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doi: 10.1152/ajpregu.00316.2007294:R528-R538, 2008. First published 21 November 2007;Am J Physiol Regul Integr Comp Physiol

Thomas M. BadgerKartik Shankar, Amanda Harrell, Xiaoli Liu, Janet M. Gilchrist, Martin J. J. Ronis andoffspringMaternal obesity at conception programs obesity in the

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Maternal obesity at conception programs obesity in the offspring

Kartik Shankar,1,2 Amanda Harrell,1 Xiaoli Liu,1 Janet M. Gilchrist,1,4 Martin J. J. Ronis,1,2

and Thomas M. Badger1,3,4

1Arkansas Children’s Nutrition Center, Little Rock; and Departments of 2Pharmacology and Toxicology, 3Physiology andBiophysics, and 4Pediatrics, College of Medicine, University of Arkansas for Medical Sciences, Little Rock, Arkansas

Submitted 6 May 2007; accepted in final form 19 November 2007

Shankar K, Harrell A, Liu X, Gilchrist JM, Ronis MJ, BadgerTM. Maternal obesity at conception programs obesity in the offspring.Am J Physiol Regul Integr Comp Physiol 294: R528–R538, 2008. Firstpublished November 21, 2007; doi:10.1152/ajpregu.00316.2007.—Riskof obesity in adult life is subject to programming during gestation. Toexamine whether in utero exposure to maternal obesity increases therisk of obesity in offspring, we developed an overfeeding-based modelof maternal obesity in rats utilizing intragastric feeding of diets viatotal enteral nutrition. Feeding liquid diets to adult female rats at 220kcal/kg3/4 per day (15% excess calories/day) compared with 187kcal/kg3/4 per day for 3 wk caused substantial increase in body weightgain, adiposity, serum insulin, leptin, and insulin resistance. Lean orobese female rats were mated with ad libitum AIN-93G-fed male rats.Exposure to obesity was ensured to be limited only to the maternal inutero environment by cross-fostering pups to lean dams having adlibitum access to AIN-93G diets throughout lactation. Numbers ofpups, birth weight, and size were not affected by maternal obesity.Male offspring from each group were weaned at postnatal day(PND)21 to either AIN-93G diets or high-fat diets (45% fat calories).Body weights of offspring from obese dams did not differ fromoffspring of lean dams when fed AIN-93G diets through PND130.However, offspring from obese dams gained remarkably greater (P �0.005) body weight and higher %body fat when fed a high-fat diet.Body composition was assessed by NMR, X-ray computerized to-mography, and weights of adipose tissues. Adipose histomorphom-etry, insulin sensitivity, and food intake were also assessed in theoffspring. Our data suggest that maternal obesity at conception leadsto fetal programming of offspring, which could result in obesity inlater life.

pregnancy; developmental programming; body composition; adiposetissue

PREVALENCE OF OVERWEIGHT and obesity in the United Statescontinues to rise steadily with one in two adults being over-weight [body mass index, (BMI) � 25] and as many as one infive adults (� 60 million individuals) being obese (BMI � 30)(14, 35). Prevalence of overweight among children has nearlydoubled in the last two decades (33). Perhaps more alarming isthe steady increase in the risk of overweight even amonginfants (0–11 mo) (32, 42). This may have critical implica-tions, as overweight in infancy and childhood significantlyincreases the risk of obesity in adulthood (32, 33, 42). Consis-tent with greater numbers of overweight individuals in thepopulation is a remarkable rise in the incidence of obesityamong pregnant women (1, 9, 10, 36, 56). Pregravid over-weight is one of the most common high-risk obstetric condi-tions (8, 25), imparting increased risk of gestational diabetes

mellitus (GDM), pregnancy-related hypertension, preeclamp-sia, neonatal death, and other labor complications (8, 9, 25, 36).

While much attention is paid to obesity-related complica-tions during pregnancy, not much is known about the subtleeffects of the obese intrauterine environment on the immediateand long-term health of the offspring. Data from 287,213pregnant women clearly reveal that overweight/obese womenare more likely to give birth to heavier babies (�90th percen-tile), consistent with the data that maternal obesity per se, evenafter controlling for gestational diabetes, is associated withtwice higher incidence of heavier babies compared with nono-bese women (8, 27, 30, 41, 44). Data from the Center forDisease Control Pediatric Nutrition Surveillance survey sug-gest that the odds ratios for obese children growing into obeseadults are 2.1 to 8.8 times that of nonoverweight children (32).Hence, greater body weight at birth and weight gain early inlife clearly increases the risk of becoming overweight or obeseas an adult. This suggests that prepregnancy overweight andgestational obesity may lead to a self-reinforcing vicious cycleof excessive weight gain and adiposity that is passed on frommother to successive offspring. While the underlying mecha-nisms of such maternal obesity-induced programming remainunclear, the hypothesis has important implications in explain-ing the rapid rise in obesity.

Studies of inheritance unambiguously show BMI of childrencorrelate more closely with maternal BMI than paternal BMI,suggesting that in addition to the genetic influences, the ma-ternal in utero environment may contribute to the developmentof obesity in the offspring (12, 54). Hence, developing animalmodels to precisely delineate in utero influences in metabolicprogramming of obesity is critical to our understanding ofunderlying mechanisms of how increased susceptibility ispassed onto the offspring. In the present report, we examinedeffects of obesity specifically at conception on the program-ming of obesity in the offspring. We have controlled for directmaternal and paternal genetic influences by developing amodel of overnutrition-driven maternal obesity so as to exam-ine only the metabolic effects of obesity on offspring health.Furthermore, by use of cross-fostering, we have excludeddirect influences of maternal obesity on nursing offspring. Ourdata strongly suggest that exposure to maternal obesity in uteroleads to metabolic programming, which results in increasedsusceptibility to obesity in the offspring.

MATERIALS AND METHODS

Animals and chemicals. Female Sprague-Dawley rats (150–175 g)were obtained from Charles River Laboratories (Wilmington, MA).

Address for reprint requests and other correspondence: Kartik Shankar,Arkansas Children’s Nutrition Center, 1212 Marshall St., Slot 512-20B, LittleRock, AR 72202 (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the paymentof page charges. The article must therefore be hereby marked “advertisement”in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Am J Physiol Regul Integr Comp Physiol 294: R528–R538, 2008.First published November 21, 2007; doi:10.1152/ajpregu.00316.2007.

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Animals were housed in an American Association for Accreditation ofLaboratory Animal Care-approved animal facility. Protocols for ani-mal maintenance and experimental treatments were conducted inaccordance with the ethical guidelines for animal research estab-lished and approved by the Institutional Animal Care and UseCommittee at the University of Arkansas for Medical Sciences.Unless otherwise specified, all chemicals were obtained fromSigma-Aldrich (St. Louis, MO).

Experimental protocol. Virgin female Sprague-Dawley rats wereintragastrically cannulated and allowed to recover for 10 days aspreviously described (3, 5, 6, 26, 45, 46). Rats were fed a liquid diet[20% protein (casein), 35% carbohydrate (dextrose and maltodextrin),and 45% fat (corn oil)] at National Research Council recommendedcaloric intake of 187 kcal/kg3/4 per day (referred to as lean dams) orwere overfed an obesegenic liquid diet (20% protein, 75% carbohy-drate, 5% fat) at 220 kcal/kg3/4 per day (15% excess, referred to asobese dams). Diets met caloric and nutritional guidelines recom-mended by the National Research Council, have been describedpreviously (3, 5, 6), and have been utilized by our group in a numberof studies (4, 19, 26, 43, 45, 46). In preliminary experiments, weobserved that while feeding at 187 kcal/kg3/4 per day mirrored bodyweight gains of ad libitum-fed controls (data not shown), feeding at220 kcal/kg3/4 per day led to substantial obesity. Infusion of liquiddiets was carried out using computer-controlled syringe pumps for 23h/day for 3 wk. Body weights were monitored three times a weekthroughout the infusion period. Animals had ad libitum access todrinking water. At the end of 3 wk of infusion, body composition wasnoninvasively estimated using NMR (EchoMRI; Echo Medical Sys-tems, Houston, TX) and X-ray computer tomography (X-ray CT,LaTheta LCT-100, Echo Medical Systems, Houston, TX) as detailedbelow. In a separate set of animals (n � 5 per group), fasting and fedlevels of serum glucose, insulin, leptin, nonesterified fatty acids(NEFA), and triglycerides were assessed. In addition, insulin sensi-tivity was examined following an oral glucose tolerance test (OGTT)as described in Serum hormones and OGTT.

To examine the long-term gestational effects of maternal obesity onthe offspring, lean (n � 7) and obese rats (n � 15) were allowed tomate with male rats for a period of 1 wk. At the time of mating (i.e.,after 3 wk of infusion), body weights clearly diverged (obese � lean).Body composition was measured using NMR and showed the sametrend as body weights. Each female rat was housed with one male andallowed ad libitum access to AIN-93G diet for this period. Followingmating, all female rats (lean and obese) received their respective dietsat 220 kcal/kg3/4 per day at the National Research Council-recom-mended caloric intake for pregnancy in rats. Body weights weremonitored three times a week. All rats were allowed to give birthnaturally. Numbers and sex of pups, birth weight, crown-to-rump andanogenital distance was measured for each pup on postnatal day(PND) 1. On PND2, four males and four female pups from each litterwere cross-fostered to dams that had been previously time-impreg-nated to give birth on the same day as the dams receiving infusiondiets. Cross-fostered dams were not cannulated and had ad libitumaccess to AIN-93G pelleted diets throughout lactation. Using thisexperimental paradigm, we ensured that offspring exposure to obese-genic influences was limited only to the maternal in utero environ-ment. Female offspring of lean and obese dams were used for separateexperiments and only data from male offspring are reported here.Male offspring from each dam group (n � 7–11 per group) wereweaned at PND21 to either regular AIN-93G diets (17% fat calories,caloric density 3.8 kcal/g) or to high-fat diets (HFD; 42% fat calories,caloric density 4.5 kcal/g) to mimic a postnatal obesegenic environ-ment. Body weights of the offspring were monitored weekly untilPND130. Body composition (NMR) was measured once a monththroughout the study. We also assessed body composition using X-rayCT scanning at PND120. Food intake was assessed at PND60 andPND120 over a two consecutive days and average caloric intake over24 h was calculated. OGTT was assessed in a subset of offspring (n �

5 per group) at PND120 following an overnight fast as describedbelow. Offspring were killed at PND130 and retroperitoneal, perire-nal, gonadal adipose tissues samples were collected for histology andwere flash frozen for subsequent analyses. Weights of organs andadipose tissues were collected. Serum levels of glucose, triglyceride,NEFA, and other hormones were assessed as described below. Adi-ponectin and resistin were assayed using commercially availableELISA (B-Bridge International, Sunnyvale, CA). Total lysates fromretroperitoneal adipose tissues were prepared in RIPA buffer (25 mMTris �HCl, 150 mM NaCl, 1% Nonidet P-40, 1% deoxycholic acid,0.1% SDS, and 2 mM EDTA) containing 1 mM PMSF and proteaseinhibitor cocktail (Sigma). Proteins (20 �g) were resolved on a 10%SDS-PAGE gel and transferred to nitrocellulose membranes. Immuno-blotting was carried out using standard procedures. Membranes wereincubated with anti-rabbit peroxisome proliferator-activated receptor-�(PPAR-�) mAB (Cell Signaling Technologies, Danvers, MA) in Tris-buffered saline with 0.05% Tween-20 containing 2.5% milk for 16 h at4°C. Following incubation with HRP-conjugated secondary IgG, mem-branes were washed with TBST, and proteins were visualized using WestPico enhanced chemiluminescence kit (Pierce, Rockford, IL) and de-tected by autoradiography. Immunoquantitation was performed bydensitometric scanning of the resulting autoradiograms using a mo-lecular imager (model GS700; Bio-Rad, Richmond, CA).

