Diet-Induced Obesity in Female Mice Leads to Offspring Hyperphagia, Adiposity, Hypertension, and Insulin Resistance: A Novel Murine Model of Developmental Programming

Diet-Induced Obesity in Female Mice Leads to OffspringHyperphagia, Adiposity, Hypertension,

and Insulin ResistanceA Novel Murine Model of Developmental Programming

Anne-Maj Samuelsson, Phillippa A. Matthews, Marco Argenton, Michael R. Christie,Josie M. McConnell, Eugene H.J.M. Jansen, Aldert H. Piersma, Susan E. Ozanne,

Denise Fernandez Twinn, Claude Remacle, Anthea Rowlerson, Lucilla Poston, Paul D. Taylor

Abstract—Maternal obesity is increasingly prevalent and may affect the long-term health of the child. We investigated theeffects of maternal diet-induced obesity in mice on offspring metabolic and cardiovascular function. Female C57BL/6Jmice were fed either a standard chow (3% fat, 7% sugar) or a palatable obesogenic diet (16% fat, 33% sugar) for 6 weeksbefore mating and throughout pregnancy and lactation. Offspring of control (OC) and obese dams (OO) were weanedonto standard chow and studied at 3 and 6 months of age. OO were hyperphagic from 4 to 6 weeks of age comparedwith OC and at 3 months locomotor activity was reduced and adiposity increased (abdominal fat pad mass; P�0.01).OO were heavier than OC at 6 months (body weight, P�0.05). OO abdominal obesity was associated with adipocytehypertrophy and altered mRNA expression of �-adrenoceptor 2 and 3, 11�HSD-1, and PPAR-� 2. OO showedresistance artery endothelial dysfunction at 3 months, and were hypertensive, as assessed by radiotelemetry (nighttimesystolic blood pressure at 6 months [mm Hg] mean�SEM, male OO, 134�1 versus OC, 124�2, n�8, P�0.05; femaleOO, 137�2 versus OC, 122�4, n�8, P�0.01). OO skeletal muscle mass (tibialis anterior) was significantly reduced(P�0.01) OO fasting insulin was raised at 3 months and by 6 months fasting plasma glucose was elevated. Exposureto the influences of maternal obesity in the developing mouse led to adult offspring adiposity and cardiovascular andmetabolic dysfunction. Developmentally programmed hyperphagia, physical inactivity, and altered adipocyte metabo-lism may play a mechanistic role. (Hypertension. 2008;51:383-392.)

Key Words: obesity � pregnancy � developmental programming � metabolic syndrome� appetite � blood pressure � mouse

Obesity among women of reproductive age is presenting acritical challenge to health care. 29% of USA women

aged 20 to 39 years are reported to be clinically obese1 andthere is serious concern in many European countries over theincreasing obesity among young women.2

While obesity is associated with increased risk of almostevery common complication of pregnancy, obesity in themother may play a direct role in transmission of an obeso-genic and diabetogenic trait from generation to generation.Increasing evidence suggests that children born of pregnan-cies complicated by either obesity or related gestationaldiabetes mellitus (GDM) are at increased risk of obesity,impaired glucose tolerance, and other facets of the metabolicsyndrome.3

Animal models have proven invaluable in interrogation ofassociations between maternal diet and body composition andoffspring phenotype.4 Those studies which have addressedeffects of maternal calorific excess, including several fromour laboratory, have generally fed rats diets rich in animalfat.4–7 Because young women of reproductive age oftenconsume excessive amounts of sugars as well as fats,8 therelevance of a diet rich in fat alone is limited. In this study, weinduced obesity by feeding mice a highly palatable diet richin sugars and animal fat, and addressed the hypothesis thatdiet-induced obesity during pregnancy can transmit a propen-sity for adiposity, glucose intolerance, and cardiovasculardysfunction to the offspring. Obesity was induced in femalemice and offspring cardiovascular and metabolic function

Received September 19, 2007; first decision October 6, 2007; revision accepted November 26, 2007.From the Division of Reproduction & Endocrinology (A.-M.S., P.A.M., M.A., M.R.C., J.M.M., L.P., P.D.T.) and Applied Biomedical Research Group

(A.R.), King’s College London, UK; the Laboratory for Health Protection Research (E.H.J.M.J., A.H.P.), National Institute for Public Health and theEnvironment, Bilthoven, Netherlands; the Department of Clinical Biochemistry (S.E.O., D.F.T.), University of Cambridge, UK; and the Institute of LifeSciences (C.R.), Universite Catholique de Louvain, Louvain-la-Neuve, Belgium.

