Non-Invasive Measurement of Corticosterone in … Non-invasive Measurement of Corticosterone in Food Restricted Rats Deborah Chava Cole Masters of Science Graduate Department of Exercise

Non-Invasive Measurement of Corticosterone

in Food Restricted Rats

by

Deborah Chava Cole

A thesis submitted in conformity with the requirements for the degree of Masters of Science

Graduate Department of Exercise Sciences University of Toronto

© Copyright by Deborah Chava Cole (2012)

II

Non-invasive Measurement of Corticosterone

in Food Restricted Rats

Deborah Chava Cole

Masters of Science

Graduate Department of Exercise Sciences University of Toronto

2012

Abstract !Blood CORT is commonly used to assess stress in rodents, but sampling can trigger a rapid

stress response. This study aims to identify whether faecal CORT metabolites (FCM) can reflect

changes in CORT induced by 7-day food restriction (FR) and an ACTH challenge. Blood and

24hr faecal samples were collected at baseline and Day 7 for control (n=8) and FR (n=10) rats.

On Day 8, after a baseline blood sample, an ACTH injection was administered and followed by

blood and fecal sampling. Results showed increased serum CORT and FCM in response to FR.

Increased adrenal sensitivity with FR was illustrated by a greater increase in serum CORT

compared to control in response to ACTH. Lastly, although it appeared that ACTH induced an

increase in FCM in FR and control, only the latter reached statistical significance. Thus FCM

might be better suited for quantifying chronic rather than acute changes in CORT.

III

Acknowledgments

It would not have been possible to write this Masters thesis without the help and

support of my supervisor, lab committee, lab partners, and my friends and family.

To my supervisor, Dr. Catherine Amara, thank you so much for your patience

and guidance in taking me through all the steps possible to make this thesis happen.

You have taught me so much about how to critique other papers and to create a story

from what may appear to the untrained eye as mere numbers. I would also like to thank

Dr. Carolyn Cummins and Dr. Jack Goodman for providing me with helpful suggestions

and positive feedback, and Dr. Rudy Boonstra for providing us with the equipment and

sharing his knowledge in the field.

I would like to thank the undergraduate and lab partners for their extra support.

Without my undergraduate volunteers as my second pair of hands in the data collection,

I would not have survived the long hours of data collection. In my daily work, I was

blessed with a friendly and cheerful group of fellow students who were always there for

support and assistance.

Finally, I would like to thank my family and friends for unconditionally being there

and encouraging me to complete my Thesis. Mom, Dad, Granddaddy, Avi, Jennifer,

Leora, and Opher, you have all provided personal support and great patience at all

times. Thank you for sitting through numbers of explanations and practice presentations

and attempting to understand what I have been doing for the past two years.

Table of Contents

ACKNOWLEDGEMENTS III

TABLE OF CONTENTS IV

LIST OF TABLES VII

LIST OF FIGURES VIII

LIST OF ABBREVIATIONS IX

LIST OF APPENDICES X

CHAPTER 1: INTRODUCTION 1

CHAPTER 2: REVIEW OF THE LITERATURE 4

2.1 The Stress Response 4

2.2 Blood Cortisol/Corticosterone Measurement 6

2.3 Importance and Application for a Non-Invasive Corticosterone 7

Measurement

2.4 Corticosterone Production 12

2.4.1 Steroidogenesis 12

2.4.2 Hypercortisolism 14

2.4.3 Hormone Regulating Factors: Leptin, Liver, and HDL 17 2.5 Coticosterone Metabolism 21 2.6 Corticosterone Measurement Techniques: RIA and EIA 23 2.7 Cortisol/Corticosterone Response to Food Restriction 25 2.8 Objectives 30

V

CHAPTER 3: METHODOLOGY 32 3.1 Data Analysis 32

3.2 Animals and Housing 34

3.3 Fecal Sampling 35

3.4 Fecal Corticosterone Extraction 35

3.5 Blood Sampling and ACTH Challenge 36

3.6 Blood Corticosterone Extraction 37

3.7 Food Restriction Protocol 38

3.8 Sample Size Justification 38

3.9 Statistical and Analytical Plan 39

CHAPTER 4: RESULTS 40

4.1 Body Composition 40

4.2 Effects of 7 Days of Food Restriction on Corticosterone Levels 41

4.2.1 Serum Corticosterone 41

4.2.2 Faecal Corticosterone Metabolites 42

4.3 Circadian Rhythms 43

4.4 ACTH Challenge 48

4.4.1 Serum 48

4.4.2 Faecal Corticosterone Metabolites 49

4.5 Sampling Effect 51

CHAPTER 5: DISCUSSION 52

5.1 Effect of 1-Week Food Restriction on Serum Corticosterone 52

5.2 Effect of 1-Week Food Restriction on FCM 55

VI

5.3 Underlying Mechanisms of Hypersecretion of Corticosterone 57

5.4 Circadian Rhythms 59

5.5 Adrenal Sensitivity 61

5.6 Limitations 62

5.7 Conclusion 64 References 66 Appendix 75

VII

List of Tables Table 1 - Change in Body Mass over 7 Days Measured 40

in Grams ± S.D.

Table 2 - Average Daily Food Intake over 8 Days in Control 40 and Food Restricted Animals Measured in Grams ± S.D

Table 3 – Speed of Sample Collection Measured in 41

Minutes ± S.D Table 4 - FCM in Ad Libitum and Food Restricted Animals 43 Table 5 – Number of Animals that Produced Samples at 45

a Given Time

VIII

List of Figures Figure 1 - Possible Explanation for Dissociation of ACTH and CORT 15 Figure 2 – Schematic of Rat Model Study Design 33 Figure 3 - Effect of Food Restriction on Serum Corticosterone. 42 Figure 4 - Effect of 7 Days of Food Restriction on Faecal Corticosterone 43

Metabolites. Figure 5 - Circadian Rhythm of Animals at Baseline and Day 7 with 46

the 20:00h-22:00h Time Omitted in Control (A), and FR (B). Figure 6 - Normalized FCM of Circadian Rhythm in (A) Control Group 47

and (B) FR animals.

Figure 7 - Serum CORT of Control and FR Animals After an ACTH 48 Challenge at Baseline 0, 30, 60, and 120 min Post Injection.

Figure 8 - 24 hr Average FCM Measurements at Day 7 and Day 8. 49 Figure 9 - ACTH Challenge over 32hrs in Control and FR Animals 50

After 8 Days Food Restriction Looking at FCM ng/g (A) and Normalized FCM (%) (B).

Figure 10 - Anticipatory Response of Serum CORT to the Sampling 51

Procedure in Control and Food Restricted Animals

IX

List of Appendices

Appendix 1

A. Animals Use Protocol Form / Amendment Request 75

Appendix 2

A. Parallelism and Recovery Test 76

Appendix 3: Raw Data

A. Date of Birth and Arrival of Animals 85

B. Animal Body Mass (g) and Faecal Corticosterone 86

Metabolites at Baseline, Day 7, and Day 8 ACTH

C. Average Mass of Food Consumed per Day Measured in Grams ± S.D. 86

Appendix 4

A. Circadian Rhythm of Control Animals at Baseline and Day 7 87

B. Circadian Rhythm of FR Animals at Baseline and Day 7 87

X

List of Abbreviations ABA = Activity Based Anorexia ACTH = Adrenocorticotropic hormone AgRP = Agouti-related Protein AN = anorexia Nervosa ARC = Arcuate Nucleus AVP = Argenine Vasopressin CBG = Corticosteroid Binding Globulin CON = Control CORT = Corticosterone CRH = Corticotropin-releasing Hormone CSF = Cerebral Spinal Fluid DMH = Dorsomedial Nucleus EIA = Enzyme Immunoassay FCM = Faecal Corticosterone Metabolites FEO = Food Entrainable Oscillator FR = Food Restriction GC = Glucocorticoid GR = Glucocorticoid Receptor HDL = High Density Lipoprotein HPA = Hypothalamic-pituitary-adrenal Axis HRP = Horseradish Peroxidase LC-NE = Locus Ceruleus Noradrenergic LDL = Low Density Lipoprotein LEW = Lewis Rats LHA = Lateral Hypothalamic Area LXR = Liver X Receptor MR = Mineral Corticoid Receptor NPY = Neruopeptide Y POMC = Pro-opeomelanocortin PVN = Paraventricular Nucleus RIA = Radioimmunoassay RF = Restricted Feeding SD = Sprague-Dawley SR-B1 = Scavenger Receptor Class B StAR = Steroidogenic Acute Regulatory Protein TMB = Tetramethylbenzidine VMH = Ventromedial Nucleus

1

Chapter 1 Introduction

Plasma corticosterone (CORT) is commonly measured to detect stress responses in rodents.

However, measuring CORT levels in circulation is complicated as it varies in both diurnal and

ultradian rhythms and the blood sampling procedure can trigger a rapid stress response. Most

human interactions with laboratory rats, including handling, restraint [1], and anesthesia [2]

result in an activation of the hypothalamic-pituitary-adrenal (HPA)-axis. The influence of

moving a rat in its cage from shelf to floor or table in the animal room was shown to increase

CORT within 5 minutes [3]. Vahl and colleagues [4] demonstrated that a stress response can

occur in as little as 3 minutes as shown by an increase in adrenocorticotropic hormone (ACTH).

However, when blood samples were collected in under 3 minutes, ACTH levels were consistent

with basal values. Similarly, Siswanto et al. [5] demonstrated that rats treated with 10ug/kg of

adrenocorticotropic hormone (ACTH) exhibited a significant increase in serum CORT compared

to basal levels between 3 and 90 minutes, suggesting that the time required from a stressful event

to a significant CORT increase is limited to a few minutes.

A number of investigators [5-7] have used faecal samples to determine the concentration of

faecal corticosterone metabolites (FCM) because it is noninvasive and samples can be collected

with minimal disturbance to the animal [8]. Faecal pellets provide an alternative method to

measuring CORT as rodents can defecate several pellets every 1-2 hrs [9], making faecal pellets

a convenient measuring tool for analysis of circadian rhythms of hormones. Analysis of FCM is

also effective for documenting changes in CORT production over a long period of time, since rat

2

faeces have relatively high concentrations of corticosterone metabolites [10]. In addition, faecal

sampling is preferable, because small rodents have limited blood volumes, thereby limiting the

frequency, volume, and /or duration with which blood samples can be collected for repeated

measures within a single animal. Jugular cannulation, which is typically used for repeated blood

sampling, has been associated with high failure and complication rates after a week [11]. The

cannulation may also act as an additional stressor to the animal thereby altering normal

glucocorticoid levels and secretion pattern [12].

Faecal measurements are reflective of plasma CORT, and this is supported by observations of a

diurnal pattern with low levels in the evening and higher values in the morning [6, 10]. Most

mammalian species experience a marked circadian rhythm in circulating glucocorticoid

concentrations, with peak circulating concentrations occurring just prior to the daily active

period or the end of the light period in nocturnal species [13]. This rhythm is asymmetrical in

many mammalian species, including rats, where circulating CORT concentrations rise rapidly at

the end of the inactive sleep period, peak just prior to the active period, and then levels slowly

decrease across the day to the nadir at the end of the active period [13-16].

Serum CORT increases in both short-term [17] and long-term [18] food restricted (FR) rats. A

prior study from the present paper’s lab observed the temporal changes in body composition,

IGF-1, leptin, and voluntary wheel running activity during 4 weeks of a FR protocol that brought

body mass down to 75% baseline [19]. No substantial increase in CORT levels were found in the

FR groups compared to the ad libitum fed animals. This unexpected finding might have been a

result of the protocol used to collect the faecal samples. Samples were collected once per day and

3

this sampling time may not have captured the peak CORT levels in FR animals as intended. FR

alters the way CORT is metabolized, and may have shifted the temporal relationship between

blood CORT and FCM such that the sampling time captured the peak CORT levels in ad libitum

fed animals (as intended), but missed the peak in the FR animals. Previous studies demonstrate

that a stressor can be measured in FCM 4-12 hours later in a number of species, including cats

and dogs [20], mice [21-22], and rats [5, 10]. Stressors included an intraperitoneal injection of

radioactive CORT [10], and an intravenous injection of CORT [6, 22]. This sampling window of

4-12 hours is large, thus sampling at one specific time point might not be sufficient to account

for inter-individual variability.