Body composition analyses. Body composition was assessed viathree independent methods, namely, whole animal body compositionby NMR (Echo Medical Systems, Houston, TX), computerized to-mography (X-ray CT scanning; LaTheta LCT-100; Echo MedicalSystems, Houston, TX), and postmortem dissected weights of retro-peritoneal, perirenal, and gonadal fat pads from rats. Echo NMR is aquantitative magnetic resonance body composition analyzer that uti-lizes the resonance energy of hydrogen nuclei in a magnetic field tocompute the density of the tissue. NMR is performed in consciousunanesthetized rats, and, unlike dual-energy X-ray absorptiometry, theNMR measurements are radiation-free and do not require the animalsto remain still. Each NMR measurement takes �1 min/rat, and allmeasurements were performed in duplicate. Indices of %fat and leanmass were derived using this technique. For CT analyses, �90sections, 1 mm apart, were acquired encompassing the entire visceralregion of the animal under isoflurane anesthesia. Approximate timefor acquisition of each slice is 4.5 s. Densitometric calculations of fatand muscle were performed using CT software (Aloka, Tokyo, Japan)using attenuation number thresholds of �120 to �500 for fat and�120 to 350 for muscle. Indices of %fat ratio (ratio of volumeoccupied by fat/volume occupied by lean tissue), %fat mass, and

Fig. 1. Body weights of female rats fed diets via total enteral nutrition (TEN)at 187 kcal/kg3/4 per day (lean, n � 7) or 220 kcal/kg3/4 per day (obese, n �15) for 21 days prior to mating. Infusion of diets was carried out for 23 h/dayvia computer-controlled syringe pumps. Data are expressed as means � SE.Statistical differences were determined using a Student’s t-test. *P � 0.001compared with lean rats.

R529FETAL PROGRAMMING BY MATERNAL OBESITY

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%lean mass were calculated. Subcutaneous and visceral fat tissueswere distinguished via manual tracing of the abdominal wall in eachof the sections. Serial images collected via CT analyses were collated,and solid three-dimensional (3D) renderings were generated using theM3D module of the MCID Elite software (GE Healthcare, ChalfontSt. Giles, UK). Z-stack distance between slices was set at 1 mm.Muscle, fat, and bone tissue compartments were rendered 3D indi-vidually and later merged in separate channels using Photoshop 5.5software to generate three-color 3D visualizations of CT scans.

Serum hormones and OGTT. A separate group of female rats (n �5 per group) were challenged with an OGTT at the end of 3 wk of

overfeeding. OGTT was also conducted in the male offspring atPND120. Rats were fasted for 6 h (from 9:00 AM to 3:00 PM forfemale rats) or overnight (in the case of offspring) prior to receivinga 2.5 g/kg oral challenge of glucose (0.5 g/ml). Blood (100 �l) fromthe tail vein was collected in capillary tubes at the beginning ofthe fast and at 0, 15, 30, 60, 90, and 150 min following the glucosechallenge. Serum glucose was measured using glucose oxidasemethod (Synermed, Westfield, IN). Serum insulin concentrations wereassayed using ELISA for rat insulin (Linco Research, St. Charles,MO). We assessed fed and fasted levels of serum leptin, fed levels ofNEFA, and triglycerides in lean and obese female rats at the end of 3

Fig. 2. Body composition analyses of lean and obese female ratsat 21 days following infusion of diets. Lean and obese ratsreceived liquid diets via TEN at 187 kcal/kg3/4 per day (lean, n �7) or 220 kcal/kg3/4 per day (obese, n � 15), respectively. Fatmass (A) and lean mass (B) expressed as %body weight asestimated noninvasively by NMR analyses. X-ray computerizedtomography (CT)-based analyses of body composition was car-ried out using LaTheta LCT-100 scanner. A Scout view of theentire rat (C) and transverse slice view of representative rats (D)are depicted. Vis, visceral adipose tissue; Ab, abdominal wall;Sc, subcutaneous adipose tissue. Approximately 90 transverseslices, 1 mm apart, were acquired encompassing the entire vis-ceral region of the animal under isoflurane anesthesia (n � 5 pergroup). Densitometric calculations of fat and muscle were doneusing Aloka CT software using attenuation number thresholds of�120 to �500 for fat and �120 to 350 for muscle. Indices of%fat ratio (volume occupied by fat/volume occupied by leantissue), %fat mass, %lean mass were calculated. Subcutaneousand visceral fat tissues were distinguished via manual tracing ofthe abdominal wall in each of the sections. Serial images col-lected via CT analyses were collated and solid three-dimensional(3D) renderings performed using M3D software. E: 3D three-color renderings of bone (green), muscle (red), and fat (blue)tissue compartments of representative lean and obese rats.F: total, visceral, and subcutaneous (SC) fat mass (as %bodyweight). G: %fat ratio (i.e. relative volume occupied by fat vs.lean tissue) in lean and obese rats as computed using Aloka CTsoftware. Data are expressed as means � SE. Statistical differ-ences were determined using Student’s t-test. *P � 0.01 com-pared with lean rats.

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wk of overfeeding. Leptin levels were assayed using ELISA (LincoResearch). Serum triglycerides and NEFA levels were estimated usingcommercially available reagents (triglycerides, Synermed, Westfield,IN and NEFA C reagents, WAKO, Richmond, VA).

Adipose histomorphometry. For histomorphometric analyses 3–4mm pieces of adipose tissue from the retroperitoneal fat depots werefixed in buffered alcoholic formalin for 4 days and embedded inparaffin using routine histological procedures. Sections (6-�m thick)were stained with hematoxylin and eosin. Diameters of adipocyteswere measured using a Ziess Axiovert microscope (Carl Ziess, Thorn-wood, NY) with ZiessVision software (Carl Ziess). A minimum of300 cells at random were measured for each slide (n � 4 per group)and percentage of cells in each size range was computed using MSExcel (Microsoft, Redmond, WA).

Statistical analysis. Data are expressed as means � SE. Associa-tions between the variables, serum leptin, insulin at 15 min postglu-cose challenge, and %fat mass, respectively, were examined by linearregression. Similarly, a linear regression analysis was carried outbetween %fat ratio and body weights in the offspring of lean or obesedams at PND120. Statistical differences between lean and obese ratsprior to conception or dams during gestation were determined usingStudent’s t-test. A two-way ANOVA followed by all-pair wise com-parison by the Student-Neuman-Keuls method was used to comparethe effects of maternal obesity and postweaning HFD. Statisticalsignificance was set at P � 0.05. Statistical analyses were performedusing SigmaStat 3.3 software (Systat Software, San Jose, CA). Graph-ical representation was performed using SigmaPlot version 10.0 forWindows (Systat Software).

RESULTS

Effect of obesity on body composition and metabolic param-eters. Body weight gains for female rats fed liquid diets viatotal enteral nutrition at 187 kcal/kg3/4 per day or 220 kcal/kg3/4 per day are represented in Fig. 1. Overfeeding 15%excess calories to rats resulted in greater weight gains duringthe 3-wk infusion period. Obese rats at the end of 3 wk were21% (P � 0.01) heavier compared with lean controls. Consis-tent with greater weight gain, overfed rats showed higher bodyadiposity at 3 wk. NMR analyses revealed that obese rats hada 98% greater %fat mass and 15% lower relative lean mass(P � 0.001, Fig. 2, A and B). X-ray CT analyses confirmedNMR data. Two-dimensional whole body radiograms (Fig. 2C)and transverse sections (Fig. 2D) of representative lean andobese rats are depicted. Higher body adiposity following over-feeding was also evident from 3D renderings of collated CTslices (Fig. 2E). Muscle and fat tissues are rendered in red andblue colors, respectively. 3D renderings of obese rats showgreater blue and violet (as a result of merging red and bluecolors) compared with primarily deep red in lean rats. Quan-titation of CT analyses revealed that total, visceral, and sub-cutaneous %fat mass in obese rats was �462, 423, and 589%of controls, respectively (Fig. 2F, P � 0.001). In addition, CTanalyses also showed %fat ratio was increased to �424% oflean controls (P � 0.001) following overfeeding (Fig. 2G).Conversely, %lean mass was significantly decreased (P �0.05) in overfed-obese rats. It should be noted that while NMRassesses body composition of the entire animal, we used X-rayCT analyses to specifically assess body composition in thetrunk region of the animals, which contributes differences inthe %fat mass and the overall magnitude of increased adipositybetween the two techniques. Despite these differences, over-feeding resulted in significant elevations in body weights andadiposity.

Serum glucose, insulin, and leptin levels were assessedfollowing 3 wk of overfeeding. Significant hyperinsulinemiawas observed in the obese rats, with approximately fivefoldhigher fasting serum insulin levels (P � 0.05) indicative ofobesity-associated insulin resistance (Table 1), which wasconfirmed by OGTT. Obese rats had severe insulin resistance(Fig. 3A), which was associated with impaired glucose dispo-sition (hyperglycemia despite hyperinsulinemia, Fig. 3B). Lin-ear regression of %fat mass and serum insulin response (at 15min) showed significant positive correlation (r2 � 0.74, P �0.001). Increased adiposity was also associated with progres-sively higher fasting and fed serum leptin concentrations (Ta-ble 1). Serum leptin in the obese rats was �4.5-fold higher thanin the lean rats. Linear regression of fed serum leptin levels and%fat mass revealed significant positive correlation (r2 � 0.97,P � 0.001). Furthermore, after normalization of serum leptinlevels to %fat mass, increased adiposity was associated withhigher leptin values (P � 0.01), suggesting that progres-sively obese rats were secreting higher serum leptin thanwould be predicted for their increased fat mass, a hallmarkof leptin resistance (Table 1). Serum triglyceride and NEFAlevels were also significantly elevated (P � 0.05) in obeserats (Table 1).

Gestational weight gains and effect of maternal obesity onoffspring growth. Following resumption of infusion of liquiddiets on day 8 at 220 kcal/kg3/4 per day, the rate of pregnancy-related weight gain (gestation days 8–21) was nearly the samein both lean and obese dams (Fig. 4). The body compositiondifferences between the groups, however, remained the sameafter mating as before mating, i.e., obese dams had greater %fatmass than the lean controls throughout gestation (P � 0.01).

Offspring were reared by surrogate dams that were notcannulated and had ad libitum access to AIN-93G pelleteddiets. Hence, offspring exposure to obesity was limited togestation. Birth weights (lean dams, 6.19 � 0.3 vs. obese dams,6.44 � 0.2 g), numbers of pups (lean dams, 11.7 � 0.8 vs.obese dams, 11.6 � 0.88), male-to-female ratio (lean dams,1.1 � 0.2 vs. obese dams, 1.5 � 0.3), crown-to-rump distance(lean dams, 1.6 � 0.05 vs. obese dams, 1.7 � 0.02 in.),anogenital distance (0.13 � 0.008 vs. obese dams, 0.15 �

Table 1. Effect of obesity on endocrine and metabolicparameters

Parameter

Lean Obese

Fasting Fed Fasting Fed

Glucose, mg/dl 138�3.3 122�8 113�5.9* 133�7Insulin, ng/ml 1.0�0.1 1.7�0.15 4.1�0.7* 11.2�1.06*Leptin, ng/ml 4.8�0.6 8.09�0.6 29.4�3.3* 36.4�5.1*Leptin, ng/ml,

normalized to%fat mass 0.26�0.03 0.45�0.03 0.75�0.05* 0.93�0.1*

Triglyceride, mg/dl NA 20.2�4.5 NA 200.7�66.0*NEFA, mM NA 0.43�0.1 NA 0.98�0.1*

Data are means � SE. Data were collected from female rats fed diets viatotal enteral nutrition (TEN) at 187 kcal/kg3/4 per day (lean, n � 5) or 220kcal/kg3/4 per day (obese, n � 5) for 21 days. Infusion of diets was carried outfor 23 h/day via computer-controlled syringe pumps. Serum insulin and leptinwere estimated using ELISA. Glucose, triglyceride, and nonesterified fattyacids (NEFA) levels were assayed using colorimetric methods. %Fat mass wasdetermined using whole animal NMR. *Significantly different by Student’st-test (P � 0.05) compared to lean controls. NA, not assessed.



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0.002 in.) and body weights at weaning (lean dams, 47.9 � 1.6vs. obese dams, 45.3 � 1.4 g) of offspring did not differbetween groups as a function of maternal adiposity. Bodyweight gains of male offspring from lean or obese dams oneither control AIN-93G or HFD from PND21 to PND130 arerepresented in Fig. 5. As anticipated, consumption of obeseg-enic HFD postnatally, resulted in increased weight gain amongthe male offspring of lean dams fed HFD (Fig. 5). While thebody weights of offspring from obese dams did not differ fromoffspring of lean dams fed AIN-93G diets, male offspring ofobese dams gained remarkably more (P � 0.005) body weightson a HFD (Fig. 5). Average body weights at PND130 were803 � 24 g for the HFD-offspring of obese dams vs. 692 �23 g for HFD-offspring of lean dams. These data suggest thatexposure to maternal obesity led to programming of increasedsusceptibility to obesity in the offspring despite normal birthweight.

Increased obesity in the offspring of obese dams and changes inadipose histomorphometry. Body composition analyses (bothNMR and CT analyses) demonstrated that increased weight

gain in the obese dam offspring was essentially due to in-creased fat mass. A two-way ANOVA, followed by Student-Neuman-Keuls post hoc analyses for NMR data at PND90showed a significant effect of maternal obesity (P � 0.032) andof HFD consumption (P � 0.001). However, at PND120,offspring of obese dams on HFD were bigger than the maxi-mum allowable body mass to be assayed by NMR and hence

Fig. 3. Serum glucose (A) and insulin (B) following an oral glucose challenge(2.5 g/kg) to fasted lean or obese rats fed diets via TEN at 187 kcal/kg3/4 perday (lean, n � 5) or 220 kcal/kg3/4 per day (obese, n � 5) for 21 days. Dataare expressed as means � SE. Statistical differences were determined usingStudent’s t-test at each time point. *P � 0.05 compared with lean rats at thesame time point.

Fig. 4. Body weights of female rats fed diets via TEN at 220 kcal/kg3/4 per dayfrom gestation day (GD) 8 to GD21. Rats were fed diets via TEN for 21 daysprior to mating at 187 kcal/kg3/4 per day (lean, n � 7) or 220 kcal/kg3/4 perday (obese, n � 15) to induce obesity. Lean and obese rats were mated withad libitum-fed male rats for a period of 1 wk. Dams resumed receiving liquiddiets via TEN at 220 kcal/kg3/4 per day through the end of gestation. Data areexpressed as means � SE. Statistical differences were determined usingStudent’s t-test at each time point. *P � 0.05 compared with lean rats at thesame time point.

Fig. 5. Body weights of male offspring of lean or obese dams from weaningthrough PND130. Dams were fed diets via TEN to induce obesity as describedunder MATERIALS AND METHODS. Male offspring were cross-fostered at birth tononsurgerized dams fed AIN-93G-based pelleted diets ad libitum. At PND21offspring were weaned to either regular AIN-93G diets (14% fat calories) or tohigh-fat (HF) diets (45% fat calories) to mimic a postnatal obesegenic envi-ronment. Body weights of the offspring were monitored weekly until PND130(n � 8, 7, 11, and 9 in lean and obese dam offspring on control and HF diets,respectively). Data are expressed as means � SE. Statistical differences wereexamined using a two-way ANOVA for the effects of maternal obesity andpostweaning HF diet, followed by Student-Neuman-Keuls post hoc analyses.*P � 0.05.



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data could not be collected. Representative whole body radio-grams (Fig. 6A) and transverse slices (Fig. 6B) of offspringfrom lean or obese dams are presented. Despite no changes inbody weights, obese dam offspring on AIN-93G diets had a�1.6-fold greater %fat ratio compared with offspring of leandams on the same diet (Fig. 6C, P � 0.05). Furthermore, obesedam offspring on HFD had a 26% greater %fat ratio and �34,25, and 60% increase in total, visceral, and subcutaneous %fatmass, respectively, compared with offspring of lean dams fedthe same HFD (Fig. 6, C and E, P � 0.05). Correlationanalyses of body weights and %fat ratios (obtained from X-rayCT analyses) showed a highly positive association (r2 � 0.866,P � 0.001, Fig. 6D). Changes in body composition andincreases in body fat in the offspring of obese dams fed a HFDcompared with offspring of lean dams is also evident in 3D

reconstructed color renderings of CT slices (Fig. 6F). Again,muscle and fat tissues are rendered in red and blue colors,respectively. Postmortem adipose tissue and organ (liver andkidney) weights are represented in Table 2. As anticipated, HFfeeding increased (P � 0.05) liver and adipose tissue weights(normalized to body weight). In addition, as observed via NMRand CT analyses, a significant effect of maternal obesity (P �0.05) was apparent in liver and adipose tissue weights whennormalized to body weight.

