REPRODUCTION-DEVELOPMENT

Effect of Food Restriction and Supplementation on Fetal Programming in Mice

Kathleen A. Pennington, Jennifer L. Harper, Ashley N. Sigafoos, Lindsey M. Beffa, Stephanie M. Carleton, Charlotte L. Phillips, and Laura C. Schulz

Division of Reproductive and Perinatal Research (K.A.P., J.L.H., A.N.S., L.M.B., L.C.S.), Departments of Obstetrics, Gynecology, and Women’s Health, University of Missouri, Columbia, Missouri 65212; and Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 Department of Biochemistry (S.M.C., C.L.P.), University of Missouri, Columbia, Missouri 65212

Metabolic disease is a significant global health and economic problem. In a phenomenon referred to as fetal programming, offspring of underweight or overweight mothers have an increased incidence of adulthood and metabolic disease. Undernourished individuals have decreased levels of leptin, a regulator of energy balance, whereas obese people develop hyperleptinemia and leptin resistance. We hypothesize that alterations in circulating leptin during pregnancy contribute to programming events caused by maternal nutritional status. To test this hypothesis, pregnant mice were randomly placed in one of three treatment groups: ad libitum feed plus saline injection (control, n ϭ 5), 50% food restriction plus saline injection (restricted, n ϭ 4), or 50% food restriction plus 1 mg/kg⅐d leptin injection (restricted, leptin treated, n ϭ 4). Mice were treated from 1.5 to 11.5 d after conception and then returned to ad libitum feeding until weaning. At 19 wk after weaning, offspring were placed on a 45% fat diet and then followed up until 26 wk after weaning, at which time they were killed, and samples were collected for further analysis. Our results dem- onstrate that males are more negatively impacted by high-fat diet than females, regardless of maternal treatment. We provide evidence that differential response to leptin may mediate the sexual dimorphism observed in fetal programming in which male offspring are more affected by maternal undernutrition and female offspring by maternal overnutrition. We show that female offspring born to food-restricted, leptin-supplemented mothers are obese and insulin resistant. This may mimic fetal programming events seen in offspring of overweight women. (Endocrinology 153: 4556–4567, 2012)

besity and its associated diseases are a significant into adulthood, which can include increased risk of met- Oproblem worldwide, having major effects on mor- abolic syndrome (2, 4). This hypothesis was put forth by bidity and mortality rates (1). The World Health Organi- epidemiologists Barker and Osmond (5), who discovered zation in 2005 predicted that by 2010 in the United States, correlations between adult cardiovascular disease and low 44.2% of adult males and 48.3% of adult females would birth weight, infant mortality, and poor maternal health, be obese [ (BMI) Ն 30 kg/m2] (1). A study and nutrition. in 2010 estimated that 34.3–38.5% of all adults suffer Both maternal undernutrition and overnutrition are as- from , a condition characterized by sociated with fetal programming events, although the pre- central obesity, , hypertension, and nonalcoholic ponderance of data suggests that male offspring are more fatty liver disease (NAFLD), which accounts for 6–7% of influenced by the former and female offspring by the latter all mortalities (2, 3). The developmental origins of health (6–11). Studies from the Dutch Hunger Winter indicated and disease, or fetal programming, hypothesis states that that men born to mothers who were undernourished early the maternal environment has lasting effects on the fetus in pregnancy have a higher incidence of cardiovascular

ISSN Print 0013-7227 ISSN Online 1945-7170 Abbreviations: BMI, Body mass index; ␮CT, microcomputed tomography; dpc, d after Printed in U.S.A. conception; GLM, general linear model; HFD, high-fat diet; IPGTT, ip glucose tolerance test; Copyright © 2012 by The Endocrine Society MRI, magnetic resonance imaging; NAFLD, nonalcoholic fatty liver disease; PFA, doi: 10.1210/en.2012-1119 Received January 30, 2012. Accepted June 20, 2012. paraformaldehyde. First Published Online July 9, 2012

4556 endo.endojournals.org Endocrinology, September 2012, 153(9):4556–4567 Endocrinology, September 2012, 153(9):4556–4567 endo.endojournals.org 4557 disease and obesity in middle age (7) compared with their Materials and Methods counterparts. In these studies, women were largely not Animals and tissue collection followed up due to tracking difficulties. However, when All animal procedures were approved by University of Mis- the phenomenon was replicated in animal models, includ- souri Institutional Animal Care and Use Committee and per- ing rats and sheep, maternal undernutrition during early formed in accordance with the National Institutes of Health pregnancy often programmed greater adverse outcomes in Guide for the Care and Use of Laboratory Animals. male offspring (6, 8, 12–15), although a few studies in Swiss-Webster female mice (Harlan, Indianapolis, IN) aged sheep show a negative effect of early pregnancy food re- 13–17 wk, were individually housed, and daily food consump- striction on female offspring (16, 17). On the other hand, tion was measured for 2 wk before mating as previously de- scribed (31). Mice were mated and then randomly placed in one