Correspondence to Dr Paul Taylor, Division of Reproduction & Endocrinology, Kings College London, 10th Floor North Wing, St Thomas’ Hospital,London SE1 7EH, UK. E-mail [email protected]

© 2008 American Heart Association, Inc.

Hypertension is available at http://hyper.ahajournals.org DOI: 10.1161/HYPERTENSIONAHA.107.101477

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was assessed at 3 and 6 months of age using radio-telemetry,small vessel myography, and assessment of glucose toler-ance. Because it has been suggested that offspring adipocytemetabolism may be permanently influenced by maternalnutritional status, and contribute to adiposity,9,10 adipocytemorphology and expression of relevant adipocyte genes wasdetermined. In view of the potential role of capillary rarefac-tion in hypertension and metabolic syndrome, and previousimplication in developmental programming,11 capillary den-sity in skeletal muscle was also investigated.

MethodsExperimental AnimalsAll studies were approved by Local Ethics Committee, and con-ducted under UK Home Office License. Female C57BL/6J mice,proven breeders (one previous litter) and approximately 100 days old

(Charles River Laboratories, UK) were maintained under controlledconditions (25°C, 12-hour light/dark cycle) and fed either a standardchow diet (7% simple sugars, 3% fat, 50% polysaccharide, 15%protein [w/w] RM1, Special Dietary Services, energy 3.5 kcal/g,n�20), or a semisynthetic energy-rich and highly palatable obeso-genic diet (10% simple sugars, 20% animal lard 28% polysaccharide,23% protein [w/w], Special Dietary Services, energy 4.5 kcal/g,n�30). The pelleted obesogenic diet was supplemented by adlibitum access to sweetened condensed milk (approx 55% simplesugar, 8% fat, 8% protein, w/w, Nestle) with added micronutrientmineral mix (AIN93G, Special Dietary Services). Macronutrient andcalorific intake were calculated from measured daily intake of pelletsand milk (approx 16% fat, 33% simple sugars, 15% protein, energy4.0 kcal/g). After 6 weeks on the diet, animals were mated (day 0pregnancy signified by the appearance of a copulation plug), andmaintained on the obesogenic diet throughout gestation and suckling.Weight gain and food intake were measured throughout gestation. 48hours after delivery litters larger than 6 pups were reduced to 6(litters less than 4 not used), with equal sex ratios where possible. At

Figure 1. Maternal characteristics. Maternal body weight (A) and calorific intake (B) in dams fed either the control (open symbols, n�8)or an obesogenic diet (closed symbols, n�8). C, Maternal plasma analytes at gestational day 18 (n�9). D, Maternal plasma analytes atweaning (n�8 to 12) Open bar, control; closed bars, obese dams. E, DEXA scan of control (15% body fat) and obese dams (34% bodyfat) at weaning. ***P�0.001, **P�0.01, * P�0.05 vs control.

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3weeks, all pups were weaned onto standard chow. Samples of milkwere obtained under anesthesia (isofluorane) without recovery fromdams at weaning (for measurement of leptin) after injection ofoxytocin. Body composition analysis was also performed in asubgroup of dams at weaning by dual energy X-ray absorptiometry(DEXA, Lunar PIXImus; Integrity Medical Systems Inc).

A subgroup of dams (n�9) were euthanized at gestational day 18(G18) and nonfasted blood sampled for insulin, glucose, leptin,cholesterol, and triglyceride analysis.

Offspring body weight and food intake were recorded weekly, andmetabolic and cardiovascular parameters determined at 3 and 6months in one male and one female per litter. Heart, fat pad(inguinal), liver, and skeletal muscle (tibialis anterior) weights weredetermined in the same animals at necropsy.

Plasma AnalysisFasted plasma insulin, glucose, leptin, triglyceride, and cholesterolconcentrations were assessed after cardiac puncture was performedon OO and OC (please see http://hyper.ahajournals.org).

RadiotelemetryCardiovascular function was assessed by remote radio-telemetry inconscious freely moving mice after surgical implantation of probes(TA11PA-C10, O.D 0.4 mm, Data Science International Inc) usingcarotid artery placement (see supplementary methods for detail).Data are reported for one 24-hour period of 7 days recording.

Vascular FunctionSmall mesenteric arteries (approx 200 �m i.d.) were mounted on amyograph (MultiMyograph 610 mol/L, Danish Myo Technology) aspreviously described12 (please see the online supplemental material).

Capillary DensityTransverse sections (10 �m) were prepared from semitendinosusmuscle, snap frozen at necropsy at 3 months. Capillary imaging wascarried out by a modified ATPase staining method,13 and visualizedby light microscopy. Capillaries and muscle fibers from 3 randomareas from each of 3 sections from each muscle were counted (ScionImage software).

Figure 2. Offspring allometry and plasma analytes. Calorific intake (A) and body weight (B) in male and female offspring of control (OC,open symbols) and obese dams (OO, closed symbols) from weaning (n�8 per group). C, Offspring inguinal fat pad mass at 6 monthsof age. D, Fasted plasma insulin, glucose, leptin, triglyceride, and cholesterol concentrations in 3-month and 6-month-old offspring ofcontrol and obese dams (male and female combined). n�6 to 8 per group. Open bars, OC, closed bars, OO. ***P�0.001, **P�0.01, *P�0.05 vs OC.

Samuelsson et al Programming of Hypertension by Maternal Obesity 385



Glucose Tolerance TestGlucose tolerance was assessed after a single bolus of glucose(1g/kg, IP) after overnight fast and tail venous blood glucoseconcentration monitored at 0, 15, 30, 60, 90, and 120 minutes inconscious semirestrained animals (GlucoMen Sensor, A.MenariniDiagnostics).

Pancreatic Insulin ContentThe snap frozen whole pancreas was homogenized in acid ethanol.Insulin content was determined by RIA14 and expressed as insulincontent/mg tissue weight.

Adipose Cell SizeAdipocyte size was estimated in inguinal fat tissue from 3-monthmice. Adipocytes were fixed as described by Etherton et al15 (seesupplementary methods). Cell size was determined using ZEISS KS400 3.0 software using an Axioskop2 Mot Plus Zeiss microscope(Carl Zeiss GmbH).

Adipose Gene ExpressionReal-time polymerase chain reaction (PCR) in adipose tissue wasperformed using standard laboratory techniques in RNA extractedfrom inguinal fat tissue from 3-month-old mice (for details andprimer sequences of genes investigated please see the online datasupplement).

Statistical AnalysisData are expressed as mean�SEM and compared using ANOVA orStudent t test unless otherwise stated. P�0.05 was regarded assignificant. In all cases “n” refers to the numbers of litters in eachgroup with one male and one female from a litter used for eachexperiment.

ResultsMaternalCalorific intake and weight gain were significantly greater indams fed the obesogenic diet compared with chow fedcontrols (Figure 1A and 1B). Although maternal weightsconverged at the end of gestation, subscapular brown adiposetissue mass was increased in late pregnancy (BAT weight [g]OC, 0.10�0.005 versus OO, 0.15�0.02, P�0.05) and ab-dominal fat mass was 4-fold greater relative to controls(WAT weight, G18 [g] OC, 1.02�0.08 versus OO,4.00�0.42, P�0.0001). At G18, obese dams were normogly-caemic but were hyperinsulinaemic and hyperleptinaemic(Figure 1C). After suckling and despite a decline in WATmass from pregnancy in both groups (WAT weight atweaning [g] OC, 0.28�0.07 versus OO, 1.43�0.04,

P�0.0001) obese dams demonstrated hyperleptinaemia, hy-perinsulinaemia with hyperglycaemia, and hypercholester-olaemia (Figure 1D). The milk leptin concentration wassignificantly raised at weaning (Leptin [ng/mL] OC, 1.9�0.2versus OO, 4.7�1.5, P�0.05, n�3). Maternal obesity wasconfirmed by DEXA at weaning (2.7-fold increase in fatmass, Figure 1E). Conception rates were reduced in the obesegroup (75% versus 90% in control), but 48-hour litter sizewas similar (OC, 5.9�0.4 versus OO, 5.6�0.3, PNS). OOneonates were heavier than OC at 48 hours (males andfemales combined, weight [g] OC, 1.31�0.04 versus OO,1.66�0.05, P�0.01, n�16) and at weaning (male, weight [g]OC, 9.06�0.2 versus OO, 12.6�0.6, P�0.001, n�8,P�0.001; females P�NS).