HF feeding significantly increased serum glucose, triglycer-ide, NEFA, insulin, and leptin levels (P � 0.05) in both leanand obese offspring (Table 3). Most strikingly, serum insulinand leptin levels increased by 2.2- and 2.3-fold in offspringfrom obese dams fed control diet compared with the offspringfrom lean dams fed the same diet. The overall effect of

Fig. 6. CT-based analyses of body compositionwas carried out as described under MATERIALS

AND METHODS (n � 3–4 per group). Scout viewof the entire rat (A) and transverse slice view(B) of representative rats from lean or obesedams fed either control (Con) or HF diets aredepicted. Densitometric calculations of fat,muscle, and bone were done using Aloka CTsoftware using attenuation number thresholdsof �120 to �500 for fat and �120 to 350 formuscle. C: %fat ratio (i.e., relative volumeoccupied by fat vs. lean tissue) in offspring oflean and obese dams fed control or HF diets ascomputed using Aloka CT software. D: linearregression analysis of %fat ratio and bodyweight shows significant (P � 0.001) positivecorrelation (r2 � 0.866). E: calculated total,visceral and subcutaneous fat mass (as a per-centage of body weight) following CT analy-ses. F: representative 3D three color renderingsof bone (green), muscle (red), and fat (blue)tissue compartments of lean and obese damoffspring on HF diet. Statistical differenceswere determined using a two-way ANOVA forthe effects of maternal obesity and postweaningHF diet, followed by Student-Neuman-Keulspost hoc analyses. Data are expressed asmeans � SE. a,b,c,dDiffering superscripts indi-cate significant differences (P � 0.05).



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maternal obesity (in both control and HFD-fed groups usingtwo-way ANOVA) showed a trend toward an increase (P �0.08). Serum adiponectin concentrations did not significantlydiffer between offspring of lean and obese dams. However,adiponectin levels normalized to %body fat showed a strongnegative correlation with body adiposity (P � 0.01), suggest-ing that adiponectin secretion per unit of fat mass significantlydecreases with increasing adiposity. Finally, serum resistinlevels in offspring from obese dams showed significant eleva-tions on both control and HFDs (P � 0.01) (Table 3).

Increased obesity in offspring of obese dams fed HFD wasassociated with a threefold increase in fasting hyperinsulinemiacompared with the lean dam offspring on the same diet (Fig. 7A).In addition, HF feeding also induced significant insulin resis-tance by itself as observed by the hyperinsulinemic responsefollowing OGTT in the offspring from lean dams. However, inthe offspring of obese dams, the insulin response followingglucose challenge was prolonged suggesting further impair-ments in insulin sensitivity (Fig. 7A). Representative hematox-ylin and eosin-stained sections of adipose tissues from AIN-93G or HFD-fed offspring of lean or obese dams are depictedin Fig. 7B. Feeding of HFD to lean dam offspring increased(P � 0.05) the mean diameter of cells indicating hypertrophy,as did exposure to maternal obesity per se (on control diet)(Fig. 7C). Approximately 10% of cells in the offspring of leandams on control diet were in the 100- to 150-�m size range,with most adipocytes in the 25- to 75-�m size range. However,

in the offspring of lean dams on HFD or offspring of obesedams on control or HFD, �35–37% of adipocytes were in the100- to 150-�m size range, indicating increased hypertrophy.In addition, obese dam offspring on HFD showed an evengreater percentage of very large (�150 �m) adipocytes. Fur-thermore, interestingly, the number of very small adipocytes(�25 �m) also increased in this group (P � 0.05), perhapssuggesting an increase in the number of newly differentiatedadipocytes (Fig. 7, C and D). Finally, we assessed levels ofPPAR-�1/2 protein in adipose tissue lysates. Consistent withthe hypothesis that obese dam offspring on HFD may haveincreased adipogenesis, PPAR-� levels were increased by�40% over control (Fig. 7E).

Effect of maternal obesity on offspring food intake. Mea-surement of food intake for individual rats over two consecu-tive days were performed, and caloric intake kcal/kg3/4 per daywas estimated . Measurements at both PND60 and PND120suggested that offspring from lean and obese dams consumedthe same amount of calories (kcal/kg3/4 per day) of both controland HFD (Fig. 8).

DISCUSSION

Evidence from a growing number of clinical and animalstudies demonstrates that obesity and other metabolic diseasesmay have developmental origins (7, 15, 31, 39). If this is thecase, early intervention may prevent these disorders. The

Table 2. Characteristics of male offspring of lean or obese dams at PND130

Parameter

Offspring of Lean Dams Offspring of Obese DamsP Values

Control HFD Control HFDEffect of

Maternal ObesityEffect of

Postweaning HFD

Body weight 607�25 692�23 650�20 803�24 0.003 �0.001% Liver weight 3.2�0.07 3.4�0.06 3.4�0.06 3.6�0.08 0.016 0.009% Total fat 4.4�0.6 8.2�0.3 5.8�0.33 9.95�0.31 0.003 �0.001% RP fat 2.0�0.3 4.0�0.2 2.8�0.18 4.65�0.28 0.014 �0.001% Gonadal fat 1.7�0.25 3.3�0.17 2.1�0.2 3.83�0.11 0.032 �0.001% Perirenal fat 0.6�0.1 0.89�0.17 0.8�0.08 1.45�0.22 0.029 0.011% Kidney weight 0.59�0.02 0.55�0.01 0.57�0.01 0.52�0.01 0.13 0.017

Data are expressed as means � SE. Data were obtained at postnatal day (PND)130 from male offspring of lean or obese dams [n � 8, 7, 11, and 9 in leanand obese dam offspring on control and high-fat diets (HFD), respectively]. Dams were fed diets via TEN to induce obesity as described in MATERIALS AND

METHODS. Male offspring were cross-fostered at birth to unsurgerized dams fed AIN-93G-based pelleted diets ad libitum. At PND21 offspring were weaned offto either regular AIN-93G diets (14% fat calories) or to HFDs (45% fat calories) to mimic a postnatal obesegenic environment. Body weights of the offspringwere monitored weekly until PND130. RP, retroperitoneal adipose tissue. Statistical differences were determined using a 2-way ANOVA to examine the effectsof maternal obesity and postweaning HFD, followed by Student-Newman-Keuls post hoc analyses.

Table 3. Serum parameters of male offspring of lean or obese dams at PND130

Parameter

Offspring of Lean Dams Offspring of Obese DamsP Values

Control HFD Control HFDEffect of

Maternal ObesityEffect of

Postweaning HFD

Glucose, mg/dl 134�5.7 154�7.4 130�2.3 153�12.6 0.78 0.01Triglyceride, mg/dl 581�162 1498�228 504�125 979�365 0.21 0.006NEFA, mM 1.3�0.16 2.0�0.16 1.3�0.08 1.6�0.18 0.16 0.004Insulin, ng/ml 3.9�1.0 9.4�1.7 8.7�2.8 13.3�3.1 0.08 0.04Leptin, ng/ml 9.5�4.6 36.2�7.7 22.1�3.8 53.6�12.9 0.08 0.002Leptin, ng/ml, normalized to %fat 1.8�0.7 4.1�0.8 4.1�0.8 4.0�0.7 0.18 0.16Adiponectin, �g/ml 6.4�1.2 9.8�2.0 8.6�1.2 9.0�2.3 0.69 0.28Adiponectin, �g/ml, normalized to %fat 2.2�0.9 1.1�0.2 1.6�0.2 0.8�0.2 0.38 0.10Resistin, ng/ml 29.3�1.6 28.4�3.1 43.5�6.2 37.8�5.1 0.01 0.80

Data are expressed as means � SE. Data were obtained at PND130 from male offspring of lean or obese dams (n � 6 per group). Statistical differences weredetermined using a 2-way ANOVA to examine the effects of maternal obesity and postweaning HFD, followed by Student-Newman-Keuls post hoc analyses.



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general dogma that children born to overweight women arethemselves at higher risk of becoming overweight is upheld byseveral well-controlled studies. However, the underlying rea-sons remain unclear. It has been especially challenging todelineate the specific contributions of obesity at conception indetermining body composition of the offspring in later lifethrough epidemiological studies due to the large number ofconfounding variables. The present studies were aimed ataddressing this very question using a total enteral nutrition-based overfeeding model in the rat. We used total enteralnutrition for its ability to overfeed rats in a controlled mannerbypassing the satiety response that limits ad libitum foodintake. Using this model, we replicated many of the metabolicand endocrine features of overweight individuals. Importantly,we were able to exclude parental genetic influences, matchgestational weight gain, limit the exposure of maternal obesityin utero, and control lactation efficiency, all of which can bedifficult confounding variables in studies with human subjects.

Hence, the model described in the present report specificallyexamines the metabolic obesegenic burden on the offspring bybeing in an obese intrauterine environment per se.

Gestational and neonatal programming of obesity risk haspreviously been examined in several animal models, primarilyin rodents but also in larger animal models such as the sheepand pig (2, 18, 58). Consumption of low-protein diets ormaternal caloric restriction during gestation invariably resultsin offspring of low birth weights (13, 23, 38, 47, 52, 53).However, susceptibility to obesity of the offspring appears tobe dependent on several factors including timing, degree, andnature of macronutrient/caloric restriction; obesegenic natureof the postnatal diet; and the species being studied (rats, mice,guinea pigs or larger animals). Overall, it appears that caloricrestriction (70% of ad libitum diet) or protein restriction (8%protein during gestation) in mice (38, 57), �50–30% of adlibitum diet (13, 53) or intrauterine growth retardation due touterine artery ligation in rats (48), leads to higher body weight

Fig. 7. A: serum insulin in offspring of lean or obese dams on control or HF diets following an oral glucose challenge (2.5 g/kg) challenge. B: estimation ofadipocyte size in the retroperitoneal adipose tissue of offspring of lean or obese dams fed either control or HF diets (n � 4 per group). Photomicrographs of 6-�mhematoxylin and eosin-stained sections of adipose tissues are depicted. Average diameter of a minimum of 300 adipocytes at random was estimated using a ZiessAxiovert microscope accompanied with Ziess AxioVision software, magnification 100. C: data are expressed as the average (�SE) %adipocytes in a givensize range. In each size range, statistical differences were examined using a two-way ANOVA for the effects of maternal obesity and postweaning HF dietfollowed by Student-Neuman-Keuls post hoc analyses. a,bDiffering superscripts indicate significant differences (P � 0.05). D: representative photomicrographshowing the presence of very small (�25 �m) adipocytes in retroperitoneal adipose tissues of offspring of obese dams on HF diets. Background image is shownat magnification of 100, inset is 400 magnification. E: immunoblot analyses of peroxisome proliferator-activated receptor-� (PPAR-�1/2) in whole cell lysatesfrom retroperitoneal adipose tissues of lean or obese dam offspring fed control or HFD. Each lane represents pooled protein (20 �g) from 3 separate animals.



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and/or adiposity in the offspring, especially when challengedwith an obesegenic diet. Similarly, studies of maternal feedrestriction in the sheep or guinea pig also show increasedadiposity in the fetus (24, 50, 58). Certainly, larger animals likethe sheep may be more suitable to study late-gestation effectsand fetal adipose tissue development compared with rodents.

An important aspect common to all of the above-mentionedstudies is the emphasis on the impact of poor fetal nutrition.While undernutrition and protein deficiency are clearly impor-tant in many countries, including the United States, a growingproblem in obesity is actually overconsumption of calorieswhich is becoming increasingly common in the United Statesand is being better documented by basic studies (16, 37).Elegant work by Levin and Govek (28), using diet-resistantand diet-induced obesity-susceptible strains of rats, showedthat both genetic factors and maternal obesity interact toinfluence offspring susceptibility to obesity. Furthermore, re-cent studies by Khan and colleagues (20–22, 51) have shownthat the consumption of diets high in lard, from 10 days priorto conception through weaning, lead to increased hypertension,hyperinsulinemia, adiposity, and endothelial dysfunction in theoffspring at 6 mo of age. Feeding high-saturated fat diets adlibitum for 10 days prior to conception led to only a modestincrease in body weights at conception (21). Rats fed the larddiet showed a dramatic increase in body weight gain duringgestation, suggesting that this model may mimic the riskfactors associated with increased weight gain during pregnancyrather than prepregnancy body composition (21). Recent stud-ies by Oken et al. (37) suggest that high gestational weight gainsignificantly increases the risk of childhood obesity. Our datasuggests that maternal body composition at conception itselfhas important implications for offspring adiposity. Gestationalweight gains in both the lean and obese rats did not differ,suggesting that offspring adiposity is susceptible to program-ming during several developmental windows and greaterweight gain during gestation may add to the risk.

In the present studies, programming of obesity occurs in theabsence of changes in birth weights of offspring and otherparameters, such as litter and offspring size as a function of

maternal obesity. These findings are consistent with reportsdemonstrating that HF feeding during gestation does not affectbirth weight (51), suggesting subtle programming of obesitymay occur in the absence of obvious changes in birth weight(17). On the other hand, Yamashita et al., (55) reported that ina model of GDM using the hetrozygous C57BL/KsJ-Leprdb/

mice, increased obesity and insulin resistance in the offspringwas associated with higher birth weights, consistent withfindings from offspring of GDM mothers (40). Since we didnot observe any increase in birth weights, it is likely thatmaternal obesity in our model is not associated with GDM.Moreover, our data indicate that fetal programming caused bymaternal obesity was evident even when fed control AIN-93Gdiets. However, the effects of maternal obesity were reinforcedwhen challenged with an obesegenic HFD implicating animportant role of postnatal diet in modifying adiposity. Thesedata beg the question as to whether children born appropriatefor gestational age (in birth weight) to overweight/obese indi-viduals may still be at higher risk of developing obesity undercertain postnatal environments. Li et al. (29) suggested thatmaternal overweight (BMI: 25–29.9) doubled the odds ofchildhood overweight in a cohort, where GDM and low or highbirth weights were excluded. Similarly, Gillman et al, (16)found maternal BMI to be an influencing variable in associa-tions between maternal BMI, GDM, and offspring obesity.Taken together, our data from animal experiments and popu-lation-based studies indicate that maternal overweight at con-ception itself may contribute to offspring obesity risk.

The detailed analyses of body composition in both the damsand offspring is an important strength of the present studies. Inaddition to quantitatively estimating fat and lean mass, CTanalysis allows examination of the distribution of body fatbetween subcutaneous and visceral fat pads. Our findingssuggest that overall adipogenesis is increased in the offspringof obese animals. Consistent with increased adipogenesis,expression of the adipogenic transcription factor PPAR-� wassignificantly higher in the adipose tissue. Similar findings werereported by Muhlhausler et al., (34) who also found increasedPPAR-� levels in the fetal perirenal adipose tissue of sheep thatwere overfed during late gestation. Hence, it appears thatregulators of the adipogenic program may be susceptible tofetal programming. Furthermore, histomorphometric analysesof adipose tissues clearly revealed a greater percentage of verylarge adipocytes that have been shown to be more insulinresistant compared with smaller adipocytes (11). Increasedwhole body insulin resistance was also confirmed by OGTT,indicating greater overall insulin resistance in the obese damoffspring. Certainly, decreased adiponectin and increased se-rum resistin as observed in our studies, have been shown to beassociated with insulin resistance (49). Although speculative atthis point, programming of the offspring hypothalamic-pitu-itary-adrenal axis, adipose levels of HSD-1, and peripheralleptin signaling are plausible mechanisms. In addition, levelsof other pro-/anti-adipogenic factors, such as C/EBP-�, Pref-1,wnt-10b, and lipogenic factors, such as SREBP-1 and LPL canbe altered by programming. Interestingly, our results suggestthat maternal obesity does not appear to alter food intake andthat adiposity in the offspring of obese dams occurs in the faceof consumption of equal calories. However, since we measuredfood intake only at two periods in the study, these data mayneed to be interpreted with caution and more detailed assess-

Fig. 8. Caloric intake of offspring of lean or obese dams on control or HF dietsat PND60 and PND120. Rats (n � 8, 7, 11, and 9 in lean and obese damoffspring on control and HF diets, respectively) were housed individually, andfood consumption in grams were calculated over 2 consecutive days. Data areexpressed as means � SE of calories consumed in 24 h normalized to bodyweight0.75. Statistical differences were determined using a two-way ANOVAfor the effects of maternal obesity and postweaning HF diet, followed byStudent-Neuman-Keuls post hoc analyses. Data are expressed as means � SE.a,bDiffering superscripts indicate significant differences (P � 0.05).



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ment of food intake and energy expenditure via indirect calo-rimetry are warranted.

In conclusion, we have demonstrated that obesity at concep-tion in the rat brought about by overfeeding of nutritionallycomplete diets led to increased obesity in the offspring. Pro-gramming of obesity occurs in the absence of changes in birthweights and is associated with significant alterations in meta-bolic and endocrine parameters and adipose tissue cellularityand appears to be independent of caloric intake. Contributionsof maternal body composition at conception in increasingoffspring susceptibility to obesity may have important impli-cations in identifying effective strategies of primary preven-tion.

ACKNOWLEDGMENTS

We would like to thank Matt Ferguson, Tammy Dallari, Trae Pittman,LaDonna Chappell, Steven Rodgers, Jennifer Crow, Jamie Badeaux, ReneeTill, Crystal Combs, and Michele Perry for their technical assistance. We thankDr. John L. Marecki for critical reading of the manuscript.

GRANTS

These studies were supported, in part, by U.S. Department of Agriculture-Agricultural Research Service Grant CRIS 6251-51000-005-035.

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