The overall goal of the current study was to determine if multiple sampling over a larger

sampling window would make it possible to non-invasively determine the CORT response to

food restriction in rats. Faecal sampling is a non-invasive measure that facilitates multiple

collections in a longitudinal design and will reflect CORT responses. If feasible, these

measurements could provide insight on how CORT may be linked with a number of symptoms

of Anorexia Nervosa (AN) including hyperactivity and fat patterning upon refeeding [23]. The

current study has three objectives: To identify the CORT response to food restriction in both

faecal and blood samples; to determine the impact of food restriction on adrenal sensitivity using

an ACTH challenge; and to determine the timing offset between blood and faecal CORT to an

ACTH challenge. Answering these objectives will help determine the correct window of time to

most accurately capture peak CORT levels in faecal matter in FR rats.

4

Chapter 2 Review of the Literature

2.1: The Stress Response

Under normal conditions, the pancreatic beta-cell hormone insulin triggers the fast uptake and

oxidative catabolism of glucose in liver, muscle, and adipose tissue, and simultaneously inhibits

glycogenolysis and gluconeogenesis in liver during feeding [24]. Low plasma glucose levels

during fasting and exercise trigger a drop in insulin and an increase in the peptide hormone

glucagon from alpha-cells within the pancreatic islets and adrenal CORT is released into the

circulation [25]. Adverse situations trigger responses of the adrenals, which result in an increase

in CORT to help defend the organism against the stressful conditions. CORT counteracts

glucose-storing insulin and increases blood sugar through gluconeogenesis to provide immediate

energy for the body. During short-term stress, CORT facilitates the escape from life-threatening

situations and improves the energy availability by breaking down protein and fat to provide

metabolites that can be converted to glucose in the liver [26]. However, during severe chronic

stress, the long-term elevation in CORT can be hazardous to the body and may decrease

individual fitness by immunosuppression and atrophy of muscle tissues.

CORT is secreted with a strong diurnal rhythm where it normally peaks in the morning and

diminishes during the night [27]. Upon synthesis, it diffuses out of the adrenal cells into the

plasma, where most of it is transported by a carrier protein, corticosteroid-binding globulin

(CBG). The unbound hormone is free to diffuse into target cells. All nucleated cells of the body

5

have glucocorticoid receptors in their cytoplasm [27]. The hormone-receptor complex enters the

nucleus, binds to DNA with the aid of a hormone-response element, and alters gene expression,

transcription, and translation.

The hormonal pathway by which stress leads to glucocorticoid secretion is referred to as the

HPA axis. Briefly, the brain perceives a stressful situation, and the paraventricular nucleus

(PVN) located in the hypothalamus sends corticotropin-releasing hormone (CRH) to the

adenohypophysis, which secretes ACTH [27]. In turn, ACTH circulates to the adrenal cortex,

where it signals glucocorticoid secretion. When an animal encounters a stressor, the PVN is

stimulated, and CRH is released into the hypophyseal portal system that connects the

hypothalamus and the anterior pituitary. CRH and arginine vasopressin (AVP) stimulate the

anterior pituitary to convert pro-opiomelanocortin into ACTH. ACTH is released in the blood

stream where it stimulates the adrenal cortex to secrete glucocorticoids well above basal levels,

where it normally takes 3-5 minutes to result in measurable increases in plasma corticosterone

concentration [28]. Plasma corticosterone levels usually peak 15-30 minutes after a stressor and

return to basal levels within 60-90 minutes [29]. With an acute stressor, the feedback mechanism

operates efficiently and the system rapidly returns to normal. With a chronic stressor, feedback

signals are weak and the system remains activated for longer periods [28]. Boonstra [30]

provides an example of the difference between chronic and acute stress: an animal being chased

for a short duration will have an acute response; animals being chased for long durations

(occurring over many hours or weeks) will have a chronic response where chronically elevated

levels of CORT might impact the acute response, and thus, result in a diminished response to an

ACTH challenge, for example, in comparison with control animals.

6

Metabolic cages are often used in biomedical research and it is debated whether housing in these

cages is more stressful than single housing in standard rodent cages and how long rodents need

to acclimate to metabolic cages prior to a study. Housing rats on a grid floor compared with

housing rats in cages with saw dust bedding has been reported to be associated with higher

plasma CORT levels [31], and increases in blood pressure and heart rate [32]. However, duration

of time rats spend in metabolic cages has also been shown to affect CORT levels whereby 12

weeks resulted in notable increases in plasma CORT [31] but 3 days did not [33]. Eriksson et al.

[33] noted that when moved to a metabolic cage, rats showed signs of anxiety and stress

including reduced body weight gain and increased frequency of defecation. However, the stress

was not of sufficient magnitude or duration to result in a significant increase in CORT excretion,

indicating that housing for a few days in metabolic cages is not associated with major stress for

laboratory rats [33].

2.2 Blood Cortisol/Corticosterone Measurement

Corticosterone is the major glucocorticoid found in rats and is the analogue to cortisol in

humans. Methods of blood withdrawal and associated handling during sampling may stress

laboratory animals [34]. To reduce stress, blood sampling in laboratory animals is often

performed under anaesthesia or via cannulation. Taking blood under anaesthesia can be

problematic in itself, because most anesthetics lead to respiratory depression, cardiovascular

disturbances, and endocrine and metabolic changes [34]. There is an increasing demand for

techniques that allow easy, fast, reliable, repeated and non-invasive collection of CORT. Fluttert

et al. [35] discuss many issues involved with intravenously implanted catheters in the jugular

7

vein: When only one or two blood samples were needed, surgery was required and rats had to be

housed singly to prevent them gnawing off each other’s cannulae. Furthermore, the catheter can

be a source of infection and irritation [36], and can consequently disrupt CORT patterns [12].

Thus, longitudinal experimental designs over the course of months or years are not possible with

this technique.

A commonly used alternative is blood collection from a tail vein. An advantage of this technique

is that tail vein nicking does not require surgery and is therefore simpler and less invasive.

However, restraint of the animal is still required during this procedure, which acts as a stressor in

itself altering secretory patterns of circulating stress hormones. To avoid this confounding effect,

Vahl and colleagues [37] concluded that blood samples should be collected in <2-3 minutes so

that sampling is completed before activation of the HPA axis.

2.3 Importance and Application for a non-invasive Corticosterone Measurement

In contrast to the blood CORT measurements, which are influenced by the stressful sampling

procedures, and which reflect a momentary situation, the collection of faeces allows for the

monitoring of chronic hormone metabolite levels without handling animals. The determination of

metabolites in animal urine samples is hampered by the difficulty of obtaining the samples, and

most of the CORT metabolites in rats are excreted via the faeces [10]. Changes in FCM

concentrations have been shown to be a good indicator of changes in blood CORT

concentrations in captive, experimental, and free-ranging animals. Wasser et al. [38] found that

the transfer of a captive owl from her usual enclosure to a novel environment resulted in a

8

comparable response in both serum and faecal CORT levels. Mashburn and Atkinson [39] found

that Stellar sea lions exposed to an ACTH challenge had a 3-fold increase in serum CORT

concentrations and an 18-fold increase in FCM concentrations.

Previous studies using rats [5, 13] have found demonstrable circadian rhythms with well-defined

acrophases and nadirs in faecal corticoids. Results from a number of studies indicate that the

circadian FCM rhythm is shifted between 6-12 hours compared to circulating blood CORT, but

the temporal dynamics are very similar in both mice [40] and rats [12-13]. Royo et al. [12]

demonstrated that faecal CORT can be used as a marker of acute stress in rats, since faecal

CORT showed a similar pattern to serum CORT with a time delay between the presence of

elevated CORT levels in blood and in faeces being approximately 12 h. Thanos and colleagues

[8] examined the applicability of a non-invasive faecal CORT metabolite measure to assess the

circadian rhythm by comparing faecal CORT metabolite levels to circulating CORT levels. The

use of a minimally invasive rapid blood sampling procedure altered both faecal output rhythm

and faecal CORT metabolite levels demonstrating that even a blood collection done in under 3

minutes can trigger stress in an animal [8]. FCM circadian rhythm was time-shifted from the

plasma CORT rhythm by approximately 7-9 h and reflected circulating CORT levels. The

relationship between the two measurements was linear and strong where rats with high faecal

CORT output also had high circulating CORT levels.

FR is a long-term stressor, and therefore, faecal sampling might provide a preferred method for

measuring corticosterone over blood measurements. A previous study using FR mice, found a

significant increase in overall FCM levels at 12 weeks and 24 weeks of food restriction

9

compared to the ad libitum control group [21]. In their study, the FR mice were fed a 60% ad

libitum calorically restricted diet, where diets were nutritionally balanced for equivalent

micronutrient levels.

There are a variety of factors associated with variations in measurement of faecal glucocorticoids

and their metabolites including but not limited to diurnal and seasonal variation, reproductive

status, diet, habituation, sex, and species variation [41]. A study in mice [40] showed that both

sex and time of day impacted the metabolism and excretion of CORT such that males excreted

more FCMs than females (73% vs 53%), and the mouse wake cycle resulted in an increased 3H-

Corticosterone metabolite excretion rate compared to their sleep cycle. Furthermore, the diet of

an animal can affect FCM concentration as well. For instance, a diet high in fibre can decrease

gut transit time [42], which could result in less time for re-absorption of glucocorticoids [43]

and, therefore, increased FCM levels.

While the current study will use female rats, the results should also be applicable to male rats,

but with males having expectedly higher values [13]. Cavigelli et al. [13] looked at sex

differences between rats and found that females excreted less overall mass of corticoid

metabolites from blood into faeces than males, which might have been due to their plasma

corticosterone-binding capacity exceeding that of males and having a slower fractional clearance

rate. Thus, females may catabolize less corticosterone through the liver and / or excrete less mass

of corticosterone metabolites in faeces, and more in urine, than males. However, between-sex

similarities in the rhythms of faecal corticoid excretion (i.e. acrophase and nadir) were clear,

therefore, confirming the usefulness of corticosterone fecal measures in both sexes. Lepschy et

10

al. [6] found no sex differences concerning the route of excretion of GC metabolites.

Furthermore, most of the CMs were excreted via the faeces (75+/-9%) in both sexes. Using mice,

Touma et al [40] found similar results where most of the CORT metabolites were excreted via

the faeces in both sexes, but the amount of radioactivity recovered in the faeces of males was

higher than females. The time courses of 3H-Corticosterone metabolite excretion in urine and

faeces, however, did not differ between the sexes.

The basal activity of the HPA axis tends to increase with age in rats as revealed by increased

CORT [44]. Han et al. [45] observed the effects of 60% food restriction in male rats of three

different age groups. They found that when the rats were 9-months of age, afternoon plasma total

and free CORT concentrations of FR rats were significantly higher than those of ad libitum fed

rats. At 15- and 21-months of age, only afternoon free CORT levels in FR rats were significantly

higher than those in ad-libitum fed rats. With an ACTH injection, peak levels of total CORT

were significantly higher in FR compared with ad libitum rats at 10 and 16 months of age.

However, at 22 months of age, the difference disappeared between the two groups, but peak

levels of free CORT were maintained at higher levels in FR rats at all three ages. Their findings

indicate that the enhanced response of total CORT to ACTH in FR rats extends to 18 months of

age, and for free CORT, extends to 22 months of age. Thus, it can be expected that the FR rats in

the present study, which were 4 months of age, will show both increased total and free CORT

levels compared to ad libitum fed rats.

Although many strains of rats show increased CORT levels in response to stress, variability has

been observed between different strains. Using a Plexiglas restrainer, Dhabhar et al. [46] tested

11

stress responses of three different rat strains to 1h of acute restraint stress, once in the morning

and once in the late afternoon on two successive days. They found differences in CORT levels

between the three strains, with Fischer 344 (F344) rats showing consistently higher diurnal and

stress CORT levels than Sprague-Dawley (SD) and Lewis (LEW) rats. In the morning, basal

CORT levels of all three strains were low. In the evening, SD and F344 rats showed the diurnal

rise in CORT levels, while the LEW rats did not. The stress CORT response of F344 rats was

significantly higher than the SD and LEW rats in the morning and evening. When differences

were observed, LEW rats consistently showed lower levels of CORT than both SD and F244 rats

to the restraint test. In another study, following exposure to a novel environment, no differences

were observed between Sprague-Dawley (SD) and Wistar rats in CORT release, while basal

locomotor activity was higher in Wistar rats than SD [47]. Based on studies demonstrating

existing differences in CORT responses to stress between different rat strains, it is important to

note the strains used in similar studies when comparing values and results. Since Wistar rats

have been shown to be similar in the CORT response to SD, we can expect our Wistar rats to

show both lower diurnal and stress response levels of CORT than F344, but higher than LEW

rats.

