, Brant LJ, Ingram DK: Effects of long-term diet restriction on

aging and longevity in primates remain uncertain. J Gerontol 2004;59A:405–407.

6 Lane MA, Ingram DK, Roth GS: Calorie restriction in nonhuman primates: effects on diabetes

and cardiovascular disease risk. Toxicol Sci 1999;52(suppl):41–48.

7 Mattison JA, Black A, Huck J, Moscrip T, Handy A, Tilmont E, Roth GS, Lane MA, Ingram DK:

Age-related decline in caloric intake and motivation for food in rhesus monkeys. Neurobiol Aging

2005;26:1117–1127.

8 Ramsey JJ, Colman RJ, Binkley NC, Christensen JD, Gresl TA, Kemnitz JW, Weindruch R:

Dietary restriction and aging in rhesus monkeys: the University of Wisconsin study. Exp Gerontol

2000;35:1131–1149.

9 Colman RJ, Roecker EB, Ramsey JJ, Kemnitz JW: The effect of dietary restriction on body com-

position in adult male and female rhesus macaques. Aging Clin Exp Res 1998;10:83–92.

10 Lane MA, Ingram DK, Roth GS: Beyond the rodent model: calorie restriction in rhesus monkeys.

Age 1997;20:45–56.

11 Colman RJ, Ramsey JJ, Roecker EB, Havighurst T, Hudson JC, Kemnitz JW: Body fat distribution

with long-term dietary restriction in adult male rhesus macaques. J Gerontol 1999;54A: B283–B290.

12 Morley JE, Silver AJ: Anorexia in the elderly. Neurobiol Aging 1988;9:9–16.

13 Morley JE: Pathophysiology of anorexia. Clin Geriatr Med 2002;18:661–673.

14 Cutler RG, Davis BJ, Ingram DK, Roth GS: Plasma concentrations of glucose, insulin, and

percent glycated hemoglobin are unaltered by food restriction in rhesus and squirrel monkeys.

J Gerontol 1992;47:B9–B12.

15 Lane MA, Ball SS, Ingram DK, Cutler RG, Engel J, Read V, Roth GS: Diet restriction in rhesus

monkeys lowers fasting and glucose-stimulated glucoregulatory end points. Am J Physiol

1995;268:E941–E948.

16 Kemnitz JW, Roecker EB, Weindruch R, Olson DF, Baum ST, Bergman RN: Dietary restriction

increases insulin sensitivity and lowers blood glucose in rhesus monkeys. Am J Physiol

1994;266:E540–E547.

17 Gresl TA, Colman RJ, Havighurst TC, Allison DB, Schoeller DA, Kemnitz JW: Dietary restriction

and �-cell sensitivity to glucose in adult male rhesus monkeys. J Gerontol 2003;58A:598–610.

18 Lane MA, Tilmont EM, DeAngelis H, Handy AM, Ingram DK, Kemnitz JW, Baum ST, Roth GS:

Short-term calorie restriction improves disease-related markers in older male rhesus monkeys

(Macaca mulatta). Mech Aging Dev 2000;112:185–196.

19 Blanc S, Schoeller D, Kemnitz J, Weindruch R, Colman R, Newton W, Wink K, Baum S, Ramsey

J: Energy expenditure of rhesus monkeys subjected to 11 years of dietary restriction. J Clin

Endocrinol Metabol 2003;88:16–23.

20 Brzezinski A: Melatonin in humans. N Engl J Med 1997;336:186–195.

21 Waldhauser F, Weiszenbacher G, Frisch H, Zeitlhuber U, Waldhauser M, Wurtman RJ: Fall in noc-

turnal serum melatonin during prepuberty and pubescence. Lancet 1984;i:362–336.

22 Selmaoui B, Touitou Y: Age-related differences in serum melatonin and pineal NAT activity and in

the response of rat pineal to a 50-Hz magnetic field. Life Sci 1999;64:2291–2297.

23 Goncharova ND, Lapin BA: Age-related endocrine dysfunction in nonhuman primates. Ann NY

Acad Sci 2004;1019:321–325.

24 Stokkan KA, Reiter RJ, Nonaka KO, Lerchl A, Yu BP, Vaughan MK: Food restriction retards aging

of the pineal gland. Brain Res 1991;545:66–72.

25 Pahlavani MA, Vargas DA, Evans TR, Shu JH, Nelson JF: Melatonin fails to modulate immune

parameters influenced by calorie restriction in aging Fischer 344 rats. Exp Biol Med 2002;227:

210–207.


26 Roth GS, Lesnikov V, Lesnikov M, Ingram DK, Lane MA: Dietary caloric restriction prevents the

age-related decline in plasma melatonin levels of rhesus monkeys. J Clin Endocrinol Metab

2001;86:3292–3295.

27 Ng Ying Kin NMK, Nair NPV, Schwartz G, Thavundayil JX, Annable L: Secretion of melatonin in

healthy elderly subjects: a longitudinal study. Ann NY Acad Sci 2004;1019:326–329.

28 Small M, Gray CE, Beastall GH, MacCuish AC: Adrenal androgens and insulin-dependent dia-

betes mellitus. Diabetes Res 1989;11:93–95.

29 Barrett-Connor E, Khaw K, Yen S: A prospective study of dehydroepiandrosterone sulfate, mor-

tality, and cardiovascular disease. N Engl J Med 1986;315:1519–1524.

30 Zumoff B, Levin J, Rosenfeld RS, Markham M, Strain GW, Fukushima DK: Abnormal 24-hour

mean plasma concentrations of dehydroepiandrosterone and dehydroepiandrosterone sulfate in

women with primary operable breast cancer. Cancer Res 1981;41:3360–3363.

31 Kemnitz JW, Roecker EB, Haffa AL, Pinheiro J, Kurzman I, Ramsey JJ, MacEwen EG: Serum

dehydroepiandrosterone sulfate concentrations across the life span of laboratory-housed rhesus

monkeys. J Med Primatol 2000;29:330–337.

32 Orentreich N, Brind J, Rizer R, Vogelman J: Age and sex differences in serum dehydroepiandros-

terone sulfate concentrations throughout adulthood. J Clin Endocrinol Metab 1984;59:551–555.

33 Carlström K, Brody S, Lunell NO, Largrelius A, Mollerstrom G, Pousette A, Rannevik G, Stege R,

von Schoultz B: Dehydroepiandrosterone sulfate and dehydroepiandrosterone in serum: differ-

ences related to age and sex. Maturitas 1988;10:297–306.

34 Roth GS, Blackman MR, Ingram DK, Lane MA, Ball SS, Cutler RG: Age-related changes in

androgen levels of rhesus monkeys subjected to diet restriction. Endocrine J 1993;1:227–234.

35 Lane MA, Ingram DK, Ball SS, Roth GS: Dehydroepiandrosterone sulfate: a biomarker of pri-

mate aging slowed by caloric restriction. J Clin Endocrinol Metab 1997;82:2093–2096.

36 Urbanski HF, Downs JL, Garyfallou VT, Mattison JA, Lane MA, Roth GS, Ingram DK: Effect of

caloric restriction on the 24-hour plasma DHEAS and cortisol profiles of young and old male rhe-

sus macaques. Ann NY Acad Sci 2004;1019:443–447.

37 Sapolsky RM: Glucocorticoids, stress, and their adverse neurological effects: relevance to aging.

Exp Gerontol 1999;34:721–732.

38 Marriotti S, Franceschi C, Cossarizza A, Pinchera A: The aging thyroid. Endocrinol Rev 1995;16:

686–715.

39 Rubenstein HA, Butler VP Jr, Werner SC: Progressive decrease in serum triiodothyronine concen-

trations with human aging: radioimmunoassay following extraction of serum. J Clin Endocrinol

Metab 1973;37:247–253.

40 Merry BJ, Holehan AM: The endocrine response to dietary restriction in the rat; in Woodhead AD,

Blackett AD, Hollaender A (eds): Molecular Biology of Aging. New York, Plenum Press, 1985,

pp 117–141.

41 Roth GS, Handy AM, Mattison JA, Tilmont EM, Ingram DK, Lane MA: Effects of dietary caloric

restriction and aging on thyroid hormones of rhesus monkeys. Horm Metab Res 2002;34:378–382.

42 Bongaarts J: Does malnutrition affect fecundity? A summary of evidence. Science 1980;208:

564–569.

43 Roth GS, Blackman MR, Ingram DK, Lane MA, Ball SS, Cutler RG: Age-related changes

in androgen levels of rhesus monkeys subjected to diet restriction. Endocrine J 1993;1:

227–234.

44 Lane MA, Black A, Handy AM, Shapses SA, Tilmont EM, Kiefer TL, Ingram DK, Roth GS:

Energy restriction does not significantly alter bone mineral metabolism or reproductive cycling

and hormones in female rhesus monkeys. J Nutr 2001;131:820–827.

45 Black A, Lane MA: Nonhuman primate models of skeletal and reproductive aging. Gerontology

2002;48:72–80.

46 Wu J, Mattison J, Ottinger M, Zelinski-Wooten M, Handy A, Roth G, Ingram D, Tilmont E, Lane

M: Ovarian function and incidence of endometriosis in long-term calorie restriction. Geronto-

logist 2004;44:10.

47 Lujan ME, Krzemien AA, Reid RL, Van Vugt DA: Developing a model of nutritional amenorrhea

in rhesus monkeys. Endocrinol Online 2005;10:1210. http://endo.endojournals.org/cgi/rapidpdf/

en.2005–0821v1.


48 Weindruch R, Walford RL: Dietary restriction in mice beginning at 1 year of age: effect on life-

span and spontaneous cancer incidence. Science 1982;215:1415.

49 Jolly CA: Dietary restriction and immune function. J Nutr 2004;134:1853–1856.

50 Pahlavani M: Caloric restriction and immunosenescence: a current perspective. Front Biosci

2000;5:580–587.

51 Grossmann A, Rabinovitch PS, Lane MA, Jinneman JC, Ingram DK, Wolf NS, Cutler RG, Roth

GS: Influence of age, sex, and dietary restriction on intracellular free calcium responses of CD4�

lymphocytes in rhesus monkeys (Macaca mulatta). J Cell Physiol 1995;162:298–303.

52 Weindruch R, Lane MA, Ingram DK, Ershler WB, Roth GS: Dietary restriction in rhesus mon-

keys: lymphopenia and reduced mitogen-induced proliferation in peripheral blood mononuclear

cells. Aging Clin Exp Res 1997;9:304–308.

53 Roecker EB, Kemnitz JW, Ershler WB, Weindruch R: Reduced immune responses in rhesus mon-

keys subjected to dietary restriction. J Gerontol 1996;51A:276–279.

54 Nikolich-Zugich J, Messaoudi I: Mice and flies and monkeys too: caloric restriction rejuvenates

the aging immune system of non-human primates. Exp Gerontol 2005;40:884–893.

55 Mascarucci P, Taub D, Saccani S, Paloma MA, Dawson H, Roth GS, Ingram DK, Lane MA: Age-

related changes in cytokine production by leukocytes in rhesus monkeys. Aging Clin Exp Res

2001;13:85–94.

56 Mascarucci P, Taub D, Saccani S, Paloma MA, Dawson H, Roth GS, Lane MA, Ingram DK:

Cytokine responses in young and old rhesus monkeys: effect of caloric restriction. J Interferon

Cytokine Res 2002;22:565–571.

57 Harper JM, Galecki AT, Burke DT, Miller RA: Body weight, hormones, and T cell subsets as pre-

dictors of life span in genetically heterogeneous mice. Mech Ageing Dev 2004;125:381–390.

58 Kayo T, Allison DB, Weindruch R, Prolla TA: Influences of aging and caloric restriction on the

transcriptional profile of skeletal muscle from rhesus monkeys. Proc Natl Acad Sci

2001;98:5093–5098.


keys lowers oxidative damage in skeletal muscle. FASEB J 2000;14:1825–1836.

60 Salamone JD, Correa M: Motivational views of reinforcement: implications for understanding the

behavioral functions of nucleus accumbens dopamine. Behav Brain Res 2002;137:3–25.

61 Weed JL, Lane MA, Roth GS, Speer DL, Ingram DK: Activity measures in rhesus monkeys on

long-term calorie restriction. Phys Behav 1997;62:97–103.

62 Moscrip TD, Ingram DK, Lane MA, Roth GS, Weed JL: Locomotor activity in female rhesus

monkeys: assessment of age and calorie restriction effects. J Gerontol 2000;55:B373–B380.

63 Ramsey JJ, Roecker EB, Weindruch R, Baum ST, Kemnitz JW: Thermogenesis of adult male rhe-

sus monkeys: results through 66 months of dietary restriction. FASEB J 1996;10:A726.

64 Ramsey JJ, Roecker EB, Weindruch R, Kemnitz JW: Energy expenditure of adult male rhesus

monkeys during the first 30 months of dietary restriction. Am J Physiol 1997;272:E901–E907.

65 Duffy PH, Feuers RJ, Hart RW: Effect of chronic caloric restriction on the circadian regulation of

physiological and behavioral variables in old male B6C3F1 mice. Chronobiol Int 1990;7:291–303.

66 Ingram DK, Chefer S, Matochick J, Moscrip TD, Weed J, Roth GS, London ED, Lane MA: Aging

and caloric restriction in nonhuman primates: behavioral and in vivo brain imaging studies. Ann

NY Acad Sci 2001;928:316–326.

67 Matochick J, Chefer SI, Lane MA, Roth GS, Mattison JA, London ED, Ingram DK: Age-related

decline in striatal volume in rhesus monkeys: assessment of long-term calorie restriction.


68 Maswood N, Young J, Tilmont E, Zhang Z, Gash DM, Gerhardt GA, Grondin R, Roth GS,

Mattison J, Lane MA, Carson RE, Cohen RM, Mouton PR, Quigley C, Mattson MP, Ingram DK:

Caloric restriction increases neurotrophic factor levels and attenuates neurochemical and behav-

ioral deficits in a primate model of Parkinson’s disease. Proc Natl Acad Sci 2004;101:

18171–18176.

69 Allen AR, Starr A: Auditory brain stem potentials in monkey (M. mulatta) and man.

Electroencephalogr Clin Neurophysiol 1978;45:53–63.

70 Torre P III, Fowler CG: Age-related changes in auditory function of rhesus monkeys (Macaca

mulatta). Hear Res 2000;142:131–140.


71 Bito LZ, DeRousseau CJ, Kaufman PL, Bito JW: Age-dependent loss of accommodative ampli-

tude in rhesus monkeys: an animal model for presbyopia. Invest Ophthalmol Vis Sci 1982;23:

23–31.

72 Kaufman PL, Bito LZ, DeRousseau CJ: The development of presbyopia in primates. Trans

Ophthalmol Soc UK 1982;102:323–326.

73 Torre P III, Mattison JA, Fowler CG, Lane MA, Roth GS, Ingram DK: Assessment of auditory

function in rhesus monkeys (Macaca mulatta): effects of age and calorie restriction. Neurobiol

Aging 2004;25:945–954.

74 Brownell CL: Outer hair cell electromotility and otoacoustic emissions. Ear Hear 1990;11:89–92.

75 Fowler CG, Torre P III, Kemnitz JW: Effects of caloric restriction and aging on the auditory func-

tion of rhesus monkeys (Macaca mulatta): the University of Wisconsin study. Hear Res

2002;169:24–35.

76 Mattison JA, Croft MA, Dahl DB, Roth GS, Lane MA, Ingram DK, Kaufman PL: Acco-

mmodative function in rhesus monkeys: effects of aging and caloric restriction. Age 2005;27:

59–67.

Donald K. Ingram, PhD

Laboratory of Experimental Gerontology

National Institute on Aging, NIH, 5600 Nathan Shock Drive

Baltimore, MD 21224 (USA)




Caloric Intake and Alzheimer’s Disease

Experimental Approaches and Therapeutic Implications

Giulio Maria Pasinetti, Zhong Zhao, Weiping Qin, Lap Ho,

Yemul Shrishailam, Donal MacGrogan, Wendy Ressmann,

Nelson Humala, Xunxian Liu, Carmen Romero, Breton Stetka,

Linghong Chen, Hanna Ksiezak-Reding, Jun Wang

Neuroinflammation Research Laboratories, Department of Psychiatry, Mount Sinai

School of Medicine, New York, N.Y., and Bronx Veterans Affairs Medical Center,

Mount Sinai School of Medicine, Bronx, N.Y., USA

AbstractAlzheimer’s disease (AD) is a rapidly growing public health concern with potentially

devastating effects. Presently, there are no known cures or effective preventive strategies.

While genetic factors are relevant in early-onset cases, they appear to play less of a role in

late-onset sporadic AD cases, the most common form of AD. Due to the fact that the disease

typically strikes very late in life, delaying symptoms could be as good as a cure for many

people. For example, it is now widely accepted that if the onset of the disease could be

delayed by even 5 years, the incidence could be cut in half. Both clinical and epidemiological

evidence suggests that modification of lifestyle factors such as nutrition may prove crucial to

AD management given the mounting experimental evidence suggesting that brain cells are

remarkably responsive to ‘what somebody is doing’. Among other nongenetic factors influ-

encing AD, recent studies strongly support the evidence that caloric intake may play a role in

the relative risk for AD clinical dementia. Indeed, the effect of diet in AD has been an area of

research that has produced promising results, at least experimentally. Most importantly, as

mechanistic pathways are defined and their biochemical functions scrutinized, the evidence

supporting a direct link between nutrition and AD neuropathology continues to grow. Our

work, as well as that of others, has recently resulted in the development of experimental

dietary regimens that might promote, attenuate or even reverse features of AD. Most remark-

ably, while we found that high caloric intake based on saturated fat promotes AD type

�-amyloidosis, conversely we found that dietary restriction based on reduced carbohydrate

intake is able to prevent it. This evidence is very exciting and is, in part, consistent with cur-

rent epidemiological studies suggesting that obesity and diabetes are associated with a

�4-fold increased risk of developing AD. The clarification of the mechanisms through which

Pasinetti/Zhao/Qin/Ho/Shrishailam/MacGrogan/Ressmann/Humala/Liu/Romero/ 160

Stetka/Chen/Ksiezak-Reding/Wang

dietary restriction may beneficially influence AD neuropathology and the eventual discovery

of future ‘mimetics’ capable of anti-�-amyloidogenic activity will help in the development of

‘lifestyle therapeutic strategies’ in AD and possibly other neurodegenerative disorders.


The health-related risks associated with obesity are of great public health

concern in view of the fact that obesity may causally promote the incidence of a

number of chronic degenerative conditions. Some of these conditions, such as

Alzheimer’s disease (AD) and Parkinson’s disease, involve the nervous system

and are being increasingly linked to issues relating to nutrition. For example,

accumulating evidence indicates that certain nutrition-related issues, such as

diabetes [1–7], increasing caloric intake [8] or obesity [9, 10], may promote

neurodegeneration. In contrast, other nutritional factors, such as dietary restric-

tion [11–14] or consumption of fish oil [15, 16], may beneficially modulate

neurodegenerative disorders.

From an economic point of view, neurodegenerative disorders have the

potential to become major public health burdens as life expectancy increases.

An estimated 4.5 million people have AD in the USA, a number that has dou-

bled since 1980 and is expected to reach as much as 16 million by 2050 [17].

Most importantly, it has been calculated that even delaying the onset of AD for

a few years would decrease its prevalence and burden on public health systems

[18]. Obesity is also an important health issue when it comes to forecasting

future cash flows in the Social Security and Medicare systems. Not only does

the likelihood of having comorbidities rise with the degree of obesity, ‘but the

prevalence of having 2 or more health conditions’ has been found to increase

with weight status [19]. Moreover, in addition to the rather easily quantifiable

impact of obesity on morbidity and mortality, being overweight or obese fre-

quently compromises individuals’ quality of life [20].

Thus, it is of concern that at the beginning of the twenty-first century, the

fraction of Americans considered to be obese had reached ‘epidemic’ levels,

according to a study published in the Journal of the American Medical

Association [21]. This study, which was carried out between 1991 and 1998,

observed a steady increase in weight in all states of the union, in both sexes,

across age groups, races and educational levels, and occurred regardless of

smoking status. It found that obesity had increased from 12.0% in 1991 to

17.9% in 1998. Likewise, ‘national survey data show that between 1976–80 and

1988–94 the age-adjusted prevalence of obesity increased by 8 percentage

points, from 14.5 to 22.5%, in the US adult population ages 20–74’ [22]. This

translates into increases in mean body mass index (BMI) and in the prevalence

of overweight and obesity for US adults and children.

Dietary Restriction and Alzheimer’s Disease Neuropathology 161

In an effort to establish a basis to define what a ‘healthy weight’ is, the

Dietary Guidelines for Americans [6th ed., 2005; http://www.healthierus.gov/

dietaryguidelines/] addresses issues of weight maintenance and weight loss by

making long-term changes in physical activity and eating behavior. The health

consequences of overweight and obesity are related to adverse health conditions

such as diabetes, coronary heart disease and hypertension. One glaring fact

about the recommended range of healthy weights in the Dietary Guidelines is

that close to more than half of the adult population, in particular half of adult

males, have been above that range at least since 1960. A discussion of the pros

and cons of this latest edition shows how complex and elusive the subject of

optimal weight can be [23]. Of interest is that leptin, a hormone important in

energy homeostasis and food intake regulation, has been singled out in the

guidelines as a metabolic indicator influencing food intake. Because levels of

leptin rise when fat stores are high, leptin may play a role in public health mon-

itoring of adiposity in the future [24].

Of particular interest to this review article is the accumulating evidence

pointing to a relationship between obesity and dementia later in life. For exam-

ple, Whitmer et al. [25] have recently reported an analysis of prospective data

from a multiethnic population-based cohort obtained with the objective of eval-

uating a possible association between obesity in middle age, as measured by

BMI and skinfold thickness, and risk of dementia later in life. Dementia was

diagnosed in 713 (6.9%; in a cohort of 10,276 people) of the participants.

Obese people (BMI �30) had a 35% greater risk of dementia compared with

those of normal weight (BMI 18.6–24.9). The authors concluded that obesity in

middle age increases the risk of future dementia independently of comorbid

conditions. This evidence is very interesting especially in view of the recent

studies suggesting that certain cardiovascular risk factors (e.g. diet) may be sig-

nificant contributors to an increased risk of vascular-related dementia [1]. A

large body of evidence indicates that cardiovascular risk factors, e.g. certain

dietary ones, may also increase the relative risk of AD and clinical dementia

even when vascular dementia cases are excluded from the analysis [26, 27].

Thus, it may be the case that additional ‘nonvascular’ events associated with

certain cardiovascular risk factors may be involved in the increased risk for AD.

Most interestingly, we also note that recent evidence suggests that type 2 dia-

betes may also be associated with an increased risk of developing AD and may

affect cognitive systems differentially [2]. Thus, it is possible that potential risk

factors associated with certain dietary regimens accepted as cardiovascular risk

factors for vascular dementia may also independently contribute to the develop-

ment and progression of AD. Further exploration of this phenomenon in AD

and AD model systems will provide critical direction for future studies investi-

gating mechanisms involved in the potentiation of AD neuropathology and



possibly future therapeutic applications based on dietary modifications.

However, as discussed below, dietary regimens including dietary restriction

(DR) and weight reduction programs in neurodegenerative disorders such as

AD can be a complex endeavor because they should be made on the basis of

combined evidence from different sources such as (1) epidemiological studies,

(2) experimental models and ultimately (and most importantly) (3) from con-

trolled clinical studies.

Based on these considerations, this review article will first discuss recent

evidence indicating (1) the beneficial role of dietary regimens in health and dis-

ease and (2) recent experimental evidence suggesting that diet and possibly the

control of caloric intake may beneficially influence AD and possibly other neu-

rodegenerative disorders. Finally, based on this evidence, this review will iden-

tify potential therapeutic scenarios for eventual future interventions.

Dietary Implications in the Onset and Progression of

Clinical Alzheimer’s Disease

The possibility that reactive oxygen species are a factor in the neuronal

damage seen in AD has led to examine how antioxidants in foods, or as vitamin

supplements in the form of tocopherol (vitamin E), ascorbic acid (vitamin C)

and carotenes, can affect AD. Although the results appear promising in some

cases, the data on the value of antioxidant supplementation remain inconclu-

sive. Likewise, deficiencies in folate, vitamin B12 and vitamin B6 lead to high

concentrations of homocysteine in the brain through different pathways. This

has led to study the possible link between homocysteine and the development of

AD and Parkinson’s disease [28]. Although epidemiological studies have impli-

cated high concentrations of homocysteine in the brain in neuronal degenera-

tion [11, 28], the data relating folate and vitamin B12 and B6 supplementation as

palliatives to the cognitive decline seen in AD are inconsistent [28].

However, Lim et al. [15] have recently used a transgenic mouse model of

AD-type neuropathology to evaluate the impact of n–3 fatty acid docosa-

hexaenoic acid (DHA) in AD-type amyloid neuropathology. They found that

DHA-enriched diets significantly reduced total AD-type amyloid neuropathol-

ogy by �70% when compared with low-DHA or control chow diets. Dietary

DHA also decreased �-amyloid (A�) 1–42 levels below those seen with control

chow. The results suggest that DHA could be protective against AD-type amy-

loid deposition in the brain and eventually prevent downstream neurodegenera-

tive conditions.

Studies relating fat and fish intake to the risk for AD and cognitive decline

have failed to establish a firm causal relationship. Despite the fact that there are


no controlled clinical studies to support dietary recommendations, in an indi-

rect way, a diet low in saturated and trans-fatty acids and high in monounsatu-

rated, polyunsaturated and fish-related fats can be assumed to be beneficial in

the prevention of cognitive decline and AD by means of promoting a viable vas-

cular system [29]. Furthermore, dietary fats may also influence AD through

lipid metabolism, insulin resistance [30], high concentrations of circulating

insulin [3], oxidation [31] and the amyloid cascade hypothesis [32, 33]. In addi-

tion, APOE, the gene associated with sporadic AD, is related to lipid metabo-

lism and modulates cholesterol concentrations in response to intake from fats

[34, 35]. People with the APOE �4 allele, who are at high risk of AD, normally

have high levels of cholesterol in the blood.

In addition to nutrients, alcohol intake has also been suggested as a risk

factor for AD. This is supported by evidence that ethanol consumption might

lead to oxidative brain damage in rat models [36]. Paradoxically, accumulating

epidemiological evidence indicates that moderate consumption of alcohol in the

form of red wine may actually lower the risk of cerebrovascular disease in older

adults [37]. A study by Goldberg et al. [38] sponsored by the American Heart

Association has found that consumption of alcohol-containing beverages (e.g.

wine) actually has cardiovascular and cognitive benefits. However, numerous

other studies relating alcohol to AD have yielded mixed results. A case has been

made for the elderly to consume red wine in moderate quantities because of its

high contents of antioxidants, such as flavonoids, which may not be found in

other alcoholic beverages [4, 39, 40]. But, in and of itself, alcohol consumption

carries the potential for abuse and addiction.

Observational studies on diet and disease may incur errors in the measure-

ment of nutrients, a caveat that has been clarified by Luchsinger and Mayeux

[4]. If the measurement error is not related to outcome, it will underestimate

true associations. Also, the long latency period of AD may be the consequence

of lifelong exposure to a number of factors that are difficult to isolate and ana-

lyze in their true context. Moreover, the validity of basing clinical decisions

about individual patients on data from randomized trials remains to be settled

[41]. So far, it appears unlikely that trials can address all questions regarding

diet and AD, given the nature of AD as a chronic disease with a latency period.

It would be difficult to conduct trials long and large enough to observe results.

Luchsinger and Mayeux [4] also touched on the concept that nutritional

supplements alone (e.g. carotenoids) may not be as effective as the whole foods

in which they may be found (fruits and vegetables) such that the interactions of

nutrients within foods, or patterns of diet, is what may actually be of benefit.

Recent findings by Gardner et al. [41] appear to corroborate their viewpoint.

According to their findings, plant-based diets may be superior to low-fat diets even

if the two diets are identical in total fat, protein, carbohydrate and cholesterol



content. These authors noted that national dietary guidelines have probably

underestimated the potential low-density lipoprotein cholesterol-lowering

effect of certain diets. In a randomized clinical trial, they set out to contrast

plasma lipid responses to two low-fat diet patterns. They found that a plant-

based low-fat diet reduces levels of low-density lipoprotein twice as much as a

low-fat diet based on prepackaged foods. Such plant-based diets may provide

an effective alternative to cholesterol-lowering drugs like statins [41].

Although there is enough evidence suggesting that dietary modification,

such as low calorie intake, may prevent AD and other age-related neurodegen-

erative disorders, malnutrition in the elderly remains a concern. Hence, dietary

recommendations may need to be made on the basis of comorbidities such as

type 2 diabetes, cardiovascular disease and osteoarthritis.

Alzheimer’s Disease and Nutrition

While genetic factors are highly relevant in early-onset AD cases, their sig-

nificance diminishes in late-onset sporadic AD cases, the most common form

of AD [17]. Nongenetic factors, including modifiable lifestyle dietary regi-

mens, are receiving great attention in AD, especially because of recent epidemi-

ological studies indicating that caloric intake may influence the relative risk for

AD clinical dementia. Dietary factors have been an area of research that has

produced promising results, at least experimentally. Most importantly, the evi-

dence supporting a direct link between nutrition and AD amyloid neuropathol-

ogy discussed below [12, 13] continues to grow, as the mechanistic pathways

are defined and their biochemical functions scrutinized.

AD is a progressive neurodegenerative disorder marked by loss of mem-

ory, cognition and behavioral stability [17]. AD is defined pathologically by

extracellular neuritic plaques comprised of fibrillar deposits of �-amyloid (A�)

and neurofibrillary tangles comprised of paired helical filaments of hyperphos-

phorylated tau. Current therapies for AD, such as cholinesterase inhibitors, treat

the symptoms but do not modify the progression of the disease. The etiology of

AD is unclear, and data from familial AD mutations strongly support the ‘amy-

loid cascade hypothesis’ of AD, i.e. that neurodegeneration in AD is initiated by

the formation of neurotoxic A� aggregates, and all familial AD mutations

increase levels of A� peptide or the density of A� deposits [17].

Current therapeutic strategies to treat AD are aimed at preventing the for-

mation of amyloidogenic A� peptides [17]. For this reason, the ‘amyloidogenic’

�- and �-secretase activities necessary for the generation of amyloidogenic A�

peptides have become central targets for development of therapeutic reagents in

AD [17]. However, it has been difficult to find safe, selective �- and �-secretase


inhibitors, mainly because of the influence of these inhibitors on other cellular

substrates whose processing is vital [17]. Ongoing studies, discussed below, in

our laboratory suggest that DR regimens based on low-carbohydrate content

may beneficially influence AD-type neuropathology through the promotion of

‘nonamyloidogenic’processing of amyloid precursor protein (APP) via the promo-

tion of �-secretase activities. In addition, since the �-secretase cleavage of APP is

known to involve the release of a soluble neuroprotective form of APP (sAPP-�;

also found in our studies), it is possible that DR, while promoting the nonamy-

loidogenic pathway in the brain, may also promote brain repair activities as a con-

sequence of sAPP-� neurotrophic function [42].

As discussed below, there is increasing consensus that the production and

accumulation of A� peptides is central to the pathogenesis of AD. The continual

search for ways to manage if not reverse AD neuropathology has led to ongoing

efforts to elucidate its underlying causes and possible treatments. The likely

link between A� peptide aggregation and AD pathology emphasizes the need

for a better understanding of the mechanisms through which dietary regimens

may influence A� production.

Diabetogenic Dietary Regimens Resulting in Insulin

Resistance Coincide with Promotion of Alzheimer’s

Disease Amyloid Neuropathology

Abnormalities in insulin metabolism associated with type 2 diabetes resis-

tance are among the central factors thought to influence the onset of AD by

promoting the synthesis and/or interference of A� degradation [5, 6, 43–45].

For example, there is in vitro evidence indicating that insulin itself may signifi-

cantly promote the generation of extracellular amyloidogenic A� peptides

through mechanisms that involve the acceleration of APP/A� trafficking from

the trans-Golgi network, a major cellular site for A� generation, to the plasma

membrane [43]. While this evidence tentatively suggests that abnormal carbo-

hydrate metabolism might play an important role in AD through mechanisms

that involve A� peptide generation, experimental studies also suggest that

insulin resistance may promote AD amyloid neuropathology in the Tg25876

mouse model of AD amyloid neuropathology, possibly by limiting A� degrada-

tion via competition with insulin for degradation by insulin-degrading enzyme

(IDE) [46], a zinc-metallopeptidase that preferentially cleaves proteins with a

propensity to form �-pleated sheet-rich amyloid fibrils [47], such as

monomeric A� peptides [46].

While the role of insulin in AD has received major attention with respect to

its potential role in amyloid neuropathology, recent evidence also suggests a role



for insulin in normal memory function, supporting the hypothesis that insulin

affects many mechanisms related to neuronal activity and cognitive function by

itself. Of interest to us is the fact that chronic hyperinsulinemia and insulin resis-

tance, or reduced insulin effectiveness, may exert a negative influence on mem-

ory [5]. For example, Hoyer [44] proposed that low concentrations in circulating

insulin in the central nervous system, together with reduced expression of IR and

subsequent altered downstream signaling AD, would ultimately lead to reduced

levels of acetylcholine with a corresponding decrease in cerebral blood flow.

Based on this evidence and the fact that type 2 diabetes appears to be asso-

ciated with an increased relative risk for AD [5, 6, 44, 45], we have recently

explored the role of experimental type 2 diabetes in the Tg2576 AD mouse

model [6]. We found that a diabetogenic diet resulting in elevated circulating

levels of insulin coincided with promoted amyloidogenic A�1–40 and A�1–42

peptide generation and amyloid plaque burden in the brain of Tg2576 mice that

corresponded with increased �-secretase activities and decreased IDE activi-

ties. Moreover, the increased AD-type amyloid neuropathology also coincided

with increased and impaired spatial memory functions assessed by performance

in a spatial water maze task [6]. Further exploration of the apparent interrela-

tionship of insulin resistance to brain amyloidosis revealed a functional

decrease in IR-mediated signal transduction in the brain, as suggested by

decreased IR �-subunit (IR-�) Y1162/1163 autophosphorylation and reduced

phosphatidylinositol 3-kinase/pS473-AKT/protein kinase B in these same brain

regions [6]. Results from this study strongly suggested that one mechanism

through which diet-induced insulin resistance in Tg2576 mice can significantly

promote AD-type amyloidosis in the brain is by reducing IR signaling, resulting

in elevation of �-secretase activities. The studies also suggested that type 2 dia-

betes may further contribute to AD amyloid neuropathology attenuating degra-

dation of A� peptides through pathways associated with IDEs (fig. 1).

Collectively, these findings indicate that clinical AD is a result of early life

as well as later life risk factors, and that genetic predisposition to the disease

may modify the constellation of predictors.

Dietary Restriction Based on a Low-Carbohydrate Diet

As discussed above, a fundamental problem of AD neuropathology is the

aberrant generation of amyloidogenic A� amyloid peptides in the brain that

lead to an abnormal deposition of the neuritic plaques that are a landmark in

AD. Although evidence supports a potential neuroprotective role for DR in

neurodegeneration, until recently there was no information as to whether

reduced caloric intake could attenuate AD neuropathology. Findings of recent


prospective studies indicate that increasing caloric intake may be a risk factor

for AD [45, 48, 49]. Because of this evidence and the epidemiological evidence

indicating that DR may influence the risk for AD [8, 45], we have continued to

explore if a clinically applicable weight reduction/DR regimen based on an

approximately 30% reduced carbohydrate content could (1) attenuate AD

neuropathology and (2) decrease preexisting amyloid neuritic neuropathology

(e.g. a reduction in plaque size), eventually resulting in recovery of amyloid-

associated neuritic dystrophy as a function of time in the same strain of Tg2576

mice fed a low-carbohydrate/DR diet. Based on these considerations, we tested

the hypothesis that low-carbohydrate/DR may be a beneficial intervention in

AD through mechanisms that prevent A� generation and neuritic plaque depo-

sition in the brain using a mouse model of AD type amyloidosis [50]. The aim

of these studies was to test the hypothesis that DR may beneficially influence

AD through mechanisms that prevent the development of amyloidosis associ-

ated with AD.

To test this hypothesis, 3-month-old Tg2576 mice, which develop AD type

amyloid neuropathology by 8–10 months of age [50], were fed for 9 months

with a daily low-carbohydrate diet resulting in a 30% lower caloric intake com-

pared to that consumed by age- and gender-matched control Tg2576 mice fed

ad libitum (AL) with a standard laboratory rodent diet. Nutrient composition in

the DR diet was achieved by selectively reducing the carbohydrate content of

the diet while consumption of protein, fat, cholesterol, vitamins and minerals

was identical to that of AL fed Tg2576 mice [12]. This dietary regimen resulted

in body weight stabilization over the 9-month study period among DR Tg2576

High-fat diet

Insulin

resistance

IDE

APP

A�

Amyloid plaque

deposition

Degradation

�-Secretase

Fig. 1. Role of insulin resistance in AD-type neuropathology.



mice relative to the AL fed group, coinciding with an approximately 3-fold

lower ependymal fat pad weight and improved glucose tolerance response as

determined by an intraperitoneal glucose tolerance test. These physiological

adaptations in the DR Tg2576 mice relative to AL fed controls are consistent

with clinical evidence that low-carbohydrate DR considerably improves abnor-

mal glucose control and obesity [9, 51–53], which are risk factors not only for

diabetes but also for AD [7, 54].

Weight Reduction Dietary Restriction Results in Decreased

Alzheimer’s Disease-Type Amyloid Neuropathology

in Tg2576 Mice

When Tg2576 mice were examined for AD-type neuropathology at 12

months of age, we found that 9-month DR treatment almost completely pre-

vented cortical and hippocampal AD-type amyloid plaque development [12]

relative to animals in the AL fed group. Consistent with this evidence, we noted

commensurately lower concentrations of amyloidogenic A�1–40 and A�1–42 pep-

tides in the neocortex and hippocampus as evaluated by ELISA assay, relative

to AL fed controls [12]. No detectable change in total full-length APP level was

noted in either brain region of DR versus AL fed Tg2576 mice [12].

To further evaluate the antiamyloidogenic role of DR in the brain of

Tg2576 mice, we explored APP processing and A� peptide generation using

immunoprecipitation (IP) and mass spectrometry (MS). Consistent with the

aforementioned ELISA evidence, using 4G8 antibody for A� IP, we confirmed

decreased levels of A�1–40 and A�1–42 in the same neocortical samples we used

for the A� ELISA assay [12]. In addition, a relative proportional reduction in

A�1–37, A�1–38 and A�1–39 peptide content was also observed in the neocortex of

the DR group compared to the AL fed control group. This evidence, together

with our observation that the concentration of the approximately 7-kDa car-

boxy-terminal fragment (CTF) � cleavage product of APP, an index of �-secre-

tase activity, was unchanged in the neocortex of the DR group relative to AL fed

controls, suggested the possibility that �-secretase activity was not involved in

the DR-associated antiamyloidogenic activity.

To further identify A� carboxy-terminal peptide fragments that would

have been otherwise undetected in the 4G8 IP-MS studies, we used 6E10 anti-

body in additional A� IP-MS studies. Consistent with the 4G8 IP-MS spectra,

we noted decreased levels of A�1–40 and A�1–42 as well as A�1–37, A�1–38 and

A�1–39 peptide in the DR group relative to AL fed control animals. In addition,

we found a major elevation in A�1–16 peptide fragment concentration in the

neocortex of the DR group that was not detected in the AL fed controls.


Because �-secretase can cleave APP, eventually resulting in the generation of

A� CTFs ending at the AA residue leucine 16 of A� [42], we continued to

explore the role of DR in �-secretase activity in the brain.

Cleavage of APP by �-secretase releases the amino-terminal extracellular

domain known as sAPP-� domain coincidental with elevation in membrane-

bound �-secretase-cleaved APP CTF-�. We therefore explored the regulation of

sAPP-� and CTF-� cleavage products of APP in the brain as indices of �-secretase

activity in response to DR. Interestingly, we found that DR in Tg2576 mice

resulted in a �2-fold elevation in the concentration of neocortical sAPP-� and

membrane-associated CTF-� relative to AL fed control Tg2576 mice. The

increase in CTF-� was somewhat less, about 1.6-fold, presumably because of

further cleavage of CTF-� by �-secretase. Compared with the CTF-� fragment,

the abundance of CTF-� signal was at the limit of detection in the neocortex of

both DR and AL fed Tg2576 mice, preventing reliable quantification [12].

Weight Reduction Dietary Restriction Diet May Influence

��-Secretase Activity in the Brain in Part by Selectively

Promoting the Generation of Mature and Catalytically

Active ADAM10 Species

In light of recent evidence indicating that the proteinase ADAM10 (a dis-

integrin and metalloproteinase) may act as an �-secretase [42], we continued to

explore the regulation of ADAM10 expression in the brains of Tg2576 mice in

response to DR, relative to AL fed controls. Both mature (62-kDa) and proform

(90-kDa) ADAM10 species were detected in the neocortex of the AL fed con-

trol animals, confirming previous evidence [12]. The 62-kDa mature ADAM10

protein species is known to act as an �-secretase in vitro and to cleave

A�-derived peptides at leucine 16 [12, 42]. Excitingly, we found that the DR

diet regimen resulted in a 30% elevation of neocortical mature ADAM10

species concentration, coinciding with a commensurate elevation in neocortical

�-secretase activity, compared to AL fed control mice.

As shown in figure 2, this evidence supports the hypothesis that low-

carbohydrate DR may prevent AD-type amyloid neuropathology through mecha-

nisms that influence �-secretase activity in the brain, possibly by promoting the

generation of mature, catalytically active ADAM10 species. Since �-secretase pro-

teolysis of the APP sequence within the A� peptide would preclude the generation

of amyloidogenic A� peptides, our studies suggest that DR may provide an attrac-

tive antiamyloidogenic strategy by promoting �-secretase activity in the brain.

In addition to promoting �-secretase activity, we found that DR led to a

small, but significant elevation of IDE content in the brain of Tg2576 mice. The



role of IDE in A� degradation was demonstrated by recent studies showing that

mice deficient for IDE exhibit increased cerebral accumulation of endogenous

A� peptides. Thus, it is possible that the attenuation of A� burden in the brain

of DR Tg2576 mice might also derive from enhanced IDE-mediated clearance

of A� peptides in addition to the promotion of the nonamyloidogenic �-secretase

cleavage of APP [5, 12, 46]. In view of a recent study from Patel et al. [13]

reporting the A�-lowering efficacy of a DR diet in additional mouse models of

AD amyloid neuropathology, it is likely that the beneficial effect of a ‘low-

carbohydrate/DR’ diet on A� neuropathology and cognitive function in the

Tg2576 AD mouse model [12] may reflect the impact of DR, per se. However,

it is possible that the low carbohydrate content in the ‘low-carbohydrate/DR’

dietary regimen may promote additional disease-modifying activities.

As discussed above, current strategies to treat AD are aimed at preventing

formation of amyloidogenic A� peptides. Therefore, �- and �-secretases that

generate A� peptides by sequential cleavage of the APP or degrade released A�

peptides are obvious and central targets for the development of therapeutic

APP

�-,�-SeDRetase

�-SeDRetase

A�

Amyloidosis

Degradation

DR

Proform

ADAM10

Mature

ADAM10

?

Fig. 2. Role of DR in the prevention of AD-type amyloid neuropathology.


reagents. Our evidence showing that DR may positively influence �-secretase,

possibly through mechanisms that may involve the generation of mature, catalyt-

ically active ADAM10 species in the brain, might prove in the future the basis of

potential novel preventative measure aimed at delaying the onset of AD neu-

ropathology. In addition, since �-secretase cleavage of APP releases sAPP-�,

which is well known for its neuroprotective properties, our study tentatively sug-

gests that promoting a low-carbohydrate DR dietary regimen may also result in

increased brain repair activities as a consequence of sAPP-� neurotrophic func-

tion. However, we cannot exclude the possibility that DR might also influence

other mechanisms, eventually resulting in decreased amyloid deposition in the

brain by promoting �-site cleavage of APP or degradation of released A� by

other proteases such as plasmin and neprilysin, respectively. In addition to pro-

moting A�-lowering activity, DR may also benefit AD through mechanisms not

directly related to generation and/or degradation of A� of peptides. In particular,

DR is known to reduce inflammation [55] and oxidative stress [56], two of the

major contributing factors in AD-type neurodegeneration [57, 58]. Therefore, it

would not be unexpected that DR may beneficially modulate the onset and/or

progression of neuropathology and neurodegeneration in AD through multiple

mechanisms. Thus, the relationship between caloric intake and AD could have

important implications in the prevention and/or therapy of AD [17].

DR is well known to improve insulin sensitivity responses, especially in

insulin resistance conditions such as type 2 diabetes [59, 60]. Based on the

observation that diet-induced insulin resistance promotes the generation of A�

peptides, it would not be unexpected that A�-lowering activity of DR may be

related to promotion of insulin sensitivity responses. However, evidence indi-

cates that insulin resistance and DR may have independent impacts on A� gen-

eration and that diabetogenic and DR diet appears to activate independent signal

transduction pathways ultimately influencing APP processing and generation of

A� peptides [6, 12]. While a diabetogenic diet induces A� generation by promo-

tion of the AKT-GSK pathway [6], ongoing studies showed that DR may reduce

A� generation by activating �-secretase activity (perhaps activation of

ADAM10 activity), in part, via promotion of the MAPK-PKC signaling path-

way. Further studies in our laboratory are presently aiming to better understand

whether DR in obese-diabetic Tg2576 mice may reverse AD-type amyloidogenic

activities via modulation of these specific signal transduction pathways.

Conclusion

Study findings support existing epidemiological evidence indicating that

caloric intake is positively associated with the increased incidence of AD and



raises the possibility that changes in dietary regimens may be used in future

preventative measures aimed at delaying the onset of AD amyloid neuropathol-

ogy. Investigations in experimental mouse models of AD neuropathology, such

as ours, are of great potential benefit in terms of public health because they pro-

vide insights into possible interventions to prevent or ameliorate conditions

associated with those over 65 years of age in the USA. This is the age group

with the highest incidence of excess weight, obesity and diabetes, and it is the

largest group associated with the highest risk to develop AD dementia.

We want to point out, however, that decisions on diet recommendations in

AD can be a complex endeavor because they should be made on the basis of

combined evidence from different sources such as (1) experimental models, (2)

prospective epidemiological studies and ultimately (3) controlled clinical stud-

ies. While we believe that the ultimate evidence to support such recommenda-

tions should come from controlled clinical trial studies, we are also aware of the

potential limitation of this approach. For example, we point out that, in view of

the chronic nature of AD dementia with a relatively long latency period, it may

be difficult to execute appropriate clinical studies for enough time and in large

enough samples to draw accurate and repeatable conclusions.

However, despite these limitations, we believe the recent prospective stud-

ies showing that increased caloric intake is a risk for AD [8] and the recent

observation that that DR [12, 13] in AD mouse models may beneficially influ-

ence AD neuropathology provide strong impetus to ascertain the validity of a

DR diet in AD patients.

Acknowledgments

This study is supported by the Dr. Robert C. Atkins Foundation, the Dana Foundation

for Brain Research Initiative and the NIH AG14766, AG02219 and NCCAM AT002602 to

G.M.P. We thank Ms. Isabela Diaconescu for editorial revision of the manuscript.

References

1 Kuusisto J, Koivisto K, Mykkanen L, Helkala EL, Vanhanen M, Hanninen T, Kervinen K,

Kesaniemi YA, Riekkinen PJ, Laakso M: Association between features of the insulin resistance

syndrome and Alzheimer’s disease independently of apolipoprotein E4 phenotype: cross-sectional

population based study. BMJ 1997;315:1045–1049.

2 Franco KS, Bronson D: Diabetes mellitus and Alzheimer’s disease. Arch Neurol 2005;62:330.

3 Luchsinger JA, Tang MX, Shea S, Mayeux R: Hyperinsulinemia and risk of Alzheimer’s disease.

Neurology 2004;63:1187–1192.

4 Luchsinger JA, Mayeux R: Dietary factors and Alzheimer’s disease. Lancet Neurol 2004;3:579–587.

5 Craft S, Watson GS: Insulin and neurodegenerative disease: shared and specific mechanisms.

Lancet Neurol 2004;3:169–178.


6 Ho L, Qin W, Pompl PN, Xiang Z, Wang J, Zhao Z, Peng Y, Cambareri G, Rocher A, Mobbs CV,

Hof PR, Pasinetti GM: Diet-induced insulin resistance promotes amyloidosis in a transgenic

mouse model of Alzheimer’s disease. FASEB J 2004;18:902–904.

7 Ristow M: Neurodegenerative disorders associated with diabetes mellitus. J Mol Med 2004;82:

510–529.

8 Luchsinger JA, Tang MX, Shea S, Mayeux R: Caloric intake and the risk of Alzheimer’s disease.

Arch Neurol 2002;59:1258–1263.

9 Gustafson D, Rothenberg E, Blennow K, Steen B, Skoog I: An 18-year follow-up of overweight

and risk of Alzheimer’s disease. Arch Intern Med 2003;163:1524–1528.

10 Sriram K, Benkovic SA, Miller DB, O’Callaghan JP: Obesity exacerbates chemically induced

neurodegeneration. Neuroscience 2002;115:1335–1346.

11 Mattson MP: Will caloric restriction and folate protect against AD and PD? Neurology

2003;60:690–695.

12 Wang J, Ho L, Qin W, Rocher AB, Seror I, Humala N, Maniar K, Dolios G, Wang R, Hof PR,

Pasinetti GM: Caloric restriction attenuates �-amyloid neuropathology in a mouse model of

Alzheimer’s disease. FASEB J 2005;19:659–661.

13 Patel NV, Gordon MN, Connor KE, Good RA, Engelman RW, Mason J, Morgan DG, Morgan TE,

Finch CE: Caloric restriction attenuates A�-deposition in Alzheimer transgenic models. Neurobiol

Aging 2005;26:995–1000.

14 Maswood N, Young J, Tilmont E, Zhang Z, Gash DM, Gerhardt GA, Grondin R, Roth GS,

Mattison J, Lane MA, Carson RE, Cohen RM, Mouton PR, Quigley C, Mattson MP, Ingram DK:

Caloric restriction increases neurotrophic factor levels and attenuates neurochemical and behav-

ioral deficits in a primate model of Parkinson’s disease. Proc Natl Acad Sci USA 2004;101:

18171–18176.

15 Lim GP, Calon F, Morihara T, Yang F, Teter B, Ubeda O, Salem N Jr, Frautschy SA, Cole GM: A

diet enriched with omega-3 fatty acid docosahexaenoic acid reduces amyloid brain burden in an

aged Alzheimer mouse model. J Neurosci 2005;25:3032–3040.

16 Puri BK, Leavitt BR, Hayden MR, Ross CA, Rosenblatt A, Greenamyre JT, Hersch S, Vaddadi KS,

Sword A, Horrobin DF, Manku M, Murck K: Ethyl-EPA in Huntington disease: a double-blind,

randomized, placebo-controlled trial. Neurology 2005;65:286–292.

17 Cummings JL: Alzheimer’s disease. N Engl J Med 2004;351:56–67.

18 Brookmeyer R, Gray S, Kawas S: Projections of Alzheimer’s disease in the United States and the

public health impact of delaying disease onset. Am J Public Health 1998;88:1337–1342.

19 Must A, Spadano J, Coakley EH, Field AE, Colditz G, Dietz WH: The disease burden associated

with overweight and obesity. JAMA 1999;282:1523–1529.

20 Katz DA, McHorney CA, Atkinson RL: Impact of obesity on health-related quality of life in

patients with chronic illness. J Gen Intern Med 2000;15:789–796.

21 Mokdad AH, Serdula MK, Dietz WH, Bowman BA, Marks JS, Koplan JP: The spread of obesity

epidemic in the United States, 1991–1998. JAMA 1994;282:1519–1522.

22 Flegal KM, Troiano RP: Changes in the distribution of body mass index of adults and children in

the US population. Int J Obes Relat Metab Disord 2000;24:807–818.

23 Flegal KM, Troiano RP, Ballard-Barbash R: Aim for a healthy weight: what is the target? J Nutr

2001;131:440S–450S.

24 Auwerx J, Staels B: Leptin. Lancet 1998;351:737–742.

25 Whitmer RA, Gunderson EP, Barrett-Connor E, Quesenberry CP Jr, Yaffe K: Obesity in middle

age and future risk of dementia: a 27-year longitudinal population based study. BMJ

2005;330:1360.

26 Petrovitch H, White LR, Izmirilian G, Ross GW, Havlik RJ, Markesbery W, Nelson J, Davis DG,

Hardman J, Foley DJ, Launer LJ: Midlife blood pressure and neuritic plaques, neurofibrillary tan-

gles, and brain weight at death: the HAAS, Honolulu-Asia Aging Study. Neurobiol Aging

2000;21:57–62.

27 Skoog I, Kalaria RN, Breteler MM: Vascular factors and Alzheimer’s disease. Alzheimer Dis

Assoc Disord 1999;13:S106–114.

28 Seshadri S, Wolf PA: Homocysteine and the brain: vascular risk factor or neurotoxin? Lancet

Neurol 2003;2:11.



29 Hu FB, Willet WC: Optimal diets for prevention of coronary heart disease. JAMA 2002;288:

2569–2578.

30 Bray GA, Lovejoy JC, Smith SR, DeLany JP, Lefevre M, Hwang D, Ryan DH, York DA: The influ-

ence of different fats and fatty acids on obesity, insulin resistance, and inflammation. J Nutr

2002;132:2488–2491.

31 Cohn JS: Oxidized fat in the diet, postprandial lipaemia and cardiovascular disease. Curr Opin

Lipidol 2002;13:19–24.

32 Hardy JA, Higgins GA: Alzheimer’s disease: the amyloid cascade hypothesis. Science

1992;256:184–185.

33 Sparks DL, Martins R, Martin T: Cholesterol and cognition: rationale for the cholesterol-lowering

treatment trial and sex related differences in beta-amyloid accumulation in the brains of sponta-

neously hypercholesterolemic Watanabe rabbits. Ann NY Acad Sci 2002;977:356–366.

34 Berglund L: The APOE gene and diets – food (and drink) for thought. Am J Clin Nutr 2001;73:

669–670.

35 Petot GJ, Traore F, Debanne SM, Lerner AJ, Smyth KA, Friedland RP: Interactions of apolipopro-

tein E genotype and dietary fat intake of healthy older persons during mid-adult life. Metabolism

2003;52:279–281.

36 Agar E, Demir S, Amanvermez R, Bosnak M, Ayyildiz M, Celik C: The effects of ethanol con-

sumption on the lipid peroxidation and glutathione levels in the right and left brains of rats. Int J

Neurosci 2003;113:1643–1652.

37 Mukamal KJ, Longstreth WT Jr, Mittleman MA, Crum RM, Siscovivk DS: Alcohol consumption

and subclinical findings on magnetic resonance imaging of the brain in older adults: the cardio-

vascular health study. Stroke 2001;32:1939–1946.

38 Goldberg IJ, Mosca L, Piano MR, Fisher EA: Wine and your heart: a science advisory for health-

care professionals from the nutrition committee, council on epidemiology and prevention, and

council on cardiovascular nursing of the American Heart Association. Circulation 2001;103:

472–475.

39 Mukamal KJ, Kuller LJ, Fitzpatrick AL, Longstreth WT Jr, Mittleman MA, Siscovick DS:

Prospective study of alcohol consumption and risk of dementia in older adults. JAMA 2003;289:

1405–1413.

40 Galanis DJ, Joseph C, Masaki KH, Petrovitch H, Ross GW, White L: A longitudinal study of

drinking and cognitive performance in elderly Japanese American men: the Honolulu-Asia Aging

Study. Am J Public Health 2000;90:1254–1259.

41 Gardner CD, Coulston A, Chatterjee L, Rigby A, Spiller G, Farquhar JW: The effect of a plant-

based diet on plasma lipids in hypercholesterolemic adults. Ann Intern Med 2005;142:725–733.

42 Postina R, Schroeder A, Dewachter I, Bohl J, Schmitt U, Kojro E, Prinzen C, Endres K, Hiemke C,

Blessing M, Flamez P, Dequenne A, Godaux E, van Leuven F, Fahrenholz F: A disintegrin-

metalloproteinase prevents amyloid plaque formation and hippocampal defects in an Alzheimer

disease mouse model. J Clin Invest 2004;113:1456–1464.

43 Gasparini L, Gouras GK, Wang R, Gross RS, Beal MF, Greengard P, Xu H: Stimulation of

�-amyloid precursor protein trafficking by insulin reduces intraneuronal �-amyloid and requires

mitogen-activated protein kinase signaling. J Neurosci 2001;21:2561–2570.

44 Hoyer S: The aging brain: changes in the neuronal insulin/insulin receptor signal transduction

cascade trigger late-onset sporadic Alzheimer’s disease. J Neural Transm 2002;109:991–1002.

45 Hendrie HC, Ogunniyi A, Hall KS, Baiyewu O, Unverzagt FW, Gureje O, Gao S, Evans RM,

Ogunseyinde AO, Adeyinka AO, Musick B, Hui SL: Incidence of dementia and Alzheimer’s dis-

ease in 2 communities: Yoruba residing in Ibadan, Nigeria, and African Americans residing in

Indianapolis, Indiana. JAMA 2001;285:739–747.

46 Farris W, Mansourian S, Chang Y, Lindsley L, Eckman EA, Frosch MP, Eckman CB, Tanzi RE,

Selkoe DJ, Guenette S: Insulin-degrading enzyme regulates the levels of insulin, amyloid-�

protein, and the �-amyloid precursor protein intracellular domain in vivo. Proc Natl Acad Sci

USA 2003;100:4162–4167.

47 Miller BC, Eckman EA, Sambamurti K, Dobbs N, Chow KM, Eckman CB, Hersh LB, Thiele DL:

Amyloid-� peptide levels in brain are inversely correlated with insulysin activity levels in vivo.

Proc Natl Acad Sci USA 2003;100:6221–6226.


48 Mattson MP, Chan SL, Duan W: Modification of brain aging and neurodegenerative disorders by

genes, diet, and behavior. Physiol Rev 2002;82:637–672.

49 Zhu H, Guo Q, Mattson MP: Dietary restriction protects hippocampal neurons against the death-

promoting action of a presenilin-1 mutation. Brain Res 1999;842:224–229.

50 Hsiao K, Chapman P, Nilsen S, Eckman C, Harigaya Y, Younkin S, Yang F, Cole G: Correlative

memory deficits, A� elevation, and amyloid plaques in transgenic mice. Science 1996;274:

99–102.

51 Yancy WS Jr, Olsen MK, Guyton JR, Bakst RP, Westman EC: A low-carbohydrate, ketogenic diet

versus a low-fat diet to treat obesity and hyperlipidemia. Ann Intern Med 2004;140:769–777.

52 Stern L, Iqbal N, Seshadri P, Chicano KL, Daily DA, McGrory J, Williams M, Gracely EJ, Samaha

FF: The effects of low-carbohydrate versus conventional weight loss diets in severely obese adults:

one-year follow-up of a randomized trial. Ann Intern Med 2004;140:778–785.

53 Vanhanen M, Soininen H: Glucose intolerance, cognitive impairment and Alzheimer’s disease.

Curr Opin Neurol 1998;11:673–677.

54 Wang R, Sweeny D, Gandy SE, Sisodia SS: The profile of soluble amyloid � protein in cultured

cell media. J Biol Chem 1996;271:31894–31902.

55 Cao SX, Dhahbi JM, Monte PL, Spindler SR: Genomic profiling of short- and long-term caloric

restriction effects in the liver of aging mice. Proc Natl Acad Sci USA 2001;98:10630–10635.

56 Merry BJ: Oxidative stress and mitochondrial function with aging – the effects of caloric restric-


57 Moreira PI, Honda K, Liu Q, Santos MS, Oliveira CR, Aliev G, Nunomura A, Zhu X, Smith MA,

Perry G: Oxidative stress: the old enemy in Alzheimer’s disease pathophysiology. Curr Alzheimer

Res 2005;2:403–408.

58 Rosemuller AJ, van Gool WA, Eikelenboom P: The neuroinflammatory response in plaques and

amyloid angiopathy in Alzheimer’s disease: therapeutic implications. Curr Drug Targets CNS

Neurol Disord 2005;4:223–233.

59 Granberry MC, Fonseca VA: Insulin resistance syndrome: options for treatment. South Med J

1999;92:2–15.

60 Wing RR, Blair EH, Bononi P, Marcus MD, Watanabe R, Bergman RN: Caloric restriction per se

is a significant factor in improvements in glycemic control and insulin sensitivity during weight

loss in obese NIDDM patients. Diabetes Care 1994;17:30–36.

Giulio Maria Pasinetti, MD, PhD

Neuroinflammation Research Laboratories, Department of Psychiatry

Mount Sinai School of Medicine, 1 Gustave L. Levy Place, Box 1230

New York, NY 10029–6574 (USA)




Can Short-Term Dietary Restrictionand Fasting Have a Long-TermAnticarcinogenic Effect?

Simon Klebanov

Obesity Research Center, St. Luke’s Roosevelt Hospital Center, New York, N.Y., USA

AbstractLong-term dietary restriction (DR) robustly inhibits various types of carcinogenesis in

rodents. Because malignancies are a major cause of death in humans, reducing the incidence

or, at least, delaying the time of onset of neoplasia may significantly increase longevity of a

large proportion of the human population. Long-term DR may not however be practical in

humans and, judging from religious practices, several days of fasting to several weeks of DR

is what a large segment of the human population can adhere to. In contrast to long-term DR,

a single episode of fasting or several fasting-refeeding cycles did not have any long-lasting

beneficial and usually had even a deleterious effect on carcinogenesis in rodent models. On

the other hand, DR of a relatively short (1–3 months) duration often significantly increased

latency and reduced the incidence of cancer over the entire life span. These results suggest

that the immediate anticarcinogenic action of DR is to slow down the expansion of initiated

clones, but that several months of DR may be sufficient for the elimination of a significant

portion of initiated precancerous clones through apoptosis. The development of optimized

DR regimens for humans will be contingent on further advances in our understanding of the

mechanisms of cancer suppression by DR.


Dietary restriction extends life span regardless of feeding pattern, diet

composition and the age of onset. However, the level and duration of a dietary

restriction regimen determine its effectiveness.

Dietary restriction (DR) is a robust antiaging intervention; DR regimens

almost uniformly result in the extension of life span. Life extension was

achieved regardless of whether food was presented as a single meal a day with

a reduced caloric content [1], as 2 [2] or even 6 [1] smaller meals a day, or ad

libitum one day followed by a day of fasting [3]. Diet composition also had little

Anticarcinogenic Effect of Brief Dietary Restriction 177

effect on the degree of life extension because DR worked even when diets were

enriched with protein [4], or fat and mineral contents were independently

manipulated [5]. Finally, DR did not have to be initiated at a very early age to be

effective. A preweaning initiation of DR did not incur any additional life exten-

sion compared with a postweaning initiation [6], while restricting caloric intake

from 6 months [7], 12–14 months [6, 8–10] and even 18 months [10] extended

life span.

While the antiaging effect can be achieved with a broad variety of DR reg-

imens, two parameters strongly influenced the outcome. First, the degree of

DR, in contrast to the pattern of feeding, significantly affected life span [11]. A

suppressive effect of DR on induced carcinogenesis was also enhanced at more

severe levels of restriction [12, 13]. Second, the duration of DR, rather than the

timing of initiation, determined the degree of life extension [6–8]. The require-

ment of maintaining a rigorous DR regimen for an extended period of time

severely limits direct applicability of DR to humans. At the same time, the defi-

ciency in our understanding limits the development of mechanism-based DR-

mimetic interventions for life extension. Under these circumstances, human life

extension still remains the ultimate, but distant goal.

Life extension is the most striking, but not the only beneficial health effect

of DR. A broad range of age-related diseases is ameliorated by DR [14]. If at

least some of these ameliorating effects could be achieved with a milder and/or

shorter DR regimen, presumably more easily tolerated by humans, it would be

of a great benefit.

Suppression of Carcinogenesis Is Very Prominent and May

Contribute Substantially to Life Extension

The ability of DR to inhibit transplanted tumor growth [15] had been

shown well before DR was reported to extend longevity [16]. A substantial

body of work has been accumulated since then that documents the inhibition of

spontaneous carcinogenesis in normal [17, 18] and genetically modified [19,

20] animals, of chemically induced carcinogenesis [21, 22] and of transplanted

tumors [23, 24]. Thus, the inhibition of various types of carcinogenesis in

numerous organs seems to be as universal and robust a feature of DR regimen

as life extension.

Although specific cancers have different incidence rates in different rodent

laboratory strains, overall, malignancies are a major cause of death in mice and rats

(for reviews, see Crispens [25], pp 159–181, and Weindruch and Walford [14], pp

73–101). Inhibition of spontaneous carcinogenesis is believed to be a major con-

tributor to the life-extending effect of DR. For example, two extensive studies on

Klebanov 178

the effect of DR in two long-lived mouse populations, a standard C57BL6 mouse

strain and a B6C3 F1 hybrid, reported that DR substantially increased life span and

that that increase could mostly be accounted for by a 30–40% decrease in the inci-

dence of lethal neoplasia and by a significant delay in the neoplasia onset [18, 26].

An extensive review of the effect of DR on spontaneous malignancies in rodents

has been provided by Weindruch and Walford [14], pp 73–101.

Malignancies are a major cause of death in humans as well. According to

the National Center for Health Statistics, cancer mortality accounts for more

than 20% of all deaths in the USA (http://www.cdc.gov/nchs/fastats/deaths.

htm). Thus, reducing the incidence or, at least, delaying the time of onset of

neoplasia will have a significant effect on longevity of a large proportion of the

human population. While the mechanisms of DR anticarcinogenesis are not

fully understood, this question is probably more tractable than the DR overall

antiaging effect.

Carcinogenesis: Current Understanding. Promotion as the Most

Promising Stage for Anticarcinogenic Interventions in Humans

The development of neoplasia in mammals involves at least three defin-

able stages: initiation, promotion and progression [27, 28]. Tumor initiation

involves DNA damage induced by endogenous or exogenous carcinogens. Such

mutations may alter the behavior of initiated cells, providing them with growth

advantage. While DR may inhibit certain aspects of initiation in models of

chemical carcinogenesis [29], this is likely to have little direct applicability to

humans. Spontaneous initiation events are ubiquitous and even if a DR regimen

were effective in reducing initiation levels, only potential initiation events that

would have occurred during the period of DR would be prevented, while the ini-

tiation events that had occurred before DR would not. Thus, to have a signifi-

cant anti-initiation effect in humans, a DR regimen will have to be applied for a

period of time comparable with human life span.

The promotion stage of carcinogenesis is characterized by clonal expan-

sion of initiated cells. Unlike initiation and progression, this stage does not

involve alterations in the structure of the genome, but rather in its expression

[27]. A very important and clinically promising feature of this stage is its

reversibility, i.e. the loss or regression of preneoplastic lesions under certain cir-

cumstances. For example, a withdrawal of a promoting agent [30] or an appli-

cation of DR [31] significantly suppresses proliferation, increases apoptosis

and leads to a selective reduction in the volume and the number of preneoplas-

tic lesions in the liver. Another clinically important consideration is that all cells

that have been initiated and moved to the stage of promotion by the time that a


DR regimen is applied, will be affected by DR. If promotion can indeed be

reversed by DR, even relatively short-term DR interventions applied regularly

may prove to be an effective anticancer intervention in humans.

The progression stage involves additional clonal expansion and progres-

sive genetic damage due to genomic instability [27]. During this stage, preneo-

plastic lesions develop into invasive tumors. While in some respect the effect of

DR on the progression stage may be similar to that on the promotion stage,

there is also a significant difference. Even if DR does not eliminate all, but only

a significant portion of promoted cells, it can still significantly retard carcino-

genesis. On the other hand, some cells in the stage of progression are already

neoplastic and even a small portion of cells surviving DR will be able to

develop into a tumor relatively quickly. Thus, a short-term DR will unlikely be

able to postpone carcinogenesis significantly at this stage, necessitating long-

term DR hardly sustainable in humans.

In light of the arguments presented above, most attention in the further dis-

cussion will be given to the effect of DR on the promotion stage in several mod-

els of induced carcinogenesis.

Effect of Dietary Restriction on Cellular Proliferation

and Apoptosis

An initiated cell, while not a tumor by itself, may give rise to a tumor. A

selective growth advantage over normal tissue is one of the crucial properties of

malignancy [30]. The rate of cellular proliferation is an important determinant

of the growth of preneoplastic and neoplastic lesions during the promotion and

progression stages of carcinogenesis. Fasting and various short- and long-term

DR protocols almost uniformly result in reduced rates of cellular proliferation

in the majority of tissues and organs, including the liver [31–35], bladder [36,

37], skin [32, 36], kidney, heart [32], mammary gland, esophagus, jejunum [36]

and colorectum [36, 38] (table 1). This reduction in proliferation may in part

explain the anticarcinogenic effect of DR. Proliferation, however, is just one

determinant of the overall growth rate. The growth rate of preneoplastic and

neoplastic lesions is determined by the difference between the proliferation rate

and the rate of cell death [30]. Most studies focused on the effect of fasting and

DR on the rate of an active cell death, apoptosis, in the liver. Apoptosis seems to

be enhanced by DR or fasting under most circumstances [31, 33, 35, 39, 40],

although a mild DR regimen applied to young, rapidly growing animals may

not be sufficient to induce apoptosis [34] (table 1). Induction of apoptosis by

DR has also been reported for the bladder [37], colon [38] and mammary gland

[41] (table 1). Thus, in addition to reducing cellular proliferation, DR may

Klebanov 180

Table 1. Effects of DR on cellular proliferation and apoptosis

Tissue Species Dietary regimen Effect References

Cellular proliferation

Liver rat 50% of ad libitum, lifelong reduced mostly early [32]

rat fasting, 5 days; refeeding, 2 days; reduced [33]

fasting, 5 days

rat 70% of ad libitum, 7 weeks reduced [34]

mouse 60% of ad libitum, 9 months reduced [35]

rat 40% of ad libitum, 4 days reduced [31]

Bladder mouse 75% of ad libitum, 1 month reduced [36]

mouse 80% of ad libitum, 5 weeks reduced [37]

Skin rat 50% of ad libitum, lifelong reduced mostly early [32]

mouse 75% of ad libitum, 1 month reduced [36]

Kidney rat 50% of ad libitum, lifelong reduced mostly early [32]

Heart rat 50% of ad libitum, lifelong reduced lifelong [32]

Mammary mouse 75% of ad libitum, 1 month reduced [36]

Esophagus mouse 75% of ad libitum, 1 month reduced [36]

Jejunum rat 50% of ad libitum, lifelong no effect [32]

mouse 75% of ad libitum, 1 month reduced [36]

Colorectum mouse 75% of ad libitum, 1 month reduced [36]

rat fasting, 4 days reduced [38]

Apoptosis

Liver rat fasting, 5 days; refeeding, increased [33]

2 days; fasting, 5 days

rat fasting, 3 days increased [39]

rat 70% of ad libitum, 7 weeks no effect [34]

mouse 60% of ad libitum, 9 months increased [35]

rat 40% of ad libitum, 4 days increased [31]

rat 60% of ad libitum, 2 months increased [40]

Bladder mouse 80% of ad libitum, 5 weeks increased [37]

Mammary rat 60% of ad libitum, 6 weeks increased [41]

Colon rat fasting, 4 days increased [38]

reduce initiated cell growth rate and inhibit carcinogenesis by increasing the

rate of cell death.

It is likely that DR effects on cell proliferation and apoptosis will vary

through the course of DR treatment. In mature animals, during the initial stage of

DR, body and most organ weights will rapidly decline, suggesting the prevalence

of cell death over cell replication. At this stage, there is net cell loss. Eventually,


a new steady state will be achieved. At this stage, there is no additional decrease

in organ sizes, and cell proliferation and cell death ought to balance each other

out. This seems in apparent conflict with the reports that long-term DR simulta-

neously decreases cell proliferation [31, 32, 35] and enhances apoptosis [35, 40].

However, apoptosis is only one kind of cell death, and it has been proposed that

some necrotic cell death, characteristic of ad libitum fed animals, is completely

replaced by apoptotic cell death in DR animals [35]. This transition from the

necrotic to the apoptotic type of cell death, if confirmed, was proposed to be

important for the anticarcinogenic effect of DR as apoptosis might be more

selective than necrosis in eliminating preneoplastic cells.

The importance of apoptosis in carcinogenesis is not, however, limited to

its quantitative negative drag on the overall growth rate. Mathematical model-

ing predicts that, with a finite rate of cell death, there is a probability of extinc-

tion of an initiated clone. This probability depends on the initial clone size,

observation (treatment) period and the ratio of cell death to cell proliferation

[42, 43]. This notion is very important as it predicts that because DR increases

the ratio of cell death to cell proliferation it may, if applied long enough and

early enough (i.e. while initiated clones are small), completely eliminate some

initiated clones.

Reversibility of Preneoplastic Lesions: Evidence for the Selective

Elimination of Initiated Cells by Dietary Restriction

Initiation involves irreversible alterations in the cellular genome [27].

During the promotion stage, such initiated cells clonally expand. It has often

been reported that the persistence of preneoplastic lesions is dependent on a

continuous administration of a promoting agent. Upon promoter withdrawal, a

majority of preneoplastic and even neoplastic lesions may spontaneously disap-

pear [30, 44–46]. This behavior of preneoplastic lesions has led to the under-

standing that the promotion stage is reversible [27]. The eventual fate of the

preneoplastic cells that disappeared has not been unequivocally determined but

may include death through apoptosis [30, 46] and redifferentiation to morpho-

logically normal cells [47, 48].

DR has been reported to impose a strong inhibitory effect on the promotion

and progression stages of carcinogenesis in the liver [31], bladder [37], skin [49]

and mammary gland [13]. When preneoplastic lesions can be traced, like in the

liver, their growth is not only retarded, but a majority of such lesions disappear

or significantly decrease in size, and the percentage of the liver volume occupied

by such lesions is significantly reduced [31]. A selective increase in the

apoptosis rates in the preneoplastic lesions, rather than in the surrounding tissue,

Klebanov 182

may be one of the mechanisms for selective elimination of preneoplastic lesions

in response to DR [31]. Thus, it is possible that DR may lead to the actual elimi-

nation of preneoplastic cells. It remains, however, to be determined whether

apoptosis is responsible for a significant cell elimination or whether a substantial

portion of such cells simply loses some of the characteristic markers, i.e. under-

goes redifferentiation to the apparently normal cellular phenotype [47, 48].

Effectiveness of Short-Term Dietary Restriction Regimens in

Inhibiting Carcinogenesis: Long-Term Outcomes of Short-Term

Dietary Restriction Interventions

Religious practices may provide guidance for dietary interventions that can be

adhered to by a large segment of the human population. They include complete

fasting for 1 (Jewish Yom Kippur) to several days and milder DR regimens for up

to several weeks (Christian Lent and Islamic Ramadan). A significant number of

studies explored the effect of comparable dietary manipulation in diverse rodent

models of induced carcinogenesis. Fasting and DR may quickly reduce the number

and the size of preexisting preneoplastic lesions [31, 33, 50], however, the longer-

term outcomes of fasting or short-term DR regimens may not be so positive.

Fasting

A number of studies explored the effect of fasting-refeeding during the

promotion stage of carcinogenesis (table 2). Almost uniformly, a single episode

of fasting or several fasting-refeeding cycles did not have any long-lasting ben-

eficial effect and usually had even a negative effect.

In the liver of rats subjected to partial hepatectomy and initiated with

diethylnitrosamine intraperitoneally, 2 periods of 5 days of fasting separated by 2

days of ad libitum feeding led to an immediate reduction of the number and the

volume of preneoplastic lesions by several fold [33]. However, just 2 weeks after

refeeding, lesion number and volume were restored and 3 months later, the

fasted-refed group had even a larger number of preneoplastic lesions than the

group fed ad libitum throughout the entire study. In another study, rats were also

initiated with diethylnitrosamine intraperitoneally and, a week later, subjected

either to 1 or 3 periods of 3 days of fasting followed by 11 days of refeeding [39,

51]. Seven weeks after diethylnitrosamine, rats were given 2-acetylaminofluorene

intragastrically, to block the proliferation of the majority of normal hepatocytes,

followed by a necrogenic dose of CCl4, to stimulate surviving hepatocyte prolif-

eration. Under these circumstances, only resistant, i.e. initiated hepatocytes would

proliferate, giving rise to altered hepatocyte foci. When sacrificed 4 weeks later,

fasted-refed rats had significantly larger preneoplastic lesions in the liver, and the


negative effect of 3 cycles of fasting-refeeding was more pronounced than that of

1 cycle [51]. When rats were sacrificed 1 year after these dietary manipulations,

the incidence of hepatocellular carcinoma was doubled, and tumor size and histo-

logical grade were increased in the fasted-refed group [39].

In the colon of rats initiated with azoxymethane subcutaneously, 5 periods

of 4 days of fasting interspersed with periods of 7–10 days of refeeding did not

Table 2. Effects of fasting-refeeding and short-term DR on carcinogenesis

Tissue Species Dietary regimen Carcinogen Effect References

Fasting-refeeding

Liver rat fasting, 5 days; diethylnitrosamine initially reduced; later [33]

refeeding, 2 days; (3 months) increased

fasting, 5 days

rat 1 or 3 cycles of diethylnitrosamine increased; more by [51]

fasting (3 days) and 3 cycles than by 1

refeeding (11 days)

rat 3 cycles of fasting diethylnitrosamine increased 1 year later [39]

(3 days) and

refeeding (11 days)

Mammary rat 3 cycles of fasting methylnitrosourea increased multiplicity [53]

(3 days) and dimethylbenz[a] of tumors (42

refeeding (10 days) anthracene weeks later)

rat fasting, 3 days increased later [54]

rat 17 cycles of fasting dimethylbenz[a] no effect [55]

(2 days) and anthracene

refeeding (2 days)

Colon rat 5 cycles of fasting azoxymethane no effect [52]

(4 days) and immediately;

refeeding increased

(7–10 days) 4 weeks later

Short-term DR

Liver rat 60% of ad libitum, spontaneous tumors reduced initially; still [31]

3 months promoted by reduced 17 months

nafenopin later

Mammary rat 50% of ad libitum, dimethylbenz[a] reduced 22 weeks [56]

5 weeks anthracene later

60% of ad libitum, 1-methyl-1- initially reduced; no [57]

5 weeks nitrosourea effect 3 weeks later

Hematopoietic mouse variably restricted, spontaneous reduced in the Klebanov,

for up to 10 weeks lymphomas in long term unpublished

p53-null mice

Klebanov 184

increase the number of aberrant crypt foci, but increased crypt multiplicity, a

good predictor of colon cancer outcome [52]. This effect was not present right

after the last fasting period, but was detectable a week after that and was even

more pronounced 4 weeks later.

Mammary carcinogenesis was also usually enhanced or at least not inhib-

ited by short-term fasting-refeeding protocols. In female rats, 3 cycles of 3 days

of fasting and 10 days of ad libitum feeding, started a week after initiation with

methylnitrosourea intraperitoneally, increased multiplicity of mammary tumors

[53]. Even a single 3-day period of fasting, imposed a week after initiation with

dimethylbenz[a]anthracene, enhanced mammary tumor growth and reduced

tumor latency [54]. However, a more restrictive 10-week-long regimen of 2 days

of fasting followed by 2 days of refeeding had no effect on mammary tumor inci-

dence in rats initiated with a gavage of dimethylbenz[a]anthracene [55].

The failure of fasting to inhibit carcinogenesis probably indicates that sev-

eral days are not sufficient to eliminate any significant number of initiated cells

and that a longer period of a continuous negative energy balance is necessary to

eliminate clones of initiated cells [42, 43]. An additional enhancement of car-

cinogenesis in fasted-refed animals may in part be due to a compensatory

increase in proliferation and a decrease in apoptosis in response to refeeding

[33, 39], which may even be more pronounced in preneoplastic lesions [52].

Suppression of the refeeding response is an obvious target in the search for an

efficient anticarcinogenic intervention [58].

Short-Term Dietary Restriction

Only few studies explored the effect of short-term DR on the long-term

carcinogenesis outcomes (table 2). The results are not as uniform as with fast-

ing, and additional studies will be required for optimizing DR anticancer inter-

ventions.

One study explored the effect of 3 months of DR, at 60% of ad libitum

intake, on the amount of liver tumors after additional 17 months of ad libitum

feeding [31]. The tumor promoter nafenopin was fed for the entire period after

DR to expose all preneoplastic lesions not eliminated by DR. Three months of

DR reduced the number and the volume of spontaneous putative preneoplastic

liver foci to just 15% of control values. Only 1 week of refeeding was sufficient

to increase this number back to 65% of control values. However, the 35%

difference persisted then for the remaining 17 months. The total tumor yield,

including hepatocellular adenoma and carcinoma, was reduced by approxi-

mately 50% by DR administered for a period of 3 months, 17 months prior to

sacrifice.

When DR was administered 1 week prior to the initiation with dimethyl-

benz[a]anthracene intravenously, and during the first 4 weeks of promotion,


mammary tumor size and number were significantly reduced 26 weeks later

[56]. In another study, mammary carcinogenesis was induced by 1-methyl-1-

nitrosourea intraperitoneally [57]. DR, started 1 week after initiation, signifi-

cantly suppressed tumor development over the 5-week period. However, just

3 weeks of ad libitum feeding almost completely abolished the effect of prior

DR on tumor incidence and multiplicity. Thus, short-term DR failed to provide

any long-term protection from carcinogenesis. One clear difference between the

studies that can explain the difference in outcomes is the administration of DR

during the initiation stage in the first study. However, the difference in the

initiation agent and, in general, a much more aggressive carcinogenesis proto-

col used in the second study might have contributed to the difference in the

outcome.

Our own experience suggests that a relatively short-term DR can signifi-

cantly suppress carcinogenesis and extend longevity of p53 knock out mice.

During a routine screening at 5 weeks of age, all p53 knock out mice had normal

body weight, but at 15 weeks, we found out that 3 out of 47 mice were under-

weight. Upon further examination, the mice were diagnosed as having maloc-

clusion that led to self-restriction of their food intake. Judging from their body

weight, mice were DR by approximately 50%. After mice had had this diagno-

sis of malocclusion, their teeth were regularly clipped and their body weight

stayed in the normal range. The life span of self-restricted mice was extended

by 14 weeks, from 26 to 40 weeks (p � 0.001). Our observation is noteworthy

for two reasons. First, no more than 10 (and probably fewer) weeks of DR

extended life span by 14 weeks! Second, DR administered over the entire dura-

tion of life has been reported to extend the life span of p53 knock out mice by

only 9 weeks [20]. Thus, it seems that a relatively short-term DR may yield the

same anticarcinogenic benefits as life-long DR.

The Endocrine System as a Mediator of the Anticarcinogenic

Effect of Dietary Restriction and as a Convenient Target for

Anticarcinogenic Pharmacological Interventions

While some optimism is warranted because of the effectiveness of short-

term DR against some forms of cancer, further progress in devising DR regi-

mens that will be both effective in inducing the ‘therapeutic’ response and

tolerable for a majority of humans will depend on improving the understanding

of molecular and cellular mechanisms of cancer suppression by DR. Several

endocrine systems affected by DR seem to be both likely mediators of the DR-

induced cancer suppression and convenient targets for future pharmacological

interventions.

Klebanov 186

The somatotropic axis is involved in the regulation of life span (for

reviews, see Barbieri et al. [59] and Bartke [60]), and insulin-like growth factor-1

(IGF-1) reduction by DR was proposed to be an important factor in ameliorat-

ing many age-related diseases, including various malignancies (for reviews, see

Kari et al. [61] and Sell [62]). The link between IGF-1 levels and various

aspects of carcinogenesis has been well established (for reviews, see Yu and

Rohan [63], Ibrahim and Yee [64] and Pollak et al. [65]). IGF-1 is known to

stimulate cellular proliferation and inhibit apoptosis in a wide array of tissues

(for reviews, see Jones and Clemmons [66], Butt et al. [67] and Gallaher et al.

[68]). In humans, higher levels of IGF-1 have been linked to an increased inci-

dence of several kinds of cancer, including that of the breast and prostate

[69–72], and, in animals, carcinogenesis is suppressed at very low and is

enhanced at high IGF-1 levels [73–80] (for a review, see Yakar et al. [81]).

While all these lines of evidence support the role of IGF-1 in modulating car-

cinogenesis, replenishing IGF-1 in DR animals abrogates the protective effect

of DR only in some [37], but not in other [82] models. Thus, it still remains to

be determined whether a modest reduction in IGF-1 levels observed in DR ani-

mals is indeed a significant factor in the anticarcinogenic effect of DR.

Leptin is another hormone whose suppression by DR [83–85] has been

hypothesized to contribute to life extension [86] and which has a procarcino-

genic effect (for reviews, see Garofalo and Surmacz [87] and Somasundar et al.

[88]). In vitro, leptin enhances cellular proliferation in a number of tissues and

cell lines [89–96]. In humans, several types of cancer appear to be linked to ele-

vated leptin levels (for a review, see Garofalo and Surmacz [87]), while sponta-

neous and induced carcinogenesis is suppressed in many tissues of leptin-null

ob/ob mice [97–100]. All this evidence is compatible with a role of leptin

reduction in the suppression of carcinogenesis by DR.

Glucocorticoids are yet another class of hormones that may contribute to

the anticarcinogenic action of DR [101, 102]. Total and/or free glucocorticoid

levels are increased by DR [103–105]. Glucocorticoids suppress cellular prolif-

eration and enhance apoptosis in a number of cell types, including osteoblasts,

lymphocytes and keratinocytes (for reviews, see Weinstein [106], Herold et al.

[107] and Budunova et al. [108]). In humans, glucocorticoids are effectively

used for treating lymphoid neoplasms [109]. Importantly, adrenalectomy abol-

ishes the protective effect of DR on skin and pulmonary carcinogenesis, while

glucocorticoid replacement restores this protection [110–112]. As with IGF-1,

the DR-induced shifts in both glucocorticoids and leptin are compatible with

their role in the DR-induced suppression of carcinogenesis. However, similarly

to IGF-1, it still remains to be determined whether, quantitatively, the changes

in glucocorticoid and leptin levels are significant contributors to the anticar-

cinogenic action of DR.


Conclusion and Future Directions

Spontaneous and induced carcinogenesis is significantly postponed by

long-term DR interventions. Long-term DR may not, however, be well tolerated

by humans. Therefore, the major question addressed in this chapter was

whether relatively short-term DR might have a significant long-lasting anticar-

cinogenic effect.

The majority of the studies suggests that fasting does not have a long-

lasting anticarcinogenic effect and that the subsequent refeeding can even pro-

mote carcinogenesis. On the other hand, DR of a relatively short (1–3 months)

duration may significantly affect cancer incidence and latency over the entire

life span. These results suggest that the immediate anticarcinogenic action of

DR is to slow down the expansion of initiated clones through shifting the

balance from proliferation to apoptosis. The long-lasting effects of DR of

1–3 months duration indicate, however, that several months of DR may be

sufficient for elimination of a significant portion of initiated precancerous

clones through apoptosis.

The anticarcinogenic mechanisms of DR, discussed in the current paper,

may be relevant only to the dividing cell populations and may not therefore

explain the entire spectrum of protective effects afforded by DR. While under-

standing the mechanisms of DR for human life extension remains the ultimate

goal, garnering the anticarcinogenic effect of DR may be in and of itself of a

significant value. This task is also likely to be more tractable than finding uni-

versal life-extending mechanisms and may be an important practical step

towards extending human life span.

The heterogeneity of humans in their susceptibility to cancer, their body

composition and their endocrine response to DR may preclude the development

of a single DR regimen that fits all. The development of optimized DR regi-

mens will be contingent on further advances in our understanding of the mech-

anisms of cancer suppression by DR.

References

1 Nelson W, Halberg F: Meal-timing, circadian rhythms and life span of mice. J Nutr 1986;116:

2244–2253.

2 Masoro EJ, Shimokawa I, Higami Y, McMahan CA, Yu BP: Temporal pattern of food intake not a

factor in the retardation of aging processes by dietary restriction. J Gerontol A Biol Sci Med Sci

1995;50A:B48–B53.

3 Goodrick CL, Ingram DK, Reynolds MA, Freeman JR, Cider NL: Effects of intermittent feeding

upon growth and life span in mice. Gerontology 1982;28:233–241.

4 Masoro EJ, Iwasaki K, Gleiser CA, McMahan CA, Seo EJ, Yu BP: Dietary modulation of the pro-

gression of nephropathy in aging rats: an evaluation of the importance of protein. Am J Clin Nutr

1989;49:1217–1227.

Klebanov 188

5 Iwasaki K, Gleiser CA, Masoro EJ, McMahan CA, Seo E-J, Yu BP: Influence of the restriction of

individual dietary components on longevity and age-related disease of Fischer rats: the fat compo-

nent and mineral component. J Gerontol 1988;43:B13–B21.

6 Cheney KE, Liu RK, Smith GS, Meredith PJ, Mickey MR, Walford RL: The effect of dietary

restriction of varying duration on survival, tumor patterns, immune function, and body tempera-

ture in B10C3F1 female mice. J Gerontol 1983;38:420–430.

7 Yu BP, Masoro EJ, McMahan CA: Nutritional influences on aging of Fischer 344 rats. I. Physical,

metabolic, and longevity characteristics. J Gerontol 1985;40:657–670.

8 Beauchene RE, Bales CW, Bragg CS, Hawkins ST, Mason RL: Effect of age of initiation of feed

restriction on growth, body composition, and longevity of rats. J Gerontol 1986;41:13–19.

9 Weindruch R, Walford RL: Dietary restriction in mice beginning at 1 year of age: effect on life-

span and spontaneous cancer incidence. Science 1982;215:1415–1418.

10 Goodrick CL, Ingram DK, Reynolds MA, Freeman JR, Cider NL: Differential effects of intermit-

tent feeding and voluntary exercise on body weight and life span in adult rats. J Gerontol

1983;38:36–45.

11 Weindruch R, Walford RL, Fligiel S, Guthrie D: The retardation of aging in mice by

dietary restriction: longevity, cancer, immunity and lifetime energy intake. J Nutr 1986;116:

641–654.

12 Tannenbaum A: The dependence of tumor formation on the composition of the calorie-restricted

diet as well as on the degree of restriction. Cancer Res 1945;5:616–625.

13 Klurfeld DM, Welch CB, Davis MJ, Kritchevsky D: Determination of degree of energy restriction

necessary to reduce DMBA-induced mammary tumorigenesis in rats during the promotion phase.

J Nutr 1989;119:286–291.

14 Weindruch R, Walford RL: The Retardation of Aging and Disease by Dietary Restriction.


15 Rous P: The influence of diet on transplanted and spontaneous mouse tumors. J Exp Med

1914;20:433–451.

16 McCay CM, Crowell MF, Maynard LA: The effect of retarded growth upon the length of life span

and upon the ultimate body size. J Nutr 1935;10:63–79.

17 Thurman JD, Moeller RB Jr, Turturro A: Proliferative lesions of the testis in ad libitum-fed and

food-restricted Fischer-344 and FBNF1 rats. Lab Anim Sci 1995;45:635–640.

18 Sheldon WG, Bucci TJ, Hart RW, Turturro A: Age-related neoplasia in a lifetime study of ad libi-

tum-fed and food-restricted B6C3F1 mice. Toxicol Pathol 1995;23:458–476.

19 Fernandes G, Chandrasekar B, Troyer DA, Venkatraman JT, Good RA: Dietary lipids and calorie

restriction affect mammary tumor incidence and gene expression in mouse mammary tumor

virus/v-Ha-ras transgenic mice. Proc Natl Acad Sci USA 1995;92:6494–6498.

20 Hursting SD, Perkins SN, Phang JM: Calorie restriction delays spontaneous tumorigenesis in p53-

knockout transgenic mice. Proc Natl Acad Sci USA 1994;91:7036–7040.

21 Reddy BS, Wang CX, Maruyama H: Effect of restricted caloric intake on azoxymethane-induced

colon tumor incidence in male F344 rats. Cancer Res 1987;47:1226–1228.

22 Sugie S, Tanaka T, Mori H, Reddy BS: Effect of restricted caloric intake on the development of the

azoxymethane-induced glutathione S-transferase placental form positive hepatocellular foci in

male F344 rats. Cancer Lett 1993;68:67–73.