OffspringEnergy intake and body composition calorific intake wasgreater in OO versus OC from 4 to 6 weeks, before asignificant increase in body weight in OO (males at 12 weeks;females transiently form 5 weeks; Figure 2A and 2B). Maleand female OO showed a marked increase in abdominalinguinal fat pad mass at 3 and 6 months (Figure 2C; Table).There was a significant reduction in mass of the tibialisanterior muscle at 3 and 6 months (Table).

Plasma AnalytesAt 3 months, OO males and females had significantlyelevated plasma trigylceride, insulin, and leptin concentra-tions relative to OC. By 6 months, OO were hyperglycaemicand plasma insulin was significantly reduced. Leptin re-mained significantly elevated and cholesterol was also raised.Triglycerides were not different from OC (Figure 2D).

Blood Pressure, Heart Rate, and ActivityAt 3 months, night-time (active phase) systolic blood pressure(SBP) was significantly higher in male and female OO versusOC (Figure 3A). Diastolic blood pressure (DBP) was in-creased in male OO only (data not shown). Daytime SBP andDBP did not differ significantly in either group. Nighttimemean arterial pressure (MAP) was significantly raised inmales and female OO versus OC (male OO, MAP [mm Hg]111�2 versus OC, 98�1, P�0.001, female OO, 115�2versus OC, 107�3, P�0.02, n�6 to 8). Male and femalenighttime heart rate was reduced in OO versus OC (Figure3B). Locomotor activity during the nighttime was signifi-

Table. Body Weight, Inguinal Fat Pad Mass, Heart, Liver, and Muscle Weights (tibialis anterior) of 3- and 6-Month-Old Offspring ofControl (OC) and Obese (OO) Dams (n�8 per Group)

Sex/Group

Body Weight (g) Fat Pad (mg/g BW) Heart (mg/g BW) Liver (mg/g BW) Muscle (mg/g BW)

3 Months 6 Months 3 Months 6 Months 3 Months 6 Months 3 Months 6 Months 3 Months 6 Months

Male

OC 23.2�0.4 26.5�0.4 12.1�0.4 14.8�0.5 7.9�0.4 6.5�0.5 36.5�2.8 31.8�0.9 6.5�0.2 7.1�0.2

OO 25.5�0.5† 32.5�0.4† 16.4�1.5‡ 19.8�0.5* 7.4�1.1 5.5�0.4 43.9�2.0‡ 38.5�1.8‡ 4.4�0.6‡ 4.3�0.2*

Female

OC 20.8�0.4 21.6�0.4 12.3�1.1 8.8�0.6 7.1�0.3 5.6�0.6 38.7�1.2 35.7�0.6 6.0�0.2 5.9�0.2

OO 21.8�0.2 23.6�0.3† 15.3�0.6‡ 12.2�1.1† 7.9�0.3 5.3�0.8 38.2�1.9 30.3�0.6 5.2�0.2‡ 4.5�0.3†

*P�0.001, †P�0.01, ‡P�0.05 vs offspring of control dams (ANOVA t test). OC indicates offspring of control dams; OO, offspring of obese dams. Values aremean�SEM, n�8 per group.




cantly reduced in 3-month male and female OO versus OC(Figure 3C). At 6 months, nighttime, but not daytime, SBPwas significantly higher in male and female OO versus OC(Figure 4A). ANOVA showed an association with gender andsystolic blood pressure in OO, with a greater effect in femaleOO. Nighttime mean arterial pressure (MAP) was signifi-cantly raised in males and female OO versus OC (male OO,MAP [mm Hg] 120�3 versus OC 111�2, P�0.05, femaleOO 119�2 versus OC 113�1, P�0.05, n�6 to 8). Male andfemale OO heart rate was increased during day and night(Figure 4B). At 6 months only female OO were less active(Figure 4C). Because estrus cycle stage has been related toactivity in mice we examined activity over the full week’srecording and found a sustained reduction in activity.

Resistance Artery FunctionAt 3 months, increased reactivity to NA was observed in maleand female OO versus OC (Figure 5A). ACh-induced relax-

ation was significantly impaired (Figure 5B). Endothelium-independent responses to NO were similar in OO and OC(RM ANOVA, P�NS). At 6 months, resistance arteryfunction was similar between groups (data not shown),because of deterioration of the ACh-induced relaxation withage in controls (max relaxation to ACh [%NA-inducedtension] male and female combined, OC, 40.3�6.4 n�10versus OO, 41.4�6.6 [n�12] P�NS).

Capillary DensityCapillary:fiber density was similar in OO and OC whenexamined across the whole muscle area (capillaries permuscle fiber mean�SEM, OC, 1.13�0.07 n�10 versus OO,1.14�0.09, n�9, P�NS).

Glucose Tolerance and Pancreatic Insulin ContentIn addition to abnormal fasting glucose and insulin, glucoseintolerance was evident at 3 and 6 months in male and female

Figure 3. Cardiovascular parameters at 3 months. Systolic blood pressure (A), heart rate (B), and locomotor activity (C) in 3-month-oldmale and female offspring of obese dams (OO, closed circles) compared with offspring of control dams (OC, open circles). **P�0.01,*P�0.05 RM ANOVA for dark phase (bar), n�6 to 8 per group.




OO versus OC (Figure 6A and 6B), with a prolongedrecovery phase in OO, although only male OO showed asignificantly different area under the glucose concentrationcurve. Pancreatic insulin content was raised in male andfemale OO versus OC at 3 months. At 6 months female OC(P�0.05), but not male OC insulin content, had declined withage. Male OO insulin content was significantly lower thanOC (Figure 6C).

Adipose Cell Size DistributionAt 3 months, adipocyte hypertrophy in OO was evident froma rightward shift in cell size distribution compared with OOin adipocytes of inguinal fat tissue (Figure 7A), and asignificant increase in mean adipocyte diameter (mean cellsize [microns] OC, 48.1�1.60 versus OO, 55.7�1.65, n�3,P�0.05).

Adipose Gene ExpressionAt 3 months mRNA expression of �2 (P�0.05) and �3

(P�0.05) adrenoreceptors and 11-�HSD type 1 (P�0.01)

was significantly reduced in OO versus OC mice. PPAR�type 2 expression was significantly increased in OO (P�0.05,Figure 7B).

DiscussionIn this study we explored the long-term effects of obesityduring pregnancy and lactation on cardiovascular and meta-bolic function of the offspring. Although the detrimentalconsequences of maternal undernutrition have been exten-sively studied, little attention has been directed towardmaternal overnutrition, with previous emphasis being placedon the consequences for the offspring of a maternal diet richin fat. In contrast to man, rodents reduce their food intakewhen fed a fat-rich diet, and in these studies the dams havegenerally not become overtly obese.6,16–19 Mice also demon-strate strain differences in response to high-fat diets,20 androdent models of fat feeding in pregnancy therefore havelimited relevance to human obesity. Our current approachwas to use a highly palatable diet to induce maternal obesity.

Figure 4. Cardiovascular parameters at 6 months. Systolic blood pressure (A), heart rate (B), and locomotor activity (C) measured byradio-telemetry in 6-month-old male and female offspring of obese dams (OO, closed circles) compared with offspring of control dams(OC, open circles). **P�0.01, *P�0.05 RM ANOVA for day or nighttime (bar) n�6 to 8 per group.




Mice were fed an obesogenic diet, in which 16% of calorieswere provided by saturated fat, and 30% from simple sugars,equivalent to high values within the normal dietary range inman and hence reflective of human diet and obesity.8 Off-spring displayed hyperphagia, increased adiposity, and anadult metabolic syndrome–like phenotype. As we have re-ported previously in offspring of fat fed rat dams,12 genderdifferences were apparent, particularly in relation to glucosehomeostasis. To our knowledge this is the first report ofpersistent effects of maternal diet-induced obesity in mice onoffspring cardiovascular and metabolic function. Two previ-ous studies have addressed the effects of a maternal hyper-calorific diet in mice on the offspring, but neither wasassociated with maternal obesity.7,21

The offspring phenotype could arise directly from influ-ences of the maternal high energy diet or may be theconsequence of the increased maternal fat mass per se.Previous studies, including some from our own laboratory,suggest an influence of dietary fat, as offspring of nonobeserats fed a fat-rich diet during pregnancy and suckling developincreased body fat mass6,12,19,22,23 and insulin resis-tance,16,23,24 despite eating a balanced diet from weaning.However, not all of these report an increase in offspring fatmass25 and the fatty acid composition of the fat enriched dietappears critical, with excessive maternal saturated fat intakebeing key.16,18 An influence of maternal obesity per se hasbeen suggested by recent studies of Yamashita et al26 andLambin et al,10 which have shown increased fat mass in adultwild-type progeny of obese and insulin resistant heterozygousleptin receptor–deficient (Leprdb/�) mice. Complex interac-tions between dietary and obesity related metabolic sequelaeare therefore likely to set in train events leading to offspring

obesity. Genotypic susceptibility could play an additive role,as suggested by Levin and colleagues; using rats bred to beobesity prone or resistant, these authors have shown clearinteractions between maternal adiposity and offspring geno-type in the development of offspring adiposity.27

Several mechanistic pathways may contribute to develop-ment of adiposity in the offspring, and we present evidencefor both hyperphagia and reduced energy expenditure in theyoung animals with daily energy intake being increasedbefore increased weight gain, suggesting hyperphagia as thedriving factor. Bayol et al recently demonstrated increasedenergy intake in offspring of “junk food”–fed rats, but incontrast to the present study, this was only evident whenoffspring were presented with a highly palatable hypercalo-rific diet, indicative of programming of food preference forsugary and fatty items, rather than hyperphagia.28 The suck-ling period in rodents has been identified as a critical timewindow for development of hyperphagia. Postnatal overfeed-ing induced by restriction of litter size immediately postpar-tum29 or by cross-fostering growth restricted offspring tonormal dams9 is associated with increased food intake inadult offspring, and offspring of diabetic rats exposed totransient neonatal hyperinsulinaemia develop hyperphagiaand adult obesity.30 The mice dams in the present study werehyperinsulinaemic and hyperglycaemic at weaning, whichwould be likely to trigger a neonatal insulin response via milkingestion. Leptin levels during the suckling period havepreviously been implicated in developmental programming ofappetite31 with exogenous administration of leptin to neonatalmice promoting an obese phenotype.32 Further studies usingaccurate methods of assessment will determine whethermetabolic rate is also “programmed” in the OO offspring,particularly because the large increase in energy intake wasassociated with only a modest rise in body weight.

Male and female offspring of the obese mice were lessphysically active at 3 months of age. Severe maternal under-nutrition in the rat results in offspring with reduced loco-motor activity and obesity,33 but little attempt has beenmade to measure physical activity in hypernutritionalprogramming models, although offspring of “junk food”–fed rat dams show no evidence of reduced activity asassessed during the light phase.28 The current findingssuggest programmed inactivity is likely to contribute to theincreased adiposity observed.

Adipocyte hypertrophy was also evident in offspring ofobese dams, as previously reported in weanling rats fromdams fed an obesogenic diet34 but not previously observed inadulthood. Persistent alteration in mRNA expression ofPPAR�2 and the �2 and �3 adrenoceptors as reported herecould increase adipocyte adipogenesis and decrease lipolysis,respectively.35,36 Whether acquired through permanent mod-ulation of adipose metabolism in early development orsecondary to increased fat mass, these changes could contrib-ute to increased fat mass. The observed decrease in11�HSD-1 would be anticipated to decrease adipocyte dif-ferentiation and is contrary to the observation that transgenicmice selectively overexpressing 11�HSD-1 are obese.37

The development of glucose intolerance with age, par-ticularly in the male offspring of the obese dams, was

Figure 5. Vascular function in isolated mesenteric arteries at 3months. Responses to noradrenaline (NA) (A) and acetylcholine(ACh) (B) in isolated small mesenteric arteries. Offspring of con-trol (OC open symbols) and obese dams (OO, closed symbols)n�8 per group *P�0.05, RM ANOVA.




associated with altered plasma and pancreatic insulincontent. Fasting plasma insulin was elevated in males andfemales at 3 months, suggesting insulin resistance. By 6months the males showed frank diabetes with fasting

hyperglycemia, reduced fasting plasma insulin, and re-duced pancreatic insulin content, indicative of beta cellexhaustion, as reported previously in offspring of rats feda lard-rich diet.38 It remains to be determined whether fetal

Figure 6. Glucose tolerance tests at 3 and 6 months. Blood glucose concentrations after administration of a bolus dose of glu-cose (1g/kg, i.p.) in 3-month-old male (left panel) and female (right panel) offspring (A); and male (left panel) and female (rightpanel) offspring of control (OC) and obese dams (OO) at 6 months (n�6 to 8) (B). Offspring of control (OC, open symbols) andobese dams (OO, closed symbols) * P�0.05 area under the glucose curve vs control. Pancreatic insulin content at 3 months (C)and 6 months (D) of age (n�5 to 10). *P�0.05 vs control, †P�0.05, 3 month vs 6 month within group comparison.

Figure 7. Adipose tissue in 3-month offspring Adipose cell size distribution and gene expression in inguinal fat. Cell diameterexpressed as numbers of cells for each size range (P�0.01, Kolmogorov-Smirnov nonparametric test) (A) and mRNA expressionof �2 and �3 adrenoreceptors and 11-� hydroxysteroid dehydrogenase (�HSD-1) and PPAR� type 2 (n�6 to 8) 0ffspring of control(OC, open bars) and obese dams (OO, closed bars) (B). *P�0.05 vs control. mRNA expressed as copy number divided by thegeometric mean of 3 reference genes (HPRT, Actin beta, TATA box), multiplied by 1000.




and neonatal pancreatic development is compromised, asshown by others, in neonates of dams fed a fat-rich diet39

or whether this occurs as a result of offspring adiposity.To our knowledge this is the first study to demonstrate

hypertension in offspring of obese mice. Using the samemethod of radiotelemetry we previously reported raised bloodpressure in rats prenatally exposed to a maternal diet rich infat.12,17 Endothelium-dependent relaxation to acetylcholinewas impaired in offspring of obese mice suggesting endothe-lial dysfunction, and as suggested in other rodent models ofdevelopmental programming,5 this could contribute to thehypertension observed. However, raised blood pressure couldalso be a direct consequence of the increased fat mass.40 Theobserved fall in heart rate at 3 months relative to controlscould reflect the normal baroreceptor response to increasingblood pressure, whereas at 6 months the abnormally highheart rate may be indicative of diminished baroreceptorsensitivity with age.

Impaired angiogenesis and microvascular rarefaction hasbeen linked to hypertension in undernutrition models ofdevelopmental programming11 and may also contribute to thedevelopment of insulin resistance. Because we found noevidence for reduced capillary density in the skeletal muscletissue in the offspring of the obese, vascular rarefaction isunlikely to play a role in this model, although the observedreduction in skeletal muscle mass could contribute to insulinresistance.

PerspectivesThis study provides the first evidence that maternal diet–induced obesity in the mouse is associated with increased riskof metabolic and cardiovascular disease in the offspring.Mechanistically, persistent changes in hypothalamic energybalance regulatory centers, locomotor behavior, and adipo-cyte metabolism are likely to play a role in development ofoffspring adiposity, which in turn could contribute to thehypertension and insulin resistance observed. The underlyingmolecular basis, including the potential for persistent modu-lation of relevant gene expression through epigenetic mech-anisms, remains to be determined. Whatever the underlyingpathways, obesity in mouse pregnancy arising from consump-tion of a sugar- and fat-rich diet of the proportions oftenconsumed by obese individuals can have persistent anddeleterious consequences for the offspring, independent ofgenetic susceptibility. Corroboration in other mouse strainsand species is important, but this model offers the potentialfor mechanistic and intervention studies relevant to obesity inpregnant women.

AcknowledgmentsWe thank William Jefferson for his assistance in the assay ofpancreatic insulin content.

Sources of FundingThis study was supported by EARNEST (EU Framework 6),Tommy’s the baby Charity (UK), the British Heart Foundation,Biotechnology and Biological Sciences Research Council and Dia-betes UK.

DisclosuresNone.

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Twinn, Claude Remacle, Anthea Rowlerson, Lucilla Poston and Paul D. TaylorMcConnell, Eugene H.J. M. Jansen, Aldert H. Piersma, Susan E. Ozanne, Denise Fernandez

Anne-Maj Samuelsson, Phillippa A. Matthews, Marco Argenton, Michael R. Christie, Josie M.Programming

Hypertension, and Insulin Resistance: A Novel Murine Model of Developmental Diet-Induced Obesity in Female Mice Leads to Offspring Hyperphagia, Adiposity,

Print ISSN: 0194-911X. Online ISSN: 1524-4563 Copyright © 2007 American Heart Association, Inc. All rights reserved.

is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231Hypertension doi: 10.1161/HYPERTENSIONAHA.107.101477

2008;51:383-392; originally published online December 17, 2007;Hypertension.

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Diet-Induced Obesity in Female Mice Leads to Offspring Hyperphagia, Adiposity, Hypertension, and Insulin Resistance: A Novel Murine Model of Developmental Programming