global maternal nutrient restriction throughout preg- Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 of three treatment groups: ad libitum feed plus saline injection nancy results in multiple aspects of metabolic syndrome in (control, n ϭ 5), 50% food restriction plus saline injection (re- both male and female offspring (18–20). In maternal over stricted, n ϭ 8), or 50% food restricted plus 1 mg/kg ⅐ d ip leptin nutrition studies, some studies indicate maternal obesity injection (restricted, leptin treated, n ϭ 8). Mice were placed on can have a negative impact on both sexes (21–24), whereas treatment at 1.5 d after conception (dpc) for 10 d, with injections other studies show a greater, or exclusive, negative impact at 1400 h and food introduced at 1600 h. After treatment, mice were returned to ad libitum feeding. Litter size was recorded and on female offspring born to overweight mothers, resulting then reduced to eight by culling randomly selected pups to con- in obesity, hypertension, and diabetes (9–11). However, trol for litter size during lactation. The sex ratio was determined no studies that have examined both sexes have shown either by visualization at weaning or by PCR for sex determining greater impact of maternal obesity on male offspring. region Y on tail clip DNA taken at culling, as previously de- Taken together these studies highlight the differences and scribed (32). Litters were weighed weekly until weaning at 3 wk similarities between maternal under- and overnutrition as of age. At weaning, pups were group housed by sex and treat- ment with up to four males or five females per cage and indi- well as the critical role timing and offspring sex have on the vidually weighed weekly. At 22 wk of age, all offspring were outcomes of fetal programming. placed on a 45% fat diet (Research Diets, New Brunswick, NJ) One key hormonal regulator of nutritional status and until the animals were killed. Ad libitum-fed offspring were energy homeostasis is leptin (25, 26). Circulating leptin killed by carbon dioxide inhalation at 29 wk of age for tissue levels are positively correlated to BMI (27), with hyper- collection. leptinemia and leptin resistance occurring in obesity (25). Blood samples were taken from all offspring at the time the animals were killed, and serum was stored at Ϫ20 C. Liver sam- In addition to its role in energy homeostasis, leptin affects ples were collected and placed in trireagent (Sigma, St. Louis, development in tissues including placenta, hypothalamus, MO) or fixed in 4% paraformaldehyde (PFA) overnight and then bone (28), and muscle (29). Furthermore, postnatal leptin embedded in optimum cutting temperature compound (Fisher levels influence the programming of adult disease (see the Scientific, Pittsburgh, PA) and stored at Ϫ80 C. Subcutaneous review in Ref. 30). Thus, we hypothesize that alterations fat, visceral fat, and hypothalamus samples were snap frozen in liquid nitrogen. All samples were stored at Ϫ80 C. The left kid- in circulating leptin levels during pregnancy contribute to ney was collected, fixed in 4% PFA, bisected midsagitally, em- fetal programming events caused by changes in maternal bedded in paraffin, and stored at room temperature. A sample of nutritional status. To test this, we have replicated the pancreas was also collected, fixed in 4% PFA, embedded in par- model of early pregnancy caloric restriction (7, 12) by affin, and stored at room temperature. Femurs were collected, feeding mice 50% of ad libitum consumption levels from cleaned, wrapped in gauze, and stored at Ϫ20 C in PBS. d 1.5 to 11.5 of pregnancy, a regimen that, as we previ- ously demonstrated, severely reduces serum leptin con- Small animal magnetic resonance imaging (MRI) Body composition was determined in 54 male offspring at 21 centrations (31). A subset of the food-restricted mice were wk of age and 54 male and 44 female offspring at 27 wk of age given daily leptin injections. We predicted that offspring after 5 wk exposure to a high-fat diet (HFD). Body fat percentage programming would be related to maternal hyper- or hy- was measured using a Micro-MRI high performance 7T MR poleptinemia, not to nutrient status per se. In other words, imaging and spectroscopy system (Varian Inc., Santa Clara, CA) metabolic programming would be evident in male off- at the Veterans Affairs Biomolecular Imaging Center at the spring of nutrient-restricted mothers but not when restric- Harry S. Truman Veterans Affairs Hospital and University of Missouri-Columbia, as previously described (33). Animals were tion was accompanied by leptin replacement. Similarly, anesthetized using isoflurane for the duration of the study. MRI female offspring of undernourished, leptin-treated moth- was performed from the diaphragm to tail base. Data analysis ers would exhibit the metabolic programming character- was performed using Mnova7software (Mestrelab Research, istic of offspring of overnourished mothers. Santiago de Compostela, Spain). 4558 Pennington et al. Effect of Leptin on Fetal Programming Endocrinology, September 2012, 153(9):4556–4567