Oitzl and colleagues [48] conducted a study looking at a series of differences in HPA-axis

regulators between LEW and Wistar rats. They found that LEW rats displayed an increased

capacity of mineralcorticoid receptors (MR)s in the hippocampus and hypothalamus and a

decreased capacity of glucocorticoid receptors (GR)s in the pituitary while the binding affinity

for MRs and GRs in the hippocampus was comparable. Lower concentrations of CRH mRNA

were detected in the PVN of the LEW hypothalamus. While adrenal weight was similar between

12

the two strains, LEW rats had about 30% less adrenocortical cells. Subjecting adrenocortical

cells to increasing doses of ACTH in vitro resulted in about a 60% smaller release of CORT in

LEW rats. LEW rats responded with lower ACTH and CORT levels than Wistar rats to a variety

of stimuli including a tail nick and restraint. Their results may offer explanations to the

variability seen between rat strains. The authors suggest that the shift of the central MR/GR

balance of LEW rats is the central regulating mechanism of the hyporeactive HPA axis in this rat

strain.

Wistar rats have previously been used in studies examining plasma CORT, faecal corticosterone,

and the correlation between food restriction and glucocorticoids [49-51], because similar to

humans, caloric restriction induces an increase in cortisol/corticosterone. Wistar rats have also

been used in activity based anorexia (ABA) studies [52] as they show similar hormonal patterns

to AN patients: decreased leptin levels co-occurring with weight loss and an increase in leptin

and insulin sensitivity in the hypothalamus of exercised rats in an IL-6 dependent manner [53].

Therefore, for this study, Wistar rats provide a good model to examine the effects of food

restriction on CORT levels.

2.4 Corticosterone production 2.4.1 Steroidogenesis

Corticosteroids are synthesized from cholesterol that is derived from receptor-mediated uptake of

lipoproteins, intracellular stores of esterified cholesterol or de novo synthesis from acetate [54].

The initial step in steroidogenesis is the conversion of cholesterol to the first steroid

pregnenolone. Pregnenolone is the precursor to every steroid hormone and constitutes a rate-

13

limiting and hormonally regulated step in steroidogenesis. The transfer of cholesterol from the

inner- to outer-mitochondria is rate-limiting, because the hydrophobic cholesterol cannot traverse

the aqueous intermembrane space of the mitochondria and reach the P450scc enzyme rapidly

enough by simple diffusion to support acute synthesis [55]. ACTH induces the expression of

StAR to increase the entry of cholesterol across the inner and outer mitochondrial membranes to

be metabolized by P450scc to pregnenolone [55].

Following ACTH stimulation of the cells, hormone sensitive lipase (HSL) mediates cholesterol

ester hydrolysis to free cholesterol, which is then released from lipid droplets to the mitochondria

[56]. The steroidogenic acute regulatory protein (StAR) is a transport protein that mediates the

delivery of cholesterol from the cytosol to the mitochondria to the site of its first enzymatic

conversion of cholesterol to pregnenolone by P450scc [56]. Inside the mitochondria, the action

of the cytochrome P450 side-chain cleavage (P450scc), converts the cholesterol to pregnenolone

[57-58]. Pregnenolone then exits the mitochondria and 3beta-hydroxysteroid dehydrogenase

(3beta-HSD) converts it to progesterone in the microsomal compartment [55].

There are reported cases of dissociation between CORT and ACTH, such as those observed in

depression, AN, and Alzheimer’s Disease [59-61]. Therefore, there must be other mechanisms

controlling CORT levels aside from the HPA axis. It appears that there are ACTH-independent

factors regulating the adrenal cortex including the immune system and neural input. Sympatho-

adrenal control may regulate both adrenal medullary secretion of catecholamines and also

cortical function by adrenal innervation [62].

14

2.4.2 Hypercortisolism

Several mechanisms have been postulated for the hypercortisolism documented in AN patients,

including decreases in cortisol clearance, affinity for cortisol binding globulin, and increased

glucocorticoid-receptor concentration [60]. However, defects in clearance or protein binding

alone cannot produce hypercortisolism in patients with normal hypothalamic and pituitary

responsiveness to the negative feedback of cortisol [60]. The dissociation between plasma ACTH

and CORT observed in some cases may result from changes in adrenal responsiveness to ACTH

induced by stress. ACTH is the only naturally occurring agonist for the ACTH receptor, which is

principally expressed in cells of the adrenal cortex [63]. This receptor signals the cells in the

zona fasciculata of the adrenal cortex to synthesize and secrete CORT. If adrenal cortical

secretion is controlled exclusively by plasma ACTH, then the changes in plasma CORT should

occur in parallel with changes in plasma ACTH [63]. However, as already discussed, there are

several reports of dissociation between changes in plasma ACTH and CORT, suggesting that

factors in addition to ACTH could contribute to adrenal CORT production.

15

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Figure 1 - Possible Explanation for Dissociation of ACTH and CORT

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16

Although decreased ACTH response to CRH observed in AN patients indicates the presence of

feedback at the pituitary level, inappropriately normal baseline ACTH levels and failure of

dexamethasone to fully suppress cortisol suggest possible impairment in feedback at a level

above the pituitary [64]. An elevated CRH level in the cerebral spinal fluid in AN has been

observed and implies a CRH-driven process [64].

In addition to the HPA-axis, the stress system also includes central activation of the sympathetic

neurons leading to activation of both the systemic sympathetic nervous system and, through the

splanchnic nerves, the adrenal medullae. ACTH-independent factors, including the immune

system and neural input, are other explanations for the dissociation between the HPA-axis and

the adrenal cortex. Sympatho-adrenal control may regulate both adrenal medullary secretion of

catecholamines and also cortical function by adrenal innervation [65]. Studies indicate that a

large number of neuropeptides, neurotransmitters, opioids, growth factors, cytokines, adipokines,

and bacterial ligands are capable of modulating adrenal CORT release independent of pituitary

ACTH [66]. Adrenocortical cells express a variety of receptors for these factors, enabling them

to act directly on CORT release [66].

Many studies indicate the prevalence of CORT release controlled at the level of the adrenals

themselves. The adrenal gland is composed of two glands: an inner medulla comprised mainly of

chromaffin cells and an outer cortex comprised of steroidogenic tissue, all of which is

surrounded by the adrenal capsule [63]. The adrenal cortex receives abundant innervation by

nerve fibers of both extrinsic and intrinsic origin. Along with the direct innervation of cortical

cells, the adrenal medulla itself may indirectly govern cortical function by secreting

17

catecholamines or neuropeptides. Cortical cells are interspersed within the adrenal medulla, and

clusters of chromaffin cells lie adjacent to cortical cells in the outer cortex [67]. Since secretion

by chromaffin cells is regulated predominantly by sympathetic preganglionic innervation,

sympathetic innervation of the adrenal could regulate secretion of medullary factors that act

locally to influence cortical function [68]. FR to a certain time of day has previously been shown

to affect steroidogenesis at the level of the adrenals by controlling StAR gene expression within

the adrenocortex [66].

2.4.3 Hormone Regulating Factors: Leptin, Liver, and HDL

Both AN and malnutrition are characterized by a marked decrease in circulating leptin

concentration and an increase in cerebral spinal fluid neuropeptide Y (NPY) concentration [69].

NPY was shown to both suppress and stimulate sympathetic activity depending on location in the

brain [70]. These hormone and protein levels may provide an explanation as to why the HPA

axis in these subjects is activated in the presence of a profoundly hypoactive locus ceruleus

noradrenergic (LC-NE)-sympathetic system [69]. Stress is involved in the regulation of appetite

by influencing the appetite-satiety centers in the hypothalamus. Fasting-stimulated increases in

NPY enhance CRH secretion, while they concomitantly inhibit the LC-NE-sympathetic system

and activate the parasympathetic system, thereby facilitating digestion and storage of nutrients

[69].

Leptin is a satiety-stimulating polypeptide secreted by the white adipose tissue. Leptin inhibits

hypothalamic NPY [69] and has been shown to also have an inhibitory effect on steroidogenesis

[69]. There is an inverse relationship between leptin and CORT observed during food restriction

18

and recovery. The decrease in leptin levels occurring during starvation may lead to increased

CORT due to lack of leptin’s inhibitory effects on StAR, P450scc, and 3betaHSD. Lin and

colleagues [69] demonstrated how leptin inhibits 8-bromo cAMP-stimulated progesterone

production in a concentration-dependent manner. Leptin also inhibits the expression of cAMP-

stimulated StAR protein [69]. A leptin-induced inhibition of expression of the steroidogenic

enzymes cytochrome P450 C21-hydroxylase (P450C21), side-chain cleavage (P450SCC), and C17

!-hydroxylase (P45017!) has been demonstrated in the bovine adrenal gland [70-71], in rat [71],

and human adrenocortical cell in vitro. Furthermore, in mice, the stimulation of CORT secretion

induced by starvation or restraint stress can be partially counteracted by concomitant

administration of leptin [73]. Cherradi et al. [72] showed that the physiological induction of

StAR protein by ACTH is significantly reduced by leptin treatment. Their data indicate that

leptin can counteract ACTH-stimulated steroidogenesis by preventing the hormone-induced

increase in StAR mRNA steady state levels. Leptin’s effect on CORT production is independent

of weight loss as demonstrated by Gairdner and Amara [19] who recently showed that the

reductions in leptin in response to severe food restriction was not correlated with body fat when

fat mass was low.

The liver is fundamental to the metabolism of biomolecules including carbohydrates and lipids.

Hypothalamic and midbrain nuclei are connected via vagal and splanchnic nerves to the liver,

allowing the organ to participate in the control of food intake by sensing and regulating the

energy status of the body. The liver is considered an important constituent of the food-

entrainable oscillator (FEO) [73]. Before food access, there is a prevalence of oxidized

cytoplasmic and mitochondrial redox states, an increase in adenine nucleotide levels, an

19

enhanced mitochondrial capacity to generate ATP, and a hypothyroidal-like condition that is not

systemic but exclusively hepatic [74]. However, after feeding, the hepatic redox state becomes

reduced in both cytoplasmic and mitochondrial compartments, the levels of ATP decline, and the

level of T3 within the liver increases. It has been found that imposing a restricted daylight

feeding time will uncouple the rat liver circadian activity from the SCN rhythmicity along with

inducing adaptations in the size, ultrastructure, and glycogen and triacylglycerol content in

hepatocytes [75]. Diaz-Munoz et al. [75] showed that the main adaptations caused by the

restricted feeding schedule occurred during the food anticipatory activity, and could be

accounted for as a cellular and metabolic anticipation by the liver in preparation for processing

more efficiently the ingested nutrients.

During chronic stress, there is a sustained import of cholesterol in the cell and mitochondria.

Cummins et al. [76] showed that the liver X receptors (LXRalpha and LXRbeta) prevent

accumulation of free cholesterol in mice adrenal glands by controlling expression of genes,

including StAR, involved in all aspects of cholesterol utilization. Under chronic dietary stress,

adrenal glands from LXR-alpha and LXR-beta deficient mice accumulated free cholesterol

implying that these liver receptors act as a protective mechanism to control elevated cholesterol

levels. Liver X receptors stimulate expression of genes that lower cholesterol. There are two

forms of identified LXR: LXR-alpha and LXR-beta. LXR-alpha is expressed at high levels in

liver but also at more modest levels in cells that are involved in cholesterol transport and

metabolism. LXR-beta are known to control the expression of genes involved in transport of

excess cholesterol from peripheral tissues to the liver and hepatic metabolism of this cholesterol

by cytochrome P450 7A1 to bile acids. The hyperproduction of CORT in the adrenal cortex is

20

associated with very high levels of cholesterol transport, lipoprotein receptors (LDL receptor and

the HDL receptor known as scavenger receptor-B1 (SR-B1), stored cholesterol esters, and

enzymes that metabolize cholesterol.

StAR has been shown to be up-regulated by cholesterol containing lipoproteins, including both

low density lipoproteins (LDL) and high density lipoproteins (HDL) [77-78]. HDL provides one

of the only ways to clear cholesterol from the body by returning cholesterol from the tissues back

to the liver [79]. Increased expression of StAR, together with increased cholesterol substrate

availability, enables adrenocortical cells to rapidly increase steroid hormone synthesis [78]. Both

HDL and LDL have been shown to increase expression of StAR mRNA 2-3-fold [78].

In general, FR lowers serum cholesterol and triglycerides relative to controls. Specifically, HDL

cholesterol levels, however, in rodents have been reported to be both lower [80] and higher [81]

in FR than in controls. Energy restriction leads to a significant decrease in cholesterol and

triglyceride levels, with an increase in levels of HDL2, particularly HDL2b cholesterol [82-83].

Verdery and colleagues [82] found monkeys restricted to 70% of ad libitum calorie intake for 6-7

years was accompanied by an increase in HDL sub-fractions HDL2b and HDL1+2b. The HDL

cholesterol has been reported as both unchanged [84] and higher in AN patients [85-87] with

normal lipid levels or increased [86-87] levels of LDL.

21

2.5 Corticosterone Metabolism

Circulating steroid hormones are metabolized by the liver and excreted as conjugates via the

kidneys into the urine or via the bile into the gut [88]. Steroids may enter the enterohepatic

circulation to be reabsorbed into the blood stream and are extensively metabolized by the

microbial flora, but the sterane skeletal structure is not degraded [89]. This allows for detection

of steroid metabolites in the faeces of mammals. As mentioned previously, a lag time occurs

between the instantaneous blood CORT levels and the faecal CORT metabolites. This lag time

depends mainly on the intestinal transit time from the duodenum to the rectum and is largely

species-specific [89]. Rather than the actual steroid concentration, faecal hormone metabolite

levels reflect the production rate or the cumulative secretion and elimination of hormones over

several hours [89].

Corticosterone is metabolized by the liver prior to excretion both through the urine and the

faeces via the bile [90]. Only free CORT that is not bound to CBG is degraded by the liver.

CORT is normally tightly bound to the carrier protein, CBG, and only 5-10% of CORT is in the

free form, unbound and biologically active [91-92]. Since only the free CORT fraction from the

blood is available for metabolism and excretion, FCM concentrations more accurately reflect the

biologically active portion of free CORT [90, 92-93].

Radiometabolism studies have allowed us to understand the metabolism and excretion of

corticosterone including the route, the time course of excretion, and the types of metabolites

formed [6, 40]. Radiometabolism studies have been conducted on a variety of animals including

mice [40], rats [94], and snowshoes hares [7]. The route and delay of excretion as well as the

22

metabolites formed with faecal glucocorticoids differ largely between species [40, 95-96]. Mice

were found to have a lag time of 10 hours, whereas rats can have a lag time of 6-9 h following an

injection of 3H-corticosterone [13]. Time of day of administration is shown to influence the

excretion rate of CORT metabolites due to physical activity levels [39]. Touma et al. [40]

demonstrated that concentrations of 3H-corticosterone metabolites were already recorded after 4h

post-injection. Mice injected in the morning at 9am dispalyed peak CORT levels 10h post

injection and those injected in the evening at 9pm displayed peak CORT 4h post injection thus

proving an effect of the time of day. Therefore, when measuring faecal CORT, it is important to

recognize that increased activity levels can speed the gut passage time, and thus the excretion

time.

Interpretations of faecal assays are based on the assumptions that FCM reflect free, biologically

active, CORT levels in the plasma, and that differences in FCM levels are an accurate reflection

of an animal’s physiological state and thus of its ability to respond to a stressor [7]. Sheriff et al.

[7] verified these assumptions in a population of free-ranging snowshoe hares. Plasma free

CORT levels mirrored FCM levels, but plasma total CORT levels did not. Differences in FCM

concentrations among hares predicted their response to a hormonal challenge where hares with

higher FCM concentrations showed a greater resistance to the suppression of their free plasma

CORT following a dexamethasone injection. They also showed a marked increase of free plasma

CORT and FCM concentrations following an ACTH injection.

23

2.6 Corticosterone Measurement Techniques: RIA and EIA

There is a large variation between species in the kind of glucocorticoid metabolites that are

excreted. Therefore, selection of the proper antibody for use in an immunoassay test is a crucial

step in validation of the assay [97]. Many antibodies have cross-reactivity for other steroids or

steroid metabolites in the sample. Thus, it is important to use an antibody with little cross-

reactivity to prevent measuring additional corticosteroids and metabolites, which can lead to

false interpretations [41].

Immunoassays, including radioimmunoassay (RIA) and enzyme immunoassay (EIA or ELISA),

are commonly used for analyzing CORT levels. Both RIA and EIA can be competitive binding

assays and are highly sensitive. Competitive binding assays require an antibody directed against

certain parts of the steroid molecule of interest. RIAs rely on a radioactive isotope to generate a

radioactive signal to quantify CORT levels. RIAs have the disadvantage of using radioisotopes,

and the disposal of radioactive material can be difficult [28]. CORT determination from blood

samples can be obtained using either plasma or serum since both give the same result [98]. The

EIA corticosterone antiserum that will be used in the current study (CJM006) has previously

been used for both serum and faecal CORT measurement in ferrets [99] and rhinoceroses [100].

Physiological validation of FCM measurements can involve pharmacologically inducing

physiological changes in circulating glucocorticoid levels and evaluating whether these changes

are reflected in measured concentrations of FCMs afterward [89]. A number of strategies can be

used to determine whether CORT is responding in the expected physiological manner. These

24

validation methods include injecting animals with radioactive cortisol to recover radioactive

metabolites (as previously discussed), investigating diurnal rhythms, and exposing animals to a

known stressor.

Measuring the naturally occurring diurnal variation of CORT from faecal samples in a given

species can indicate biological relevance [12, 89]. Bamberg et al. [10] were unable to

demonstrate that changes in adrenocortical activity were well reflected by concentrations of

FCMs measured by their applied in-house corticosterone immunoassay and found their CORT

EIA to be unsuitable. The strong diurnal rhythm of the FCM observed might be an explanation

for the findings of that experiment since their attempt to detect higher CM concentrations caused

by ACTH stimulation might have been masked by the diurnal variation [6, 10]. Without

investigating the natural diurnal variation via a rigorous sampling regime, it might not be

possible to distinguish between the diurnal variation peak of CMs and a peak caused by an

ACTH injection or a stressor [6].

An ACTH challenge can be used to validate the CORT assay and to assess adrenal sensitivity.

Faecal samples should be ideally collected frequently before and after the injection of ACTH,

and they should reflect the sharp increasing and decreasing CORT levels after a certain lag time.

Out of 140 articles published in peer-reviewed journals dealing with faecal CORT in more than

70 species of mammals and birds, Touma and Palme [89] only found convincing physiological

and biological validation experiments in a few studies dealing with FCMs. An ACTH challenge

has previously been used in the literature to examine CORT in blood and faeces in ad libitum fed

rats [5]. It was shown that CORT rapidly increased in blood after ACTH stimulation, and an

25

increase in CORT excretion was detected in faeces 8 hours after ACTH injection. In contrast,

Bamberg et al. [10] demonstrated the presence of a diurnal variation (DV) and the suppression of

FCM as expected after a dexamethasone suppression test in rats. However, they could not find

any increase in the metabolites following an ACTH stimulation test. Therefore with the

antibodies used in their study, it would not be possible to monitor any stressor which acts for

only a short period of time.

An ACTH challenge has previously been used in FR rats to analyze adrenal sensitivity [101].

Han and colleagues [101] found that both in vitro and in vivo adrenal responsiveness to ACTH

was higher in FR than ad libitum rats. Garcia-Belenguer et al. [102] showed that a slight food

restriction to 85% of the ad libitum intake was sufficient to increase the pituitary reactivity to

exogenous CRF with the diurnal rise of CORT starting earlier in FR animals. Thus, it would be

expected to observe an increase in FCM in both FR and ad libitum fed animals to an ACTH

challenge but with FR rats displaying higher FCM levels than the ad libitum group. The ACTH

stimulation test has also been performed on AN patients resulting in an increased CORT

response than average weight participants [103]. These results further confirm that the adrenal

gland overproduces CORT in AN compared to healthy weight individuals.

2.7 Cortisol/Corticosterone Response to Food Restriction !Some animals have prolonged natural fasting to adapt to environments where food is either

unavailable or where feeding would disrupt activities of greater importance (e.g. hibernation,

incubation). The ability to suppress the stress response may permit fasting animals to utilize fat

26

stores and spare protein by preventing the catabolic, protein-mobilizing effects of glucocorticoids

[30]. Fasting is characterized by three phases: During phases I and II, glycogen stores and then

lipid stores, respectively, are the sources for energy with concentrations of corticosterone,

insulin, and thyroid hormone remaining low. If the fast continues until lipid stores reach some

critical lower limit, the body enters phase III and utilizes protein for its energy source.

Corticosterone and glucagon concentrations now increase markedly, and because CORT

promotes protein mobilization, protein now becomes the main energy source [30]. The net result

is muscle wasting.

It has previously been shown that fasting hypercorticosteronemia is caused predominantly by a

reduction in hormone clearance from the plasma, and that this is related to a reduced capacity for

hepatic metabolism of the hormone [94]. After a 48 h fast, rats were anaesthetized using

halothane and CORT was injected into the femoral vein. The tail artery was immediately

cannulated and blood samples of approximately 200 ul were taken into heparinized tubes at 5,

10, and 15 minutes after injection. After 20 min, a final blood sample was taken by cardiac

puncture and the animals were sacrificed. Woodward et al. [94] demonstrated that both sexes of

fasted rats had reductions in metabolic clearance rate (the volume of plasma cleared of steroid

per unit time), increases in plasma CORT concentration, and no change in plasma secretion rate.

They concluded that the increased plasma steroid levels are probably due to a reduction in the

rate of removal of hormone from the plasma, with no change in the rate of secretion from the

adrenal gland [94]. More recently, however, it has been demonstrated that the basis for the

hyperadrenocorticism in caloric restricted rats resides in the adrenal cortex as the consequence of

an enhanced sensitivity of adrenal cells to ACTH [101]. This is supported by studies showing

27

increased plasma CORT levels in the presence of decreased ACTH in FR rats [50]. Under

caloric restriction, there are elevated levels of plasma corticosterone [49-51] and it has been

demonstrated that CORT levels increase parallel to the amount of caloric restriction [51].

Most, if not all, daily rhythms are generated by an endogenous circadian oscillator, which, in

mammals, is located in the suprachiasmatic nucleus of the hypothalamus (SCN) [104]. The SCN

controls the rhythm of CORT secretion via direct and indirect neural control of CRH release and

subsequently ACTH [105], and through autonomic innervation of the adrenal gland [106]. Meal

time is a powerful external cue that can alter CORT diurnal rhythm when restricting food

availability to under a few hours per day leading to an anticipatory peak of CORT release 1-2 h

before the availability of food [107]. The daily schedule of food availability induces a feeding-

associated increase in locomotor activity prior to the expected time of feeding [108-109]. In food

restricted rats, there are two distinct peaks in plasma CORT: the light-dark (LD)-associated

component and the feeding-associated one [110-112]. This diurnal peak anticipating the time of

feeding has been correlated to a prefeeding release of norepinephrine in the paraventricular

nuclei of the hypothalamus where corticotropin-releasing hormone is produced and leads to an

activation of the HPA axis [113].

Restricted daily feeding of rats (being fed 50% of ad libitum daily food intake 2 h after the onset

of light) does not appear to affect the phase of the nocturnal peak of plasma CORT as it appears

to remain insensitive to feeding conditions [49]. After one month of restricted daily feeding, the

CORT anticipatory feeding peak was measured at 60 ng/ml compared to the nocturnal CORT

peak of 130 ng/ml [49]. But 2 months of restricted daily feeding resulted in the CORT

28

anticipatory feeding peak to be higher than the nocturnal peak (115 ng/ml vs 95 ng/ml). Thus,

one can expect both the CORT peaks to change over duration. Girotti and colleagues [107]

demonstrated that when rats are food restricted to three hours per day for 24 days, there is an

anticipatory peak in CORT secretion, but not ACTH 1h before daytime feeding.

Time of feeding may impact the ability to observe a difference between ad libitum and FR serum

CORT. The time of food availability has been shown to affect the CORT diurnal rhythm as

shown when food restriction to 2 hr [114], 3h [107], or 4h [111, 115] of the light period shifts the

CORT peak to the onset of the eating period. A CORT peak is not detected when food

availability is extended to 6 h or more [111, 116] in this condition, probably because the rats are

able to consume the amount of food normally ingested in 24h. Rats provided with food an hour

before dark phase onset also did not demonstrate a shift in peak CORT levels from the beginning

of the dark phase to time of feeding [51].

Restricting feeding to a certain time of day also affected StAR gene expression within the

adrenocortex, suggesting that timed FR resets the timing of peak steroidogenic activity of the

adrenal gland. The adrenal oscillator was affected by RF, reversing the expression profile of each

clock gene examined. Girotti et al. [107] suggest that the FR-dependent shift in CORT peak

secretion may result from both an altered response to changes in an extrinsic signal and the

resetting of the adrenocortex functional state, probably as a result of altered clock gene

expression. It is important to note that hypocaloric and not normocaloric timed FR has been

shown to change the oscillatory pattern of the master clock [117]. Therefore, it is important to

administer food during the wake cycle when comparing FR rats to ad libitum fed rats. One would

29

also expect to find a similar nocturnal peak of plasma CORT with an additional anticipatory

CORT peak prior to the onset of food.

Previous literature indicates an increase in CORT production during stress and food restriction,

with the ability to measure this hormone through blood and faecal samples. Serum and plasma

CORT measurements have shown increased levels of CORT production in FR animals. Blood

measurements are an invasive technique and the handling involved can result in increased stress.

FCMs are a non-invasive tool of collecting CORT samples and have previously been used in rats

to observe a stress response. However, it remains unclear as to whether this hormone can be

measured through FCM in FR rats. Therefore, the current study examined the possibility of

measuring FCMs in FR rats in order to find a non-invasive technique to measure CORT

production.

30

2.8 Objectives !1. To determine faecal CORT response in a rat model of food restriction

To the best of my knowledge, there is currently no research analyzing faecal CORT in food

restricted rats. Therefore, the first objective is to identify a CORT response to food restriction via

FCM. If CORT levels are increased in FR rats, and if it is possible measure CORT metabolites in

faecal matter, then it should be possible to identify a CORT response to food restriction in faeces

if sampled over a sufficient time frame. It is hypothesized that both peak and 24-hr CORT levels

will be higher in the FR group at the end of the one-week FR intervention period.

2. To evaluate the effect of FR on CORT adrenal sensitivity

The second objective is to determine the impact of food restriction on adrenal sensitivity by

comparing the CORT response to an ACTH challenge between FR and ad libitum fed animals. In

addition, the measurements at 30, 60, and 120 minutes post injection!will allow us to determine

the magnitude of the CORT response. We hypothesize that FR animals will show higher levels

of serum CORT to the ACTH challenge than Control animals as previously research indicates

that adrenal responsiveness to ACTH is higher in FR than ad libitum rats [101].

3. To compare the temporal pattern of faecal CM and serum CORT to determine whether

differences exist between ad libitum fed and FR animals

A third objective is to observe whether the FCM will show a diurnal rhythm with peak

CORT levels occurring at the beginning of the wake cycle and levels declining throughout the

day. It is hypothesized that faecal CORT will have around a 9h time lag from serum CORT in

31

the Control as reported in previous papers. This time lag will be even greater in the FR group

compared with the ad libitum, because the rate of metabolism will be slower. Similar patterns

between the two measurements are expected. It is hypothesized that if serum CORT levels

increase in FR rats, then faecal CORT will also be elevated compared to ad libitum fed controls.

Based on the knowledge that resting energy expenditure (REE) is significantly lower in AN

patients than in healthy volunteers [118], and that caloric restricted rats have slower metabolisms

[119], we postulate that faecal CORT metabolites will be time-shifted from the plasma CORT

rhythm in FR rats more than ad libitum fed rats (i.e., they will occur later).

If group differences in CORT exist between faecal and blood measurements, then it will

consequently be possible to recognize the correct window of time to most accurately capture

peak corticosterone levels in faecal matter in food restricted rats. This study will be sampling

faeces every two hours in the first 12 hours post ACTH injection and every 4 hours 24 hours

following. This ensures enough sampling times to most accurately capture peak corticosterone

levels in faecal matter in food restricted rats.

32

Chapter 3 Methodology

3.1 Data Analysis

A previous study in this lab used adolescent female Wister rats to mimic the caloric

deprivation seen in AN that mainly affects women [19]. That study was conducted over four

weeks, but demonstrated an ability to bring the animals’ body weight down to 88% of original

body mass by the end of 1 week while being fed 7g of chow per day or 75% of ad libitum fed

rats. In this current experiment, there were 12 adolescent female Wistar rats (Charles River,

Quebec, Canada) using the same degree of food restriction over one week. All animals were

housed in separate cages with food and water ad libitum. Constant temperature of 23-25°C was

maintained with a 12-h/12-h reverse light-dark cycle (lights off at 8:00 h, lights on at 20:00 h).

There was a 1-week acclimation period for animals to adjust to the opposite light/dark cycle and

to their new environments. During acclimation, animals were placed in a metabolic cage for 30

minutes everyday as these were the cages used during faecal sample collection. The rats were

given unrestricted access to water throughout the experiment and fed on standard laboratory

chow (Purina Standard Rat Chow).

After animals were acclimated for one week, an initial baseline measurement (Baseline 1) was

taken that included one blood sample from each rat and faecal samples every 2 hours for 24

hours. A second baseline measurement (Baseline 2) was taken the following morning to ensure

that the samples were true baselines. Animals were then divided into two groups, ad libitum fed

controls (CON) (n=8) and 1-week food restricted (FR) to 88% - 92% body mass of controls

33

(n=10), all singly housed. FR rats were fed 7g/day, and all rats were weighed on days 1, 3, and 7

to confirm the degree of weight loss and projection for Day 8. To assess the changes in CORT

levels, on Day 7, one blood sample from each rat and faecal samples every 2 hours for 24 hours

were acquired. On Day 8, a blood sample was drawn to determine the effect of FR on serum

CORT. The blood sample served as the baseline level prior to an intramuscular injection of

ACTH, which was followed by blood samples (300 ul) at 30, 60, and 120 minutes post injection.

On Day 8, faecal samples were obtained every 2 hours for 12 hours followed by collection every

4 hours until 32 hours post injection. Animals were decapitated as outlined in the animal use

protocol, at the end of collection in order to collect the trunk blood and organs including the

liver, adrenal glands, and heart. Future studies will be conducted to analyze the trunk blood and

organs to investigate the underlying mechanisms of increased CORT.

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34

3.2 Animals and Housing

Study rats were 4 months old upon arrival weighing between 264 and 341.2 grams. A study

conducted in the current study’s previous lab [19], showed FR rats fed 7g/day ate 75% of ad

libitum fed CON animals. Rats were housed in metabolic cages where excreta could drop

through the bars of the wire floor and be separate from urine. Fecal samples were collected

separately, stored at -20°C and time of sampling was documented. As per Bosson et al. [120], the

cages were polypropylene rodent housing cages (47cm X 26 cm X 20 cm) nested within a

polycarbonate rodent housing cage. There was a wire mesh cage that allowed for collection of

faecal samples without disturbing the animals. With this set up, faeces could pass through the

upper mesh, but not through the lower mesh, while urine was able to pass through both levels of

mesh to the bottom of the container. The cages were less comfortable for the rats, and they

therefore, were housed in standard rat cages with bedding except during sample collections.

During the week of acclimation, animals were handled by the same people each day and

familiarized with the wire mesh bottom cages for 30 minutes a day. The rats showed decreased

signs of stress from interacting with them, including less hyperactivity and noise. Animals did

not appear startled when removed from cages or upon entry into the room and were calm when

handled. Importantly, the same people who handled the animals during acclimation did all of the

handling on testing days. On Day 5, animals were transferred to the metabolic cages. Housing

for a few days in metabolic cages is not associated with major stress for laboratory rats [33].

35

3.3 Faecal Sampling

Faecal samples were collected at baseline and day 7 for 20 hours every 2 hours until 02:00h and

the final sample was collected with a 4hr window between 02:00h and 06:00h. Faecal samples

were collected in order to assess the level of change of CORT, to compare the FR group to the ad

libitum fed group, and to compare FCM to the blood samples. On Day 8, faecal samples were

collected every 2 hours for 22 hours and every 4 hours thereafter until 32 hours post initial

collection. Samples that were not contaminated with urine were collected and transferred to a 1.5

ml plastic snap cap, labeled, and immediately stored at -20°C.

3.4 Faecal Corticosterone Extraction

Faecal samples were sorted by date into 17x100 mm plastic snap cap test tubes. They were

lyophilized for 14-18 h to dry them out and to control for water content [92, 97] and

homogenized. 0.5g +/- 0.05g of faecal matter was weighed into the corresponding tube and

recorded for future calculations to convert values into ng/g faeces. When all samples were

weighed, 1.0 mL of 80% methanol in dH20 was added to each tube immediately using a repeater

pipette and the sample vial was sealed to avoid evaporation. Samples were mixed for 30 minutes

on a rotator/shaker. Samples were centrifuged at 1500 g for 15 minutes. Extracts were stored at -

20°C.

Prior to the current study, the in-house assay was biochemically validated with parallelism and

recovery tests. The sample followed a similar pattern to the standard reflecting the binding

36

characteristics of CORT (appendix 2). Based on these data, we used a 1:40 dilution for the

sample. For the CORT EIA, 41.7ul antibody stock (1:100, -20°C) was added to 5 ml coating

buffer (working dilution, 1:12000). Following that, 50ul of the mixed antibody stock with

coating buffer was added to each well using an Eppendorf repeater pipette. Plates were labeled,

tightly sealed, and incubated overnight at room temperature. High standard (25ng/ml) was

diluted serially 2-fold using 200ul standard and 200ul assay buffer to make 9 standards

consisting of 25, 12.5, 6.25, 3.12, 1.56, 0.78, 0.39, 0.195, and 0.09. Samples were diluted in

assay buffer to the appropriate dilution of 1:40, according to linearity test. 25ul HRP stock

(1:1000) was added to 5 ml assay buffer (working dilution, 1:200 000), plates were washed 5

times with wash solution and blotted on paper towel to remove excess wash solution. For plate

loading, 50 ul standard, sample or control were added to each well. Using Eppendorf repeater

pipette, 50 ul of diluted HRP were immediately added to each well. Plates were tightly covered

and incubated at room temperature for 2 hrs. For the substrate, we added 40 ul 0.5M H2O2 and

125 ul 40 mM ABTS to 12.5 ml substrate buffer and mixed well. 100 ul of substrate was added

to all wells. Substrate mixture was covered tightly and incubated at room temperature for 30-60

minutes with shaking. Plates were read at 405nm with maximum OD of 1.0. !Sensitivity of the

CORT assay was 0.09 ng/mL. The inter-assay coefficient of variance was between 3.15 and

6.19% and the intra-assay coefficient of variance was between 4.4 and 10.1%.

3.5 Blood Sampling and ACTH Challenge

Animals were swiftly removed from their cages and ~300ul of blood was taken from the tail by

making a small lateral incision with a number 11 scalpel blade (Magna Almedic, Montreal

Canada) and allowing the blood to drop directly into a 1.5ml snap cap. After the sample was

37

drawn, pressure was place on the incision point to prevent further bleeding. Animals were then

returned to their cages in the animal housing room. All samples were taken in under 3 minutes,

as this time has been shown to have little effect on CORT levels [4, 121]. Two baseline blood

measurements on consecutive mornings were taken following 7 days of acclimation. Blood

samples were taken again on Day 7 after a week of food restriction to assess the effects of food

restriction on CORT levels. On Day 8, another blood sample was taken and followed by an

intramuscular injection of 4IU/kg Adrenocorticotropic hormone (ACTH) (Synacthen Depot,

CIBA, Ontario, Canada) in the hind limb. The protocol was based on a procedure outlined in

Boonstra and McColl [121]. Briefly, after the baseline blood sample had been drawn, an

intramuscular injection (4 IU/kg) of synthetic ACTH was administered. Animals were returned

to their cages until further blood samples were taken at 30, 60, and 120 minutes post injection.

3.6 Blood Corticosterone Extraction

Approximately 300uL of blood was taken from the tail vein and stored in a sterile microtubule.

The blood was left for 1.5-2 hours to allow for clotting, and then spun in a centrifuge at 3000

rpm for 12-15 minutes. Once spun, serum components were obtained by pipetting the

supernatant into a 1.5ml plastic cap tube and samples were then stored at -80 for later EIA.

Serum CORT EIA extraction was done according to kit instructions (mouse/rat Corticosterone

ELISA; ALPCO Diagnostics, Salem, NH). Briefly, 10µl of each calibrator, sample and control

with new disposable tips were dispensed into appropriate wells. 100µl of incubation buffer was

dispensed into each well. 50µl enzyme conjugate was then added into each well. Wells were then

incubated for 2 hours at room temperature on a microplate mixer. Sensitivity of the serum CORT

assay was 4.1ng/mL The inter-assay coefficients of variation reported by the manufacturer

38

ranged between 4.8 and 12.4%, and between 2.8 and 8.3% for the intra-assay coefficient of

variation.

3.7 Food Restriction Protocol

10 animals were food restricted for one week with unlimited access to water. They were placed

on a diet which consisted of 7g/day until the goal weight of 88%-92% of control body mass was

reached. The animals were individually monitored daily to reach the goal weight, since it has

previously been reported that animals adapt to reduced food intake in an individualized manner

[122-123].

3.8 Sample Size Justification

Using 10 male ground squirrels, Boonstra et al. [121] obtained significant differences in CORT

levels between two groups of squirrels following an ACTH injection (175.9+/-15.5nM/L for

Arctic ground Squirrels vs 343.6+/-32.4nM/L for Red squirrels; p<0.0001). 12 rats (n=6) were

used in an experiment by Chacon et al. [50] observing the effects of anesthesia and blood

sampling techniques on plasma metabolites and CORT in Wistar rats. This sample size was large

enough to see significant differences across the study groups from baseline concentrations

(19.4+/-5.6 ng/ml) with sampling procedure CORT levels (F6,60=9.90, P<0.05) altering in

comparison to the control procedure (+525 and 353% vs. +13%, both Ps <0.05).

Based on the results from Levay et al. [51], sample size for the current study was calculated

using Sigma Plot (minimal detectable difference in means = 50; expected standard deviation of

residuals = 56.6; number of groups = 2; desired power = 0.8; alpha = 0.05). A minimum sample

39

size of 10 total (n=5/group) is required in order to obtain significance comparing serum CORT in

FR rats to ad libitum fed rats. Due to the small sample size, we used 12 animals (n=6/group).

However, after analysis, it appeared that inadequate power was responsible for the lack of

significant findings for the FCM response to FR on Day 8. Therefore, another four FR animals

and 2 more CON animals were used giving us a sample size of n=10 for FR and n=8 for CON.

3.9 Statistical and analytical plan

Data are presented as means ± S.E.M. The means and variances of all groups were compared

with mixed model analysis of variance (ANOVA), corrected when appropriate for repeated

measures. In case of a significant main effect or interaction, a posteriori comparison was

performed using the Bonferroni HSD test. Statistical significance was accepted at p < 0.05. For

blood analysis, a 3-way ANOVA was used using group, day, and time as variables. Between

groups comparisons were analyzed for the effect of food restriction, and within group

comparisons were used to observe changes in CORT occurring across sample times.

40

Chapter 4 Results

4.1 Body Mass and Caloric Intake

FR animals were between 86 and 92% of original body mass and controls had a 0.7 to 9% weight

gain over 7 days (Table 1). Ad libitum fed animals ate more than the rats from Gairder & Amara

[19] (24.58±3.66g vs 16.0±0.63g), but this diet still reduced animals to the anticipated goal of

88% of original body mass. CON animals consumed on average 24.6g/day, while FR animals

consumed on average 7g/day or 28.5% of CON (Table 2). Food was provided at 14:00h every

day to the FR animals and was consumed within an hour.

Table 1 Change in Body Mass over 7 Days Measured in Grams ± S.D.

Body Mass

Group Baseline (g) Day 7 (g) % of Baseline to Day 7

Control 296.75±20.53 307.84±15.16 104.78

Food Restricted 290.16±26.54 258.67±24.65 89.12

Table 2 Average Daily Food Intake over 8 Days in Control and Food Restricted Animals Measured in Grams ± S.D

Day Control Food Restricted

Average Daily Food intake (g)

1 22.95±3.72 7.13±0.17

2 22.84±5.07 7.13±0.21

3 25.01±0.14 7.27±0.33

41

4 26.93±2.62 7.32±0.35

5 21.34±0.86 7.35±0.27

6 24.58±5.03 7.69±0.34

7 27.10±10.44 7.5±0.25

8 25.88±1.42 7.67±0.45

Total Average 24.58±3.66 7.38±0.30

4.2 Effect of 7 Days Food Restriction on Corticosterone Levels 4.2.1 Serum Corticosterone

All blood measurements were collected in under 3 min (Table 3). Baseline 1 blood samples

(6:30-7:00h) for serum CORT were not different between the groups, however a significant

difference between the groups was observed on Day 7 (p=0.007) with FR animals having higher

serum CORT (Fig. 3). The increase from baseline to Day 7 for FR animals did not reach

statistical significance.

Table 3: Speed of Sample Collection Measured in Minutes ± S.D.

Group Baseline (min)

Day 7 (min)

Day 8 (Baseline)

Day 8: 30 min (min)

Day 8: 60 min (min)

Day 8: 120 min (min)

Control 02:31±0.02 02.39±0.02 02:29±0.02 02:07±0.03 02:05±0.02 02:29±0.02

Food Restricted

02.35±0.02 02:56±0.01 02:24±0.02 02:24±0.02 02:18±0.02 02:33±0.02

42

Baseline Day 70

20

40

60

80

100ControlFR

*S

erum

Cor

t (ng

/ml)

Fig 3. Effect of food restriction on serum corticosterone. Blood samples were taken between 6:30 and 7:30am before the beginning of their dark cycle at 8:00am. FR animals showed higher levels of serum CORT than CON animals on Day 7 (p=0.007). The increase from baseline to Day 7 for FR animals did not reach statistical significance. (CON n=8; FR n=10).

4.2.2 Faecal Corticosterone Metabolites

FCM was originally examined using a three-way ANOVA analyzing groups (FR, C) X Time

(using 2 h blocks over 24 h) X Day (day of study protocol), but no significance was found using

all three variables. However, with a two-way ANOVA comparing the groups and a 24hr average

of FCM for each day, FR animals demonstrated a significant increase in FCM from Baseline

(295.4 ±16.2 ng/g) to Day 7 (419.1±25.1 ng/g) (p=0.000), while CON animals showed no

significant change from Baseline (355.4±18.5 ng/g) to Day 7 (384.1±20.9 ng/g) (p=0.213) (Fig.

4). Table 4 shows the 24 hour mean values of FCM for each group at Baseline, Day 7, and Day 8

ACTH challenge. There was a significant increase in FCM for CON Day 7 (384.1±29.1ng/g) to

Day 8(521.5±26.3ng/g) (p=0.001), but not for FR animals.

43

Table 4 Faecal Corticosterone Metabolites in Ad Libitum and Food Restricted Animals Group Baseline FCM

(ng/g faeces) Day 7 FCM (ng/g faeces)

Day 8 ACTH FCM (ng/g faeces)

Control 355.4 ±128.4 384.1±133.7 496.6±256.1 ** Food Restricted

294.6±145.6 419.1±250.5* 523.8±405.2

* indicates significant increase from Baseline to Day 7 in FR animals (p=0.007). Data are presented as ng/g ±S.D. ** indicates significant difference from Day 7 to Day 8 over a 24hr sample collection for the CON animals.

Baseline 1 Day 70

100

200

300

400

500

ControlFR

*

FCM

(ng/

g)

Fig 4. Effect of 7 days of food restriction on faecal corticosterone metabolites. Measurements are averages of 24 hours collections. FCM increased with 7 days of food restriction in the FR animals. (CON n=8; FR Baseline n=10; FR Day 7 n=9). 4.3 Circadian Rhythm FCM concentration in both groups of rats displayed a diurnal rhythm with a peak occurring

between 14:00h to 16:00h for CON animals (Fig. 5A) and between 16:00h and 18:00h for FR

animals (Fig. 5B). Peak values were ~ 2.7 times higher than nadir values for control animals on

Day 7 (457.7±64.5ng/g vs 210.5±111.8ng/g) and ~1.8 times higher than nadir values for food

44

restricted animals (496.3±55.9ng/g vs 275.2±79.0ng/g). The nadir for CON animals occurred

between 08:00h and 10:00h at Baseline and 06:00h and 08:00h on Day 7 (Fig. 5A). Figures 5A

and 5B illustrate the circadian rhythms of the CON and FR animals at Baseline and Day 7 with

the 20:00h-22:00h time omitted due to an unexpected dip. This omitted time point had limited

samples for both CON (3) and for FR (2) compared to the average number of samples at all other

time points combined (5.27 for Control and 3.64 for FR) (Table 4). Table 5 shows the number of

animals that provided samples for each time point. For graphs with the 20:00h-22:00h time

included, refer to appendix 3A and 3B.

To better visualize temporal patterns in FCM throughout a 24h cycle, data were normalized for

each animal by dividing by the average hormone concentration across all time points for their

particular group and multiplying by 100 (figures 6A and 6B). A clear circadian rhythm was

observed in the CON group. Figure 6A shows a monophasic peak in CON animals with

increased FCM from nadir at 08:00-10:00h (63% of the mean) to peak FCM at 16:00-18:00h

(125%) (p=0.003) at Baseline. A diurnal pattern was seen on day 7 with a significant increase in

FCM from nadir at 06:00-08:00h (58%) to peak at 14:00-16:00h (126%) (p=0.003). In figure 6B,

FR animals demonstrate a significant increase from nadir (68.1%) at 06:00-08:00h to peak FCM

(121.0%) at 14:00-16:00h (p=0.022) at Baseline, but no significant difference could be observed

in FCM throughout a 24h period on Day 7.

45

Table 5: Number of Animals that Produced Samples at a Given Time

% /01%2% /01%9%

#*=-%@'*.)%A<B% "'.)('7% 4''5%

&-,)(*+)-5%

"'.)('7% 4''5%

&-,)(*+)-5%

CDECCFC:ECC% O! G% 8% C%

:ECCF2CECC% H% 2C% D% I%

2CECCF28ECC% :% G% D% 8%

28ECCF2IECC% :% G% H% 8%

2IECCF2DECC% 9% 2C% D% 8%

2DECCF2:ECC% :% 2C% 9% :%

2:ECCF8CECC% :% G% 9% G%

8CECCF88ECC% H% I% J% 8%

88ECCF8IECC% 9% :% D% J%

8IECCFC8ECC% H% :% I% I%

C8ECCFCDECC% 9% G% D% I%

46

0

200

400

600

Baseline1Day 7A

FCM

(ng/g

)

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:00

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:000

200

400

600

Baseline1Day 7

B

Time (hrs)

FCM

(ng/g)

Fig 5. Circadian rhythm of animals at Baseline and Day 7 with the 20:00h-22:00h Time Omitted in CON (A), and FR (B). Black bars on X-axis represent the 12hr dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00h. Black arrows indicate time of feeding at 14:00hr. Data have been double-plotted for clarity. CON n=8; FR Baseline n=10; FR Day 7 n=9).

47

0

50

100

150 BaselineDay 7** *

A

Norm

alize

d FCM

(%)

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:00

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:00

-50

0

50

100

150

200 BaselineDay 7

*

B

Time (hrs)

Norm

alize

d FCM

(%)

Fig 6. Normalized FCM of Circadian Rhythm in (A) CON Group and (B) FR animals. Black bars represent the 12hrs of dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 7:00hrs. Data have been double-plotted for clarity. A) * represents significant increase from nadir at 08:00-10:00h (63%) to peak FCM at 16:00-18:00h (125%) (p=0.003) at Baseline. ** represents significant increase from nadir at 06:00-08:00h (57.7%) to peak FCM at 14:00-16:00h (126%) (p=0.003) on Day 7 (n=8). B) * indicates significant increase from nadir (68.1%) at 06:00-08:00h to peak FCM (121%) at 14:00-16:00h (p=0.022) at Baseline (n=10).

48

4.4 ACTH Challenge

4.4.1 Serum

Both groups responded to an ACTH challenge with increased serum CORT levels compared to

Baseline before the injection. CON animals demonstrated increased serum CORT from Baseline

to 30 min (p=0.000) with CORT levels also remaining significantly higher than Baseline at 60

min (p=0.000), and 120 min (p=0.001) post ACTH injection. FR animals also exhibited an

increase in serum CORT from Baseline to 30 min (p=0.000), with this elevation also remaining

significantly higher than Baseline levels at 60 minutes (p=0.000) post ACTH injection. The

increase in serum CORT in response to the ACTH challenge was significantly greater in FR

animals compared to CON at 30 min (p=0.001) and at 60 min (p=0.002) post injection (Fig. 7).

Peak serum CORT was observed at 60 minutes, with levels declining by 120 minutes post ACTH

injection.

0 30 60 1200

200

400

600

800 ControlFR

* !

* !

* **

Time (min)

Ser

um C

ort (

ng/m

l)

Fig 7. Serum CORT of CON and FR animals after an ACTH challenge at Baseline 0, 30, 60, and 120 min post injection. (*) Significant difference in CORT from Baseline. (") significant difference between groups. (CON n=8; FR n=10).

49

4.4.2 Faecal Corticosterone Metabolites

Increased mean FCM levels over a 24hr collection period were seen in both groups after an

ACTH challenge (Fig 8). CON 24hr FCM was higher on Day 8 (521.5±26.3) compared to Day 7

(384.1±29.1) (p=0.001). Although there was a trend for higher FCM in FR on Day 8 vs Day 7,

the increase in FCM did not reach statistical significance for FR animals. When time points were

normalized over the 32hr period (Fig. 9B) on Day 8, CON animals significantly increased FCM

levels from nadir at 08:00-10:00h (76.7%) to peak at 16:00-18:00h (176.4%) (p=0.000).

Day 7 Day 80

200

400

600ControlFood Restricted

*

FCM

(ng/

g)

Fig 8. 24 hr average FCM measurements at Day 7 and Day 8. Food restriction began after Baseline measurements. There was a significant increase in FCM for CON Day 7 (384.1±29.1ng/g) to Day 8(521.5±26.3ng/g) (p=0.001) (CON n=8; FR n=9).

50

0

500

1000

1500

ControlFood Restricted

A

FCM

(ng/

g)

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:00

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:000

50

100

150

200

250

ControlFood Restricted*

Time (hrs)

Norm

alize

d FC

M (%

)

B

Fig 9. ACTH Challenge over 32hrs in Control and FR animals after 8 days food restriction looking at FCM ng/g (A) and Normalized FCM (%) (B). Black bars represent the 12hrs of dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00hrs. Black arrows indicate time of feeding at 14:00hr. B)* represents significant increase in FCM from nadir at 08:00-10:00h (76.7%) to peak at 16:00-18:00h (176.4%) in CON animals (p=0.000) (CON n=8; FR n=9).

51

4.5 Sampling Effect There was a trend in the groups to show increased CORT levels on the second day of blood

sampling following a prior sampling the morning before (Fig. 10) but this increase did not reach

statistical significance. Mean CON serum CORT tended to be higher on Baseline 2 (73.3 ±

19.44ng/ml) than Baseline 1(40.21 ± 10.55ng/ml), and higher on Day 8 Baseline (56.76 ±

19.36ng/ml) than Day 7 (31.8 ± 14.62 ng/ml). In addition, there was a trend for mean serum

CORT for FR animals to be higher at Baseline 2 (69.59 ±16.84 ng/ml) than Baseline 1 (58.57 ±

9.13ng/ml), and higher on Day 8 Baseline (82.16 ± 16.764ng/ml) than Day 7 (80.16 ±

12.66ng/ml).

Baseline 1 Baseline 2 Day 7 Day 80

50

100

150

ControlFood Restricted

Ser

um C

ort (

ng/m

l)

Fig 10. Anticipatory Spike in Control and FR Animals Observed in Serum CORT. Two baseline measures were recorded each over 24hrs. Day 8 is based on the first 24hrs of collection beginning at 6:00-8:00h. (CON n=8; FR Baseline n=10; FR Day 7 and Day 8 n=9).

52

Chapter 5 Discussion

This study investigated whether FCMs could be used as a non-invasive measurement in

rats to evaluate the chronic effects of food restriction on CORT levels. Results showed

that FCM in CON animals demonstrates a diurnal pattern with peak levels occurring

around 8-10hrs after the initiation of the wake-cycle. FR animals appear to have a

flattened diurnal rhythm with no significant difference between peak and nadir CORT

levels. However, FR animals did have an overall increase in FCM compared to CON

after 7 days of food restriction. Only CON animals showed a significant increase in a

24hr collection of FCM in response to an ACTH challenge. 7 days of food restriction

resulted in increased serum CORT and elevated plasma CORT response to exogenous

ACTH. The current study characterizes CORT response in female food restricted rats and

provides a physiological validation of the in-house EIA for the determination of FCM

concentrations in both CON and FR rats. The EIA used in this paper was able to measure

FCM enabling a non-invasive monitoring of adrenocortical activity.

5.1 Effect of 1-Week Food Restriction on Serum CORT

One week of food restriction resulted in increased serum CORT levels as has been shown

by previous studies, but the change did not reach significance. The serum CORT levels

measured in the current study (40.21±10.55ng/ml for CON and 80.16±12.66ng/ml for

FR) are lower than expected based on values from prior research [51, 75, 124]. While

levels in this current paper range between 40.21±10.55ng/ml for CON and

53

80.16±12.66ng/ml for FR, former studies report ad libitum fed animals reaching peak

serum CORT levels of 300ng/ml and 48h food restricted animals reaching peak serum

CORT levels of 475ng/ml [124]. Diaz-Munoz et al. [75] recorded similar results with

control male Wistar rats obtaining peak plasma CORT levels at 250ng/ml and 3-week

food restricted rats at 375ng/ml. Levay and colleagues [51] demonstrated even lower

results than Djordjevic et al. [124] and Diaz-Munoz et al. [75] with CON animals

reaching 175ng/ml plasma CORT and FR rats between 250 and 315ng/ml plasma CORT

depending on the severity of food restriction. The range in blood CORT levels ranging

from 175 to 300ng/ml in CON and 250 to 475ng/ml in FR animals between studies may

be due to several factors including age of animals, length of food restriction, breed and

sex of rat, time of estrous cycle in female rats, speed of sample collection, and time of

blood sample.

Age, sex, and breed of animals do not explain the lower serum in CON CORT values

observed in the current study compared to similar literature. Females were expected to

have higher serum CORT levels than data recorded on male Wistar rats (250ng/ml) [72],

but the female rats in the present paper were much lower (40.21±10.55ng/ml for CON

and 80.16±12.66ng/ml for FR) than their results. Age also does not explain the

discrepancy in the current results as rats in the study by Levay and colleagues [51] were

also 4 months old.

Speed of blood collections affect CORT levels, where samples taken in over 3 minutes

can result in increased CORT due to handling stress [4-5]. Sample collections in the

54

current study were taken in under 3 minutes and animals were acclimated for 1-week to

handling and cage moving. The precautions taken to decrease stress in the animals still

may not, however, explain the lower than expected blood CORT results obtained in the

current paper as previous studies took similar safety measures [51, 75].

The likely explanation for the lower than expected serum results observed in the current

paper may be a combination of sample collection occurring during the estrous cycle and

the time of day samples were collected. CORT levels will fluctuate based on time of

estrous cycle in female rats where daily CORT means are lowest on the day of estrus and

rise progressively during metestrus, diestrus, and proestrus [13]. It is possible that serum

CORT was collected during the estrus phase resulting in lower values.

The blood samples in the current paper were measured an hour before the wake cycle as

previous data [125-127] demonstrate a circadian rhythm of plasma CORT with peak

levels occurring shortly before the onset of the dark cycle. However, this peak appears to

have a range as other groups have reported peak CORT levels occurring 30 to 60 min

after lights-off [49, 128]. Only one blood measurement was taken per animal on a given

day, and therefore, our samples may not have captured the animals’ peak CORT.

Attempting to obtain the peak CORT in the diurnal rhythm by sampling at one time point

for all animals might not be reliable, as there appears to be variability among animals.

Additionally, the varying time of peak CORT reported in previous investigations

highlights the importance of finding a more accurate tool for assessing stress in these

animals.

55

The FR animals may have lower serum CORT than expected because of the length of

treatment and the amount of food restriction. It appears that the severity of food

restriction will affect the suprachiasmatic nuclei of the hypothalamus in a graded manner:

mild metabolic challenge was shown to affect c-fos but not the master oscillator [107],

while a more severe metabolic challenge was shown to alter central clock function [49].

Our treatment was only 1-week in duration and FR rats were fed 30.0% of ad libitum

controls. Studies looking at short-term food restriction, referred to as acute food

deprivation (AFD), are usually under 72 hrs and involve complete food deprivation,

which may explain the higher levels of CORT compared to chronic FR studies.

5.2 Effect of 1-Week Food Restriction on FCM

7 days of food restriction lead to an increase in FCM with peak levels reaching 1.4 times

higher than nadir values, while CON peak CORT levels were 2.7 times higher than nadir.

Despite FR animals having a smaller difference in change between nadir and peak,

CORT remained elevated for a longer duration resulting in an overall greater total

amount of CORT over a 24hr period.

The increase in FCM levels in the FR animals was expected as previous studies have

demonstrated increased blood CORT in response to short and long-term food restriction.

Furthermore, studies have also shown that FCMs can be used to measure stress responses

to numerous stimuli in rodents including temperature, environmental changes, and food

restriction in mice [21]. The increase in CORT during FR stimulates several processes

56

that collectively serve to increase and maintain normal concentrations of blood glucose

levels. It is theorized that the hyperadrenocorticism associated with FR is a protective

mechanism that results in a rise in locomotor activity and may reflect an increased drive

for refeeding [23]. One paper looking at FCM in FR mice took faecal samples, which

were collected as a random subsample of all faecal matter deposited over the course of a

24h period [21]. Food consumption in the FR mice was lowered 10% per week from ad

libitum until 60% of ad libitum was reached. Faecal samples were collected 1 week

before the initiation of FR and at 12-week intervals thereafter. The study on FR mice did

not, however, examine whether a circadian rhythm could be detected in FR rodents. The

current study further supports the ability to measure FCMs in FR rodents and extends the

research by Harper and colleagues [21] by tracing the circadian rhythm of FCM in food

restricted animals.

In previously reported data [13], ad libitum rats demonstrated nadir FCM values of 100

ng/3hrs and peak values of 425ng/3 h. The FCM values in the current study are expected

to be less than those for the male rats seen in Cavigelli et al. [13] because females have

lower FCM values than males as shown in their results. Male rats in their study peaked at

512.6±31.4ng/3h and dipped at 74.5±9.0ng/3h. Female rats in their study demonstrated

lower peak CORT than the male rats, but these values varied in peak CORT levels

depending on the estrous cycle (peak levels reached ~75ng/ 3h for estrus phase,

~100ng/3h for metestrus phase, ~150ng/3h for diestrus phase, and ~200ng/3h for the

proestrus phase). Peak faecal CORT levels during proestrus were two or three times

higher than peaks during estrus or metestrus. These data are measured in different units,

57

and thus values are not comparable to those found in the current study. One study [92]

observing seasonal changes in free-ranging snowshoe hares, reported FCM values in ng/g

and their values ranged between 450 and 700ng/g, depending on the season. Even though

the present study looked at a different species, changes in FCM seem comparable as

CON rats had nadir values of 210.5±111.8ng/g and peak values of 457.7±64.5ng/g.

Faecal glucocorticoid metabolite assays reflect an average level of circulating

glucocorticoids over a time period, rather than a single point in time, and therefore may

provide a more accurate assessment of chronic glucocorticoid levels [21].

5.3 Underlying mechanisms of hypersecretion of CORT

CORT triggers its effects by binding to cytoplasmic glucocorticoid receptor (GR) and

functions as a ligand-dependent transcription factor. In the absence of CORT, GR is

found in cytoplasm as part of a large multiprotein complex, which contains heat shock

proteins (HSPs) 70 and 90 that allow the proper folding of the receptor and control

entrance into the nucleus [129]. After CORT binds to GR, conformational change

releases the receptor from the complex, and it translocates to the nucleus to bind

glucocorticoid-response elements at regulatory regions of target genes, to which

repression is the most common response [129]. Increased CORT in FR may be a result of

the increased GR activation also seen in FR [128, 130-131]. Ogias and colleagues [131]

found that fasting decreased GR in the cytoplasm concomitant with an increase GR at

transcriptional and protein levels in the nucleus. Increased CORT and lower CBG would

facilitate the binding of CORT to GR and its shuttling into the nucleus.

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Many studies indicate increased CORT with no change in ACTH during food restriction

[50, 132-133]. This finding may reflect the presence of a normal pituitary corticotroph

cell caught in the balance between excessive endogenous CRH stimulation from above

and a hyperresponsive adrenal cortex from below [60]. The increased CORT levels

observed in FR rats may be due to increased StAR protein, which is the rate-limiting

factor in steroidogenesis and CORT production and increases with increasing CRH [134].

It is unclear what causes the increased StAR production in FR, but possibilities include

decreased leptin [69], increased liver X receptors [135], and increased HDL [82].

Leptin, a satiety-stimulating polypeptide secreted primarily by the white adipose tissue,

has been shown to have an inhibitory effect on steroidogenesis [136]. During food

restriction, there is a decrease in leptin correlated with weight loss. Gairdner & Amara

[19] demonstrated that 1-week of food restriction to 92.65% of original body mass

resulted in leptin decreasing to 27% of original measurements. The low levels of Leptin

during FR may aid in increased CORT production because they are no longer inhibiting

8-bromo cAMP-stimulated progesterone production and the expression of cAMP-

stimulated StAR protein [136]. In mice, the stimulation of CORT secretion induced by

starvation or restraint stress can be partially counteracted by concomitant administration

of leptin [73]. During FR, there may be an increase in HDL as seen in humans [87, 137],

monkeys [82], and aged male rats [138]. There is limited information on the effects of

chronic FR on HDL levels in young female rats. It would be interesting to see if HDL

levels increase along with increased StAR protein and CORT as HDL cholesterol levels

in rodents have been reported to be both lower [80] and higher [81] in FR than in

59

controls. The changes due to increases in HDL may occur via regulation of StAR by the

liver X receptors that are activated by increasing intracellular cholesterol. Liver X

receptors (LXRalpha and LXRbeta) increase StAR gene expression by preventing

accumulation of free cholesterol [135]. The liver X receptors controlling cholesterol

buildup may also be responsible for the increased CORT observed in the FR rats in the

present paper.

5.4 Circadian Rhythm

FCMs were excreted in a circadian rhythm with well-defined acrophases and nadirs in the

CON animals. 2-hr sampling intervals provided a high degree of temporal acuity for

characterizing the circadian CORT rhythm. Ad libitum fed rats excreted FCMs in a

diurnal pattern with a rapid rise in CORT excretion at the onset of the dark cycle

followed by a more gradual return to trough levels over the day. This asymmetrical

rhythm was similar to that previously identified in both plasma [125] and FCM [13]. The

time frames of peaks and nadirs are in accordance with previous data where FCM of both

male and female Sprague-Dawley rats fed ad libitum rose from lowest to highest values

within 9h, and took approximately 15 h for FCM to fall back to nadir [13].

One might expect a flattening of the CORT rhythm in FR animals due to the increased

CORT levels through out the day, and because FR has been shown to blunt other

hormone diurnal variations such as thyroid stimulating hormone [50]. Cano et al. [139]

demonstrated that a high fat-diet increased CORT and blunted the 24 hr secretory pattern.

Garcia-Belenguer et al. [102] demonstrated that when rats were fed 85% of the control

60

chow, there was a sharpening of the peak CORT. However, with a more severe FR of

50%, mean CORT levels were increased and the day-night difference in circulating

CORT levels was reduced due to high levels maintained throughout the day. Their study

was over 4-weeks, whereas the FR protocol in the current paper reduced body mass to

88-92% of original body mass in one week. FR animals in the present study demonstrated

a blunted circadian rhythm (after 1 week of FR) with no significant difference between

the nadir and peak values. There was, however, a trend in FR values where they appear to

peak 2hr after control occurring between 16:00 and 18:00hr instead of 14:00 and 16:00hr.

A time lag in peak CORT levels between CON and FR was expected as food restriction

causes a slowed metabolism in both animals [140] and humans [141].

There is a trend in FCM levels peaking again between 2:00 and 6:00hrs. Our FR animals

seem to have an anticipatory food peak, which is shown in the FCM 12-14 hrs after

feeding time. This time of anticipatory peak makes sense since their 24h peak FCM

occurred 10-12 hrs after the beginning of their wake-cycle. The anticipatory peak may be

a result of restricted feeding resetting the timing of peak steroidogenic activity of the

adrenal gland as previously observed by altering StAR gene expression within the

adrenocortex [66]. Restricted feeding has also been shown to alter functional (c-fos, crh,

pomc) and clock gene expression (per1, per2, and bmal) at all levels of the HPA axis

[66].

61

5.5 Adrenal Sensitivity

The use of exogenous ACTH to induce CORT secretion in rats is well established and

has previously been used to analyze both serum and faecal CORT responses [5]. In

addition to providing physiological validation for the current paper’s FCM

measurements, the aim of the ACTH injection in the present study was to investigate the

time required from ACTH administration to a detectable increase in CORT concentration

in the blood compared to an increase in CORT metabolite in the faeces and to assess

whether differences in the magnitude of response occurred between groups. Results

demonstrate serum CORT increased at 30 and 60 minutes post injection and decreased

thereafter in both groups. The FR animals reached significantly higher CORT levels than

the CON group at 30 and 60 minutes post injection as was expected based on previous

publications looking at both rats [43] and pigs [142]. It has previously been demonstrated

that peak levels of total CORT are significantly higher in 9- and 15-month chronic FR

rats compared to CON in response to an ACTH challenge [43]. Based on the enhanced

response of serum CORT to exogenous ACTH in the FR group compared to the ad

libitum animals, the current data indicate that chronic FR increases the sensitivity of the

adrenal gland to ACTH.

Faecal CORT levels displayed a diurnal rhythm after the ACTH administration. A return

to baseline levels after an induced response is important since maintaining low levels of

CORT with a flattening of the diurnal CORT rhythm may indicate a neuroendocrine

dysfunction such as that observed in depression [143]. CON animals peaked between

16:00 and 18:00hrs and FR animals peaked between 18:00 and 20:00 hrs. Following an

62

ACTH injection, Lepschy et al. [6] recorded a 4-12h increase in FCM concentrations

which remained elevated over a period of 4-19h. Similar to their study, both CON and FR

animals also peaked for 10 hrs.

There was little or no significant difference between group peak FCM values after an

ACTH challenge suggesting that FCM measurements may not be sensitive enough to

detect acute stress in chronically food restricted animals. The significant increase in

CORT observed in the serum is a reflection of an immediate biological response to a

stressor at a given time. FCM response to an ACTH challenge reflects a biological

response to a stressor over a cumulative time frame, which may have resulted in a less

dramatic increase in FCM compared to Serum CORT. These results are not surprising as

a prior study analyzing two diets found that one group of mice excreted significantly

higher levels of FCM compared to the other group throughout the experiment. However,

even though the effect of the ACTH challenge could readily be detected, it had a

considerably lesser impact when comparing FCM levels than did the difference in diet

[22]. FR rats did not show a higher peak CORT than the ad libitum fed rats in response to

an ACTH challenge, but they remained elevated for a longer duration resulting from an

overall greater total amount of CORT release.

5.6 Limitations

One limitation of this study was that the estrous cycle was not recorded in the rats. The

nadir and peak values of female rats vary with the estrous cycle: daily CORT means are

lowest on the day of estrus and rise progressively during metestrus, diestrus, and

63

proestrus [13]. Peak faecal CORT levels on prostrus were two or three times higher than

peaks during estrus or metestrus. Peak levels reached ~75ng/ 3h for estrus phase,

~100ng/3h for metestrus phase, ~150ng/3h for diestrus phase, and ~200ng/3h for the

proestrus phase. Not recording the estrous cycle could have affected values in the current

study, as serum CORT levels were lower than previously reported data possibly because

some animals were in their estrus phase.

Another limitation with this study was the fewer amount of samples produced in food

restricted animals. FR animals tended to excrete the most 2 to 4 hours after feeding time,

but at other collection times only around half the animals had samples. However, despite

having fewer samples compared to the CON animals, we were still able to find

significant increases in the overall CORT levels.

Blood sampling on consecutive days appears to have resulted in an anticipatory increase

in CON CORT levels whereby a sequential morning blood measurement resulted in

increased serum CORT. Baseline 2 was higher than Baseline 1, and Day 8 baseline

measurement was higher than Day 7. The days in between blood sampling may have

abolished the anticipatory stress as CORT levels returned to initial baseline levels.

Furthermore, while Day 8 Baseline CORT was higher than Day 7, the increase was not as

prominent as between Baseline 2 and Baseline 1. This is in line with repeated stress

studies, which indicate that the CORT response subdues with further exposure to

stress.[144-145].

64

Finally, the current study did not record the volume of water consumed by animals, and it

is possible that food restriction lead to decreased water intake as indicated by previous

data [146]. Kiss et al. [147] demonstrated that food and water deprivation can lead to

further increases in plasma CORT compared to food deprivation alone (9.2±2.2 vs

6.9±1.2ug/dl, respectively). The average CORT of the CON animals in that study was

1.8ug/dl. Thus, the addition of complete water deprivation only increased the CORT

response to food deprivation alone by 17%. While water intake was not quantified in the

current study, it was apparent that at least some water was being ingested since water had

to be replenished to fill the bottles on a daily basis. Thus, the effect of possible

dehydration in this study would likely be much less than that reported by Kiss et al.

[147]. Additionally, the observed difference between CORT in FR vs. CON animals in

the current study is 183% greater than could be expected from the influence of

dehydration.

5.7 Conclusion

The current paper was able to measure CORT non-invasively in FR rats. It was

demonstrated that 7 days FR results in increased serum CORT, and we also, for the first

time, showed that 7 days food restriction results in increased 24hr mean FCM. While FR

animals responded with significantly higher serum CORT values than CON to an ACTH

challenge, there was little or no significant difference between group peak FCM values.

The insignificant difference between the two groups suggests that FCM measurements

may not be sensitive enough to detect acute stress in chronically food restricted animals.

The in-house assay used in the current study was validated as measurements show a clear

65

circadian rhythm in CON animals, FCM values were higher in FR rats, and both groups

responded to an ACTH challenge with an elevated CORT response. Therefore, given the

non-invasive nature of the measurement, it is recommended that future studies measure

stress and /or CORT in FR rats via fecal sampling. One such application might be to

examine FCM in response to chronic food restriction to better understand the disease of

Anorexia Nervosa and its response to exercise and/or refeeding. In addition, future

studies can test how this hormone is correlated to other variables such as body mass,

leptin, and StAR protein. Unlike serum CORT measurements, FCM integrates both the

baseline and total free CORT released, and thus provides a powerful indicator of the

physiological state of the animals.

66

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Appendix 1A Animal Use Protocol Form

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!!Appendix 2 Parallelism and Recovery Test

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3A Animal Body Mass (g) and Faecal Corticosterone Metabolites at Baseline, Day 7, and Day 8 ACTH. Animals Baseline

Weight (g)

Day 7 Weight (g)

Day 7 Body Mass as a % of original body weight

24hr Avg. Baseline FCM (ng/g)

24hr Avg. Day 7 FCM (ng/g)

24hr Avg. Day 8 FCM (ng/g)

C1 314.7 318.6 101.1 339.5 336.4 550.3

C2 305.3 317.36 103.9 530.4 456.1 550.9

C3 314.7 317 100.7 438.8 572.8 653.5

C4 308.7 321.7 104.2 268.5 249.3 624.3

C5 310.0 330.0.0 106.4 364.2 363.4 501.9

C6 287.6 305.6 106.3 321.4 348.1 813.4

C7 266.0 292.2 109.8 304.8 258.2 362.3

C8 267.0 282.4 105.8 276.7 n/a 238.5

FR1 341.2 292.0 85.6 197.2 n/a n/a

FR2 307.6 285.0 92.6 308.7 460.1 422.0

FR3 318.0 294.0 92.4 288.4 480.9 606.7

FR4 293.5 258.0 87.9 394.8 477.2 531.3

FR5 301.0 270.4 89.8 376.1 635.5 970.8

FR6 275.0 241.0 87.6 546.5 800.7 1225.7

FR7 268.7 238.8 88.9 272.857 209.1 211.4

FR8 264.0 234.0 88.6 198.1 179.4 327.8

FR9 268.8 237.5 88.3 252.2 286.0 286.3

FR10 263.8 236.0 89.5 185.3 265.2 317.4

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Weight is in grams. FCMs are averaged over 24 h for each animal. Control group (C) included animals fed ad libitum and food restricted (FR) group was fed 7g at 14:00 h everyday.

3B. Date of Birth and Arrival of Animals

Batch Date of Birth Date of Arrival

1 March 10 2012 July 11 2012

2 May 5 2012 September 19 2012

3C Average mass of food consumed per day measured in grams ± S.D.

Control FR

Day Food Mass (g)

1 28.42±5.40 7.12±.0.16

2 25.96±0.97 7.13±.0.22

3 22.37±10.29 7.27±0.33

4 21.48±11.17 7.31±0.35

5 22.95±3.72 7.35±0.27

6 22.85±5.07 7.69±0.34

7 25.01±0.14 7.5±0.25

8 26.93±2.61 7.66±0.45

9 21.34±0.86

10 24.58±50.30

11 27.10±10.44

12 25.88±1.42

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4A Circadian Rhythm of Control animals at Baseline and Day 7.

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:00

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

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20:00-22:00

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24:00-2:00

2:00-4:00

4:00-6:000

200

400

600Baseline1Day 7

Time (hrs)

FCM

(ng/

g)

Black bars on X-axis represent the 12hr dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00h. Data have been double-plotted for clarity.

4B Circadian Rhythm of FR animals at Baseline and Day 7.

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:00

6:00-8:00

8:00-10:00

10:00-12:00

12:00-14:00

14:00-16:00

16:00-18:00

18:00-20:00

20:00-22:00

22:00-24:00

24:00-2:00

2:00-4:00

4:00-6:000

200

400

600

Baseline1Day 7

Time (hrs)

FCM

(ng/g

)

Black bars on X-axis represent the 12hr dark cycle from 8:00-20:00hrs. The grey arrows indicate the time of blood sampling at 07:00h. Data have been double-plotted for clarity. !

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Non-Invasive Measurement of Corticosterone in … Non-invasive Measurement of Corticosterone in Food Restricted Rats Deborah Chava Cole Masters of Science Graduate Department of Exercise