23 Matsuzaki J, Yamaji R, Kiyomiya K, Kurebe M, Inui H, Nakano Y: Implanted tumor growth is sup-

pressed and survival is prolonged in sixty percent of food-restricted mice. J Nutr 2000;130: 111–115.

24 Mukherjee P, El-Abbadi MM, Kasperzyk JL, Ranes MK, Seyfried TN: Dietary restriction reduces

angiogenesis and growth in an orthotopic mouse brain tumour model. Br J Cancer 2002;86:

1615–1621.

25 Crispens CG: Handbook on the Laboratory Mouse. Springfield, Thomas, 1975.

26 Blackwell BN, Bucci TJ, Hart RW, Turturro A: Longevity, body weight, and neoplasia in ad

libitum-fed and diet-restricted C57BL6 mice fed NIH-31 open formula diet. Toxicol Pathol

1995;23:570–582.

27 Pitot HC, Hikita H, Dragan Y, Sargent L, Haas M: Review article: the stages of gastrointestinal

carcinogenesis – Application of rodent models to human disease. Aliment Pharmacol Ther

2000;14(suppl 1):153–160.


28 Hursting SD, Kari FW: The anti-carcinogenic effects of dietary restriction: mechanisms and future

directions. Mutat Res 1999;443:235–249.

29 Pashko LL, Schwartz AG: Effect of food restriction, dehydroepiandrosterone, or obesity on

the binding of 3H-7,12-dimethylbenz(a)anthracene to mouse skin DNA. J Gerontol 1983;38:

8–12.

30 Grasl-Kraupp B, Ruttkay-Nedecky B, Mullauer L, Taper H, Bursch W, Schulte-Hermann R:

Inherent increase of apoptosis in liver tumors: implications for carcinogenesis and tumor regres-

sion. Hepatology 1997;25:906–912.

31 Grasl-Kraupp B, Bursch W, Ruttkay-Nedecky B, Wagner A, Lauer B, Schulte-Hermann R: Food

restriction eliminates preneoplastic cells through apoptosis and antagonizes carcinogenesis in rat

liver. Proc Natl Acad Sci USA 1994;91:9995–9999.

32 Merry BJ, Holehan AM: In vivo DNA synthesis in the dietary restricted long-lived rats. Exp

Gerontol 1985;20:15–28.

33 Hikita H, Nuwaysir EF, Vaughan J, Babcock K, Haas MJ, Dragan YP, Pitot HC: The effect of short-

term fasting, phenobarbital and refeeding on apoptotic loss, cell replication and gene expression in

rat liver during the promotion stage. Carcinogenesis 1998;19:1417–1425.

34 Higami Y, Shimokawa I, Ando K, Tanaka K, Tsuchiya T: Dietary restriction reduces hepatocyte

proliferation and enhances p53 expression but does not increase apoptosis in normal rats during

development. Cell Tissue Res 2000;299:363–369.

35 James SJ, Muskhelishvili L: Rates of apoptosis and proliferation vary with caloric intake and may

influence incidence of spontaneous hepatoma in C57BL/6 � C3H F1 mice. Cancer Res

1994;54:5508–5510.

36 Lok E, Nera EA, Iverson F, Scott F, So Y, Clayson DB: Dietary restriction, cell proliferation and

carcinogenesis: a preliminary study. Cancer Lett 1988;38:249–255.

37 Dunn SE, Kari FW, French J, Leininger JR, Travlos G, Wilson R, Barrett JC: Dietary restriction

reduces insulin-like growth factor I levels, which modulate apoptosis, cell proliferation, and tumor

progression in p53-deficient mice. Cancer Res 1997;57:4667–4672.

38 Premoselli F, Sesca E, Binasco V, Franchino C, Tessitore L: Cell death and cell proliferation con-

tribute to the enhanced growth of foci by fasting in rat medial colon. Boll Soc Ital Biol Sper

1997;73:71–76.

39 Tomasi C, Laconi E, Laconi S, Greco M, Sarma DS, Pani P: Effect of fasting/refeeding on the inci-

dence of chemically induced hepatocellular carcinoma in the rat. Carcinogenesis 1999;20:1979–1983.

40 Selman C, Kendaiah S, Gredilla R, Leeuwenburgh C: Increased hepatic apoptosis during short-

term caloric restriction is not associated with an enhancement in caspase levels. Exp Gerontol

2003;38:897–903.

41 Thompson HJ, Zhu Z, Jiang W: Identification of the apoptosis activation cascade induced in mam-

mary carcinomas by energy restriction. Cancer Res 2004;64:1541–1545.

42 Luebeck EG, Mollgavkar SH, Buchmann A, Schwarz M: Effects of polychlorinated biphenyls in

rat liver: quantitative analysis of enzyme altered loci. Toxicol Appl Pharmacol 1991;111:469–484.

43 Moolgavkar SH: Carcinogenesis modeling: from molecular biology to epidemiology. Am Rev

Public Health 1986;7:151–159.

44 van der Heijden CA, Dormans JAMA: Short-term induction of neoplastic nodules in the rat liver.

II. Study of their development and the effects of withdrawal of 2-acetylaminofluorene.

Carcinogenesis 1981;2:147–156.

45 Hendrich S, Glauert HP, Pitot HC: The phenotypic stability of altered hepatic foci: effects of with-

drawal and subsequent readministration of phenobarbital. Carcinogenesis 1986;7:2041–2045.

46 Kolaja KL, Stevenson DE, Walborg EF Jr, Klaunig JE: Reversibility of promoter induced hepatic

focal lesion growth in mice. Carcinogenesis 1996;17:1403–1409.

47 Enomoto K, Farber E: Kinetics of phenotypic maturation of remodeling of hyperplastic nodules

during liver carcinogenesis. Cancer Res 1982;42:2330–2335.

48 Tatematsu M, Nagamine Y, Farber E: Redifferentiation as a basis for remodeling of carcinogen-

induced hepatocyte nodules to normal appearing liver. Cancer Res 1983;43:5049–5058.

49 Birt DF, Pelling JC, White LT, Dimitroff K, Barnett T: Influence of diet and calorie restriction on

the initiation and promotion of skin carcinogenesis in the SENCAR mouse model. Cancer Res

1991;51:1851–1854.

Klebanov 190

50 Hikita H, Vaughan J, Pitot HC: The effect of two periods of short-term fasting during the promo-

tion stage of hepatocarcinogenesis in rats: the role of apoptosis and cell proliferation.


51 Laconi E, Tessitore L, Milia G, Yusuf A, Sarma DS, Todde P, Pani P: The enhancing effect of fast-

ing/refeeding on the growth of nodules selectable by the resistant hepatocyte model in rat liver.


52 Caderni G, Perrelli MG, Cecchini F, Tessitore L: Enhanced growth of colorectal aberrant crypt

foci in fasted/refed rats involves changes in TGFbeta1 and p21CIP expressions. Carcinogenesis

2002;23:323–327.

53 Tessitore L, Chiara M, Sesca E, Premoselli F, Binasco V, Dianzani MU: Fasting during promotion,

but not during initiation, enhances the growth of methylnitrosourea-induced mammary tumours.


54 Sesca E, Premoselli F, Binasco V, Bollito E, Tessitore L: Fasting-refeeding stimulates the develop-

ment of mammary tumors induced by 7,12-dimethylbenz[a]anthracene. Nutr Cancer

1998;30:25–30.

55 Mehta RS, Harris SR, Gunnett CA, Bunce OR, Hartle DK: The effects of patterned calorie-

restricted diets on mammary tumor incidence and plasma endothelin levels in DMBA-treated rats.


56 Sylvester PW, Aylsworth CF, Meites J: Relationship of hormones to inhibition of mammary tumor

development by underfeeding during the ‘critical period’ after carcinogen administration. Cancer

Res 1981;41:1384–1388.

57 Zhu Z, Jiang W, Thompson HJ: An experimental paradigm for studying the cellular and molecular

mechanisms of cancer inhibition by energy restriction. Mol Carcinog 2002;35:51–56.

58 Berrigan D, Perkins SN, Haines DC, Hursting SD: Adult-onset calorie restriction and fasting delay

spontaneous tumorigenesis in p53-deficient mice. Carcinogenesis 2002;23:817–822.

59 Barbieri M, Bonafe M, Franceschi C, Paolisso G: Insulin/IGF-I-signaling pathway: an evolution-

arily conserved mechanism of longevity from yeast to humans. Am J Physiol Endocrinol Metab

2003;285:E1064–E1071.

60 Bartke A: Minireview: role of the growth hormone/insulin-like growth factor system in mam-

malian aging. Endocrinology 2005;146:3718–3723.

61 Kari FW, Dunn SE, French JE, Barrett JC: Roles for insulin-like growth factor-1 in mediating the

anti-carcinogenic effects of caloric restriction. J Nutr Health Aging 1999;3:92–101.

62 Sell C: Caloric restriction and insulin-like growth factors in aging and cancer. Horm Metab Res

2003;35:705–711.

63 Yu H, Rohan T: Role of the insulin-like growth factor family in cancer development and progres-

sion. J Natl Cancer Inst 2000;92:1472–1489.

64 Ibrahim YH, Yee D: Insulin-like growth factor-I and cancer risk. Growth Horm IGF Res

2004;14:261–269.

65 Pollak MN, Schernhammer ES, Hankinson SE: Insulin-like growth factors and neoplasia. Nat Rev

Cancer 2004;4:505–518.

66 Jones JI, Clemmons DR: Insulin-like growth factors and their binding proteins: biological actions.

Endocr Rev 1995;16:3–34.

67 Butt AJ, Firth SM, Baxter RC: The IGF axis and programmed cell death. Immunol Cell Biol

1999;77:256–262.

68 Gallaher BW, Hille R, Raile K, Kiess W: Apoptosis: live or die – Hard work either way! Horm

Metab Res 2001;33:511–519.

69 Kaaks R, Lukanova A: Energy balance and cancer: the role of insulin and insulin-like growth fac-

tor-I. Proc Nutr Soc 2001;60:91–106.

70 Djavan B, Waldert M, Seitz C, Marberger M: Insulin-like growth factors and prostate cancer.

World J Urol 2001;19:225–233.

71 Hankinson SE, Schernhammer ES: Insulin-like growth factor and breast cancer risk: evidence

from observational studies. Breast Dis 2003;17:27–40.

72 Renehan AG, Zwahlen M, Minder C, O’Dwyer ST, Shalet SM, Egger M: Insulin-like growth fac-

tor (IGF)-I, IGF binding protein-3, and cancer risk: systematic review and meta-regression analy-

sis. Lancet 2004;363:1346–1353.


73 Yang XF, Beamer WG, Huynh H, Pollak M: Reduced growth of human breast cancer xenografts in

hosts homozygous for the lit mutation. Cancer Res 1996;56:1509–1511.

74 Deitel K, Dantzer D, Ferguson P, Pollak M, Beamer W, Andrulis I, Bell R: Reduced growth of

human sarcoma xenografts in hosts homozygous for the lit mutation. J Surg Oncol 2002;81: 75–79.

75 Ramsey MM, Ingram RL, Cashion AB, Ng AH, Cline JM, Parlow AF, Sonntag WE: Growth

hormone-deficient dwarf animals are resistant to dimethylbenzanthracine (DMBA)-induced

mammary carcinogenesis. Endocrinology 2002;143:4139–4142.

76 Wu Y, Yakar S, Zhao L, Hennighausen L, LeRoith D: Circulating insulin-like growth factor-I

levels regulate colon cancer growth and metastasis. Cancer Res 2002;62:1030–1035.

77 Bol DK, Kiguchi K, Gimenez-Conti I, Rupp T, DiGiovanni J: Overexpression of insulin-like

growth factor-1 induces hyperplasia, dermal abnormalities, and spontaneous tumor formation in

transgenic mice. Oncogene 1997;14:1725–1734.

78 DiGiovanni J, Bol DK, Wilker E, Beltran L, Carbajal S, Moats S, Ramirez A, Jorcano J, Kiguchi

K: Constitutive expression of insulin-like growth factor-1 in epidermal basal cells of transgenic

mice leads to spontaneous tumor promotion. Cancer Res 2000;60:1561–1570.

79 Hadsell DL, Murphy KL, Bonnette SG, Reece N, Laucirica R, Rosen JM: Cooperative interaction

between mutant p53 and des(1–3)IGF-I accelerates mammary tumorigenesis. Oncogene 2000;19:

889–898.

80 Tornell J, Carlsson B, Pohjanen P, Wennbo H, Rymo L, Isaksson O: High frequency of mammary

adenocarcinomas in metallothionein promoter-human growth hormone transgenic mice created

from two different strains of mice. J Steroid Biochem Mol Biol 1992;43:237–242.

81 Yakar S, Leroith D, Brodt P: The role of the growth hormone/insulin-like growth factor axis in

tumor growth and progression: lessons from animal models. Cytokine Growth Factor Rev

2005;16:407–420.

82 Zhu Z, Jiang W, McGinley J, Wolfe P, Thompson HJ: Effects of dietary energy repletion and

IGF-1 infusion on the inhibition of mammary carcinogenesis by dietary energy restriction. Mol

Carcinog 2005;42:170–176.

83 Cha MC, Jones PJ: Dietary fat type and energy restriction interactively influence plasma leptin

concentration in rats. J Lipid Res 1998;39:1655–1660.

84 Xu B, Kalra PS, Farmerie WG, Kalra SP: Daily changes in hypothalamic gene expression of neu-

ropeptide Y, galanin, proopiomelanocortin, and adipocyte leptin gene expression and secretion:

effects of food restriction. Endocrinology 1999;140:2868–2875.

85 Gazdag AC, Wetter TJ, Davidson RT, Robinson KA, Buse MG, Yee AJ, Turcotte LP, Cartee GD:

Lower calorie intake enhances muscle insulin action and reduces hexosamine levels. Am J Physiol

Regul Integr Comp Physiol 2000;278:R504–R512.

86 Shimokawa I, Higami Y: Leptin signaling and aging: insight from caloric restriction. Mech Ageing

Dev 2001;122:1511–1519.

87 Garofalo C, Surmacz E: Leptin and cancer. J Cell Physiol 2006;207:12–22.

88 Somasundar P, McFadden DW, Hileman SM, Vona-Davis L: Leptin is a growth factor in cancer.

J Surg Res 2004;116:337–349.

89 Frank S, Stallmeyer B, Kampfer H, Kolb N, Pfeilschifter J: Leptin enhances wound re-epithelialization

and constitutes a direct function of leptin in skin repair. J Clin Invest 2000;106:501–509.

90 Goren I, Pfeilschifter J, Frank S: Determination of leptin signaling pathways in human and murine

keratinocytes. Biochem Biophys Res Commun 2003;303:1080–1085.

91 Hu X, Juneja SC, Maihle NJ, Cleary MP: Leptin – A growth factor in normal and malignant breast

cells and for normal mammary gland development. J Natl Cancer Inst 2002;94:1704–1711.

92 Hardwick JC, Van Den Brink GR, Offerhaus GJ, Van Deventer SJ, Peppelenbosch MP: Leptin is a

growth factor for colonic epithelial cells. Gastroenterology 2001;121:79–90.

93 Konopleva M, Mikhail A, Estrov Z, Zhao S, Harris D, Sanchez-Williams G, Kornblau SM, Dong

J, Kliche KO, Jiang S, Snodgrass HR, Estey EH, Andreeff M: Expression and function of leptin

receptor isoforms in myeloid leukemia and myelodysplastic syndromes: proliferative and anti-

apoptotic activities. Blood 1999;93:1668–1676.

94 Okumura M, Yamamoto M, Sakuma H, Kojima T, Maruyama T, Jamali M, Cooper DR, Yasuda K:

Leptin and high glucose stimulate cell proliferation in MCF-7 human breast cancer cells: recipro-

cal involvement of PKC-alpha and PPAR expression. Biochim Biophys Acta 2002;1592:107–116.

Klebanov 192

95 Somasundar P, Yu AK, Vona-Davis L, McFadden DW: Differential effects of leptin on cancer in

vitro. J Surg Res 2003;113:50–55.

96 Stallmeyer B, Kampfer H, Podda M, Kaufmann R, Pfeilschifter J, Frank S: A novel keratinocyte

mitogen: regulation of leptin and its functional receptor in skin repair. J Invest Dermatol

2001;117:98–105.

97 Cleary MP, Phillips FC, Getzin SC, Jacobson TL, Jacobson MK, Christensen TA, Juneja SC,

Grande JP, Maihle NJ: Genetically obese MMTV-TGF-alpha/Lep(ob)Lep(ob) female mice do not

develop mammary tumors. Breast Cancer Res Treat 2003;77:205–215.

98 Heston WE, Vlahakis G: Genetic obesity and neoplasia. J Natl Cancer Inst 1962;29:197–209.

99 Thompson CI, Kreider JW, Black PL, Schmidt TJ, Margules DL: Genetically obese mice: resis-

tance to metastasis of B16 melanoma and enhanced T-lymphocyte mitogenic responses. Science

1983;220:1183–1185.

100 Vlahakis G, Heston WE: Relationship between recessive obesity and induced pulmonary tumors

in mice. J Hered 1959;50:99–102.

101 Masoro EJ: Antiaging action of caloric restriction: endocrine and metabolic aspects. Obes Res

1995;3(suppl 2):241s–247s.

102 Nelson JF, Karelus K, Bergman MD, Felicio LS: Neuroendocrine involvement in aging: evidence

from studies of reproductive aging and caloric restriction. Neurobiol Aging 1995;16:837–843.

103 Klebanov S, Diais S, Stavinoha WB, Suh Y, Nelson JF: Hyperadrenocorticism, attenuated inflam-

mation, and the life-prolonging action of food restriction in mice. J Gerontol A Biol Sci Med Sci

1995;50:B79–B82.

104 Sabatino F, Masoro EJ, McMahan CA, Kuhn RW: Assessment of the role of the glucocorticoid

system in aging processes and in the action of food restriction. J Gerontol 1991;46:B171–B179.

105 Han ES, Evans TR, Shu JH, Lee S, Nelson JF: Food restriction enhances endogenous and corti-

cotropin-induced plasma elevations of free but not total corticosterone throughout life in rats.

J Gerontol A Biol Sci Med Sci 2001;56:B391–B397.

106 Weinstein RS: Glucocorticoid-induced osteoporosis. Rev Endocr Metab Disord 2001;2:65–73.

107 Herold MJ, McPherson KG, Reichardt HM: Glucocorticoids in T cell apoptosis and function. Cell

Mol Life Sci 2006;63:60–72.

108 Budunova IV, Kowalczyk D, Perez P, Yao YJ, Jorcano JL, Slaga TJ: Glucocorticoid receptor func-

tions as a potent suppressor of mouse skin carcinogenesis. Oncogene 2003;22:3279–3287.

109 McKay LI, Cidlowski JA: Corticosteroids; in Kufe DW, Pollock RE, Weichselbaum RR, Bast RC

Jr, Gansler TS, Holland JF, Frei E III (eds): Cancer Medicine. Hamilton, Decker, 2003, chapt 62.

110 Stewart JW, Koehler K, Jackson W, Hawley J, Wang W, Au A, Myers R, Birt DF: Prevention of

mouse skin tumor promotion by dietary energy restriction requires an intact adrenal gland and glu-

cocorticoid supplementation restores inhibition. Carcinogenesis 2005;26:1077–1084.

111 Pashko LL, Schwartz AG: Reversal of food restriction-induced inhibition of mouse skin tumor

promotion by adrenalectomy. Carcinogenesis 1992;13:1925–1928.

112 Pashko LL, Schwartz AG: Inhibition of 7,12-dimethylbenz[a]anthracene-induced lung tumorigene-

sis in A/J mice by food restriction is reversed by adrenalectomy. Carcinogenesis 1996;17:209–212.

Simon Klebanov, PhD

Research Associate

Obesity Research Center

St. Luke’s Roosevelt Hospital Center

1111 Amsterdam Avenue, WH1020

New York, NY 10025 (USA)

Tel. �1 212 5231632, Fax �1 212 5231632, E-Mail [email protected]

193

Al-Regaiey, K.A. 69

Anderson, R.M. 18

Bartke, A. 69

Bonkowski, M.S. 69

Chen, L. 159

Cheng, H. 39

Finch, C.E. 83

Gems, D. 98

Ho, L. 159

Hof, P.R. VII

Houthoofd, K. 98

Humala, N. 159

Ingram, D.K. 137

Isoda, F. 39

Johnson, T.E. 98

Klebanov, S. 176

Ksiezak-Reding, H. 159

Lane, M.A. 137

Liu, X. 159

MacGrogan, D. 159

Masoro, E.J. 1

Mastaitis, J.W. 39

Masternak, M.M. 69

Mattison, J.A. 137

Mobbs, C.V. VII, 39

Morgan, T.E. 83

Pasinetti, G.M. 159

Qin, W. 159

Ressmann, W. 159

Romero, C. 159

Roth, G.S. 137

Shrishailam, Y. 159

Stetka, B. 159

Tatar, M. 115

Vanfleteren, J.R. 98

Wang, J. 159

Weindruch, R. 18

Wong, A.M. 83

Yen, K. VII, 39

Zhang, M. 39

Zhao, Z. 159

Author Index

194

ADAM10, dietary restriction effects in

Alzheimer’s disease model 169–171

Adipose tissue, dietary restriction and aging

retardation 27–29

Advanced glycation end products (AGEs),

dietary restriction reduction and

anti-inflammatory effects 90, 91

Alcohol, Alzheimer’s disease risks 163

Alzheimer’s disease (AD)

dietary restriction studies in transgenic

mouse model

amyloid precursor protein processing

effects 168–171

clinical implications 171, 172

experimental design 167, 168

rationale 166, 167

economic impact 160

insulin metabolism defects 165, 166

nutritional factors 160, 162–164

obesity as risk factor 161, 162

oxidative stress in pathology 59, 60, 162

pathology 164

therapeutic targets 164, 165

Ames dwarf mice

dietary restriction effects on insulin

signaling genes

IGF-1 findings 76–79

IRS-1 findings 75

IRS-2 findings 76

longevity response 73, 74

PPAR-� findings 75, 76

overview of model 70–73

Apoptosis, dietary restriction effects

179–181

Astrocyte, age-related activation and glial

fibrillary acidic protein role 86, 87

Behavior, dietary restriction effects in

rhesus monkey 150–152

Body composition, dietary restriction

effects in rhesus monkey 138–140

Caenorhabditis elegans

clk-1 mutant studies 43, 44, 103

dietary restriction study limitations 100,

101

DNA silencing alterations in dietary

restriction 108

growth 100–102

insulin signaling in dietary restriction

104, 105, 107, 108

metabolic rate in dietary restriction 103,

104

overview of model 99, 100

RNA interference and longevity gene

studies 22, 23, 102

stress resistance in dietary restriction

104

TOR signaling in dietary restriction

108–110

Cancer, dietary restriction effects

cell proliferation and apoptosis 179–181

endocrine system mediation 185, 186

preneoplastic lesion reversal 181, 182

promotion stage interventions 178, 179

prospects for study 187

short-term intervention effects

fasting 182–184

Subject Index

Subject Index 195

mouse studies of short-term dietary

restriction effects 184, 185

suppression of carcinogenesis 177, 178

tumor burden 56, 57

chico1, dietary restriction effects 127–132

clk-1, longevity studies 43, 44, 103

Cold stress, dietary restriction effects 9

Corticosterone, dietary restriction effects in

rat 3, 4, 8

Cortisol, dietary restriction effects in rhesus

monkey 144, 145

C-reactive protein (CRP), dietary restriction

effects 89

Cytokines, dietary restriction effects in

rhesus monkey 148, 149

DAF, dietary restriction effects 22, 23, 28,

30, 105, 107, 108

Dehydroepiandrosterone (DHEA), dietary

restriction effects in rhesus monkey

142–144

Diabetes

Alzheimer’s disease risks 165, 166

hyperglycemia in aging 46

mitochondrial dysfunction 25

oxidative stress in pathology 60

Drosophila melanogaster

dietary restriction

experimental diets 117–123

longevity effects 115, 116

mechanisms

chico1 mutant studies 127–132

gene interactions 127–132

prospects for study 133

resource allocation 123–126

mortality analysis 116, 117

longevity-regulating genes 23, 24, 126

Electron transfer chain complexes,

longevity regulation

clk-1 mutant studies in Caenorhabditis

43, 44, 103

complex I activity reduction and

increased life span 43

complex II and reactive oxygen species

production 42, 43

metabolic shifts 46, 47

overview 42

Electron transport system, dietary

restriction effects on gene expression 20,

21

FADH2, glucose switch profile 51

Fatty acid oxidation, dietary restriction

effects 20

FOXO

dietary restriction effects 26

longevity regulation in Drosophila 23,

24

Ganciclovir, response in dietary-restricted

mice 4

Glial fibrillary acidic protein (GFAP)

aging and neuroinflammation 86, 87

dietary restriction effects 87

Glucocorticoids

anti-inflammatory effects in dietary

restriction 90

dietary restriction and carcinogenesis

suppression 186

hormesis and dietary restriction 3–12

levels and longevity 10

Glucose

dietary restriction effects on levels in

rhesus monkey 140, 141

hyperglycemia in aging 46

hypothalamic neuron toxicity and

metabolism regulation 47, 48

hysteresis hypothesis 54–59

induced genes and longevity effects 53,

54

metabolic fate regulation and glucose

switch gene profile 49–53

Glucose-6-phosphate dehydrogenase,

polymorphisms and longevity 50, 51

GLUT-1, hypoglycemia induction 49, 50

Glycolysis, dietary restriction effects 44, 45

Gompertz mortality rate, dietary restriction

analysis 83

Growth hormone (GH), receptor/binding

protein knockout mice and dietary

restriction effects on insulin signaling

genes


IRS-1 findings 75

Subject Index 196

IRS-2 findings 76

longevity response 73, 74

overview of model 71–73


GSK3�, dietary restriction effects 26, 27

Heat stress, life span extension in lower

organisms 5, 10, 11

HIF-1�, longevity effects 54

Hormesis

dietary restriction mechanisms 3–12

overview 2, 3

HSF-1, stress response in C. elegans 7

Huntington’s disease, oxidative stress in

pathology 60

Hypoglycemia

antioxidant enzyme induction 51, 52

gene induction 49, 50

senescence reversal 60, 61

Hypothalamic neuron, glucose toxicity and

metabolism regulation 47, 48

Hysteresis, metabolic regulation of gene

expression 41

IGF-1, dietary restriction effects

carcinogenesis suppression 186

expression in long-lived mutant mice

76–79

Immune function, dietary restriction effects

9, 10, 148, 149

Inflammatory response, dietary restriction

effects

advanced glycation end product reduction

90, 91

C-reactive protein response 89

DNA microarray studies of anti-

inflammatory effects 88–90

glucocorticoid role 90

neuroinflammation attenuation in aging

animal models 87, 88

astrocytic activation and glial fibrillary

acidic protein role 86, 87

microglial activation and white matter

degeneration 85, 86

overview 84, 85

PPAR-� response 90, 91

Insulin

Alzheimer’s disease and metabolism

defects 165, 166, 171

Caenorhabditis signaling in dietary

restriction 104, 105, 107, 108

dietary restriction effects

secretion 45

sensitivity in rhesus monkey 141

signaling genes in mutant mice

Ames dwarf mice 70–74

growth hormone receptor/binding

protein knockout mice 71–74


IRS-1 findings 75

IRS-2 findings 76


resistance, see Diabetes

IRS-1, dietary restriction effects on

expression in long-lived mutant mice 75

IRS-2, dietary restriction effects on

expression in long-lived mutant mice

76

JNK, dietary restriction effects 26, 27

lac operon, metabolic regulation

glucose switch hypothesis 52, 53

hysteresis 41

overview 40

Leptin, dietary restriction effects

carcinogenesis suppression 186

expression 27, 28

Lipid oxidation, dietary restriction effects

44, 45

Meal number, dietary restriction response of

longevity 176, 177

Melatonin, dietary restriction effects in

rhesus monkey 142

Metabolic rate, dietary restriction effects in

rhesus monkey 141

Metabolic reprogramming, dietary

restriction 18–31

Methionine, dietary restriction effects on

longevity 55

Microglia, age-related activation and white

matter degeneration 85, 86

Growth hormone (continued)

Subject Index 197

Mitochondria

complexes, see Electron transfer chain

complexes

diabetic dysfunction 25

dietary restriction effects on function 24,

25

oxidative stress and longevity 25, 26

Monkey, see Rhesus monkey

NADH, glucose sensing 48, 49

NADPH, glucose switch profile 50

Nonhuman primates, see Rhesus monkey

Obesity

age-related dementia association 161,

162

epidemiology 160

Oxidative stress

aging pathology 59, 60

dietary restriction effects

markers in rhesus monkey 14

reactive oxygen species on production

11

electron transfer chain complexes

clk-1 mutant studies in Caenorhabditis

43, 44, 103

complex I activity reduction and

increased life span 43

complex II and reactive oxygen species

production 42, 43

metabolic shifts 46, 47

p38 mitogen-activated protein kinase,

dietary restriction effects 27, 78

Parkinson’s disease

dietary restriction effects in rhesus

monkey model 152

nutritional factors 160

PGC-1�

dietary restriction effects on adipose

tissue expression 28, 29

functions 29, 30

knockout mouse 30

PNC1, dietary restriction response role in

yeast 6

PPAR-�, dietary restriction effects on

expression

anti-inflammatory effects 90, 91

long-lived mutant mice 75, 76

overview 20

skeletal muscle expression 20

Pyruvate dehydrogenase, longevity effects

54

Reactive oxygen species, see Oxidative

stress

Reproductive function, dietary restriction

effects in rhesus monkey 145–147

Respiratory quotient (RQ)

aging effects 54

dietary restriction effects 54, 55

Rhesus monkey

dietary restriction response

behavior 150–152

body composition 138–140

cortisol 144, 145

dehydroepiandrosterone 142–144

DNA microarray studies 149

glucose levels 140, 141

immune function 148, 149

insulin sensitivity 141

melatonin 142

metabolic rate 141

oxidative stress markers 149

reproductive function 145–147

sensory function 152, 153

thyroid hormone 145

history of dietary restriction studies 137,

138

life span and limitations of model 138

RNA interference, longevity gene studies in

Caenorhabditis 22, 23, 102

Rpd3, longevity regulation in Drosophila

23, 132

Senescence, hypoglycemia and reversal 60,

61

Sensory function, dietary restriction effects

in rhesus monkey 152, 153

Sin3, longevity regulation in Drosophila

23

Sir2, dietary restriction response role

Drosophila 132

yeast 6, 7

Subject Index 198

SIRT1, dietary restriction effects 26, 28

Sirtuin, dietary restriction response role 6,

7, 53, 108

SOD, hypoglycemia induction 51, 52

Stearoyl coenzyme A desaturase 1,

longevity effects 53, 54

Thyroid hormone, dietary restriction effects

in rhesus monkey 145

TOR signaling, dietary restriction effects

22, 108–110

Tumor burden, dietary restriction effects

56, 57

UCP3, dietary restriction effects 24, 25

Wound healing, dietary restriction

effects 9

Documents

Anti-inflammatory mechanisms of dietary restriction in slowing aging processes