Behavioral assays ness (shear modulus of elasticity) are descriptors of the material Behavioral activity was monitored automatically on two male the bone is made of, independent of the size and shape of the and two female offspring from each mother at 26 wk of age using bone. After the ␮CT analyses, the femur was potted in a cylin- the Med Associates’ Open Field Test Environment (ENV-515) drical holder and placed in a test fixture designed to apply a pure monitors (Georgia, VT) in the Translational Neuroscience Be- torsional load on the femur as previously described (34). havior Testing Core at the University of Missouri-Columbia as previously described (33). Sensor break data were used to mea- Pancreatic morphology sure distance traveled. Time spent in the middle of the maze vs. Immunohistochemistry was performed on pancreatic tissue the perimeter was used to estimate behavioral anxiety. Mice were from 42 male and 40 female offspring. Briefly, five 5-␮m paraffin habituated to the testing area for 3–4 h and then placed indi- sections at intervals of 50 ␮m or greater were examined per vidually in the chamber for 60 min. Trials were run every other pancreas. After blocking in a 1:10 solution of normal goat serum day for a total of three trials per mouse.

in PBS for 30 min, each sample was dual stained overnight with Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 an ␣-cell marker, mouse-antiglucagon (K79bB10; Abcam Inc., Serum assays Cambridge, MA) and a ␤-cell marker, rabbit-anti insulin Serum leptin and insulin concentrations were measured in (ab63820; Abcam) at 1:2000 and 1:1000, respectively. Second- offspring using the mouse leptin and rat/mouse insulin ELISA, ary antibodies Alexa Flour 568 goat-antimouse and 488 goat- respectively (Millipore, Billerica, MA) following the manufac- antirabbit (Invitrogen, Carlsbad, CA) were used at 1:500. A total turer’s guidelines. True serum triglyceride concentrations were of 991 islets were examined, 515 female (145 control, 202 re- measured in each offspring using a serum triglyceride determi- stricted, 168 restricted leptin treated) and 476 male (178 control nation kit (Sigma) following the manufacturer’s guidelines. In- 157 restricted 141 restricted leptin treated), and an average of traperitoneal glucose tolerance tests (IPGTT) were performed on 12.1 Ϯ 0.4 islets were examined per pup. Islet values were av- 13 male (five control, four restricted, four leptin) and 25 female eraged per animal. The ␣- and ␤-cell numbers were determined offspring (10 control, eight restricted, seven leptin) according to using the manual counting feature of ImageJ (National Institutes the National Institutes of Health animal models of diabetic of Health). The ␤-cells were manually circled and area measured, complications consortium protocol (http://www.diacomp.org/ using the Image J region of interest feature, as previously de- shared/showFile.aspx?doctypeidϭ3&docidϭ11). Briefly, ani- scribed (35). The average ␤-cell area per islet was used as an index mals were fasted for 6 h, and then a fasting glucose level was of ␤-cell mass. obtained by venous tail sample. An ip injection of glucose was then given (1 mg/g), and blood glucose levels were obtained at 30, RNA isolation and real-time RT-PCR analysis 60, and 120 min after the injection by venous tail sample using Total cellular RNA was isolated from liver, sc fat, visceral fat, the OneTouch Ultra glucose meter (LifeScan Inc., Milpitas, CA). and hypothalamus. Samples were homogenized in trireagent (Sigma), and, after phase separation using phase lock gel heavy Kidney morphology tubes (5 Prime Inc., Gaithersburg, MD), the aqueous layer was Kidneys were sectioned and stained with hematoxylin and placed on an RNEasy minicolumn (QIAGEN, Valencia, CA), eosin, and then glomeruli number was counted in the largest and total cellular RNA was purified according to manufacturer’s kidney cross-section using the counting feature in the ImageJ instructions. software (National Institutes of Health, Bethesda, MD). For liver, visceral and sc fat, and quantitative RT-PCR, 1 ␮g of RNA was reverse transcribed using the RT2 first strand kit Bone analysis (QIAGEN) following the manufacturer’s instructions. Real-time 2 Geometric parameters were defined from right femurs of one PCR was then performed using a custom RT Profiler PCR plate male and one female offspring from each mother by microcom- array, containing multiple PCR and RT2 SYBR Green ROX puted tomography (␮CT) scan analysis (Siemens Inveon, Sie- qPCR master mix (QIAGEN) on an Applied Biosystems 7500 mens Preclinical Solutions, Knoxville, TN) before ex vivo tor- real-time PCR system (Applied Biosystems, Foster City, CA) ac- sional loading as previously described (34) with some cording to the PCR array manufacturer’s guidelines. Three exceptions. The ␮CT data acquisition scanning protocol was as housekeeping genes, 18s rRNA (18s ribosomal RNA), Actb (ac- follows: x-ray settings of 80 kVp voltage and 500 ␮A current tin-␤), and Hprt (hypoxanthine phosphoribosyltransferase 1) with a 190-msec exposure. The ␮CT image slices were recon- were examined; however, due to expression variation of 18s and structed to give a cubic voxel dimension of 0.083 mm and ana- Hprt, only Actb was used to calculated fold changes. Manufac- lyzed using Amira 5.3.3 software package (Mercury Computer tures external reverse transcription and positive PCR controls Systems/TGS, Chelmsford, MA). Marrow cavity diameter were also included. (MCD) is reported as the average of the two endosteal diameters. For hypothalamus quantitative RT-PCR, 500 ng of RNA was Cortical bone width (CBW) is reported as the average of the four reverse transcribed using the SuperScript first-strand synthesis thicknesses on the major and minor axes of the cortical midslice. system (Invitrogen) following the manufacturer’s instructions. Geometrical parameter polar moment of area (mm4) was used as SYBR Green real-time PCR was performed for Agrp (agouti re- an overall measure of the amount of bone as previously described lated protein), Cart (cocaine and amphetamine regulated tran- (34). Whole-bone parameters of strength (torsional ultimate script), Npy (neuropeptide Y), Pomc (proopiomelanocortin), strength and strain energy to failure) and stiffness (torsional stiff- Lepra and Leprb (leptin receptor-a and -b). For internal control, ness) are descriptors of the whole bone, taking into account both Actb was used. Sequences of primers (Integrated DNA Technol- the material comprising the bone and the size and shape of the ogy, Coralville, IA) can be found in Supplemental Table 1, pub- bone. Material properties of strength (tensile strength) and stiff- lished on The Endocrine Society’s Journals Online web site at Endocrinology, September 2012, 153(9):4556–4567 endo.endojournals.org 4559 http://endo.endojournals.org. Lepra and Leprb primer se- isons between male and female offspring, regardless of maternal quences were developed using Primer Express software (Applied treatment, data were analyzed by ANOVA using a GLM of SAS. Biosystems), Actb (36), Agrp, Cart, Npy, and Pomc primer se- For all analyses, P Ͻ 0.05 was considered statistically significant. quences were previously published (37). One male and one female liver sample per mother were an- alyzed by PCR. Two male and two female sc fat, visceral fat, and Results hypothalamus samples were examined per mother. Pregnancy NAFLD analysis Food restriction resulted in 50% pregnancy loss, a rate Histological analysis was completed on offspring livers (54 that was not further modified by leptin injection (Table 1). males, 44 females) for NAFLD. Frozen liver samples were sec- ␮ ␮ No pregnancy loss occurred in the control (ad libitum fed) tioned at 8 m, and three sections at 50- m intervals were placed Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 on a slide. Samples were stained with Oil red O (Sigma) accord- dams. Maternal weight was not different among groups at ing to published guidelines (38), counterstained with Mayer’s d 1.5; however, control mothers gained weight and re- hematoxylin (Sigma), and mounted with glycerin jelly. Liver sec- stricted mothers lost it, such that restricted mothers were tions were evaluated for NAFLD based on published guidelines significantly lighter, independent of leptin treatment (P Ͻ (39, 40). Briefly, common features evaluated by an operator blinded to treatment were hepatocellular ballooning, steatosis, 0.05), by d 11.5 (Table 1). In other experiments with the and portal inflammation. Mallory’s hyaline was also noted. Each same design, leptin concentrations measured at d 11.5, 2 h abnormality in each liver section was graded using a 0–3 scale after the last injection were significantly increased (P Ͻ with a value of 0 corresponding to less than 33% of the sample 0.05) in restricted, leptin-treated mothers over control and covered with the abnormality. A value of 1 corresponded to restricted mothers (Schlitt, J. M., and L. C. Schulz, un- 33–50%, 2 to 50–66%, and 3 to presence of the abnormality in published data). There were no differences in leptin con- 66–100% of the sample. The total score for each section was the sum of the abnormalities, a range of 0–9 (examples, Supplemen- centrations at d 18.5 among treatment groups (unpub- tal Fig. 1). Three section scores per animal were averaged. lished data). Maternal treatment did not alter litter size or sex ratio. Preweaning weights were not different among Statistical analysis groups (Table 1). Data were analyzed using Statistical Analysis System (SAS institute, Cary, NC). Maternal data were analyzed by ANOVA Effect of maternal treatment on offspring obesity using a general linear model (GLM) of SAS. Offspring weight Although there were no differences in body weights data were analyzed using repeated-measures ANOVA with pups nested in the mother. Offspring serum insulin, leptin, and tri- among male offspring (Fig. 1A), body fat percentage was Ͻ glyceride measurements, heart weight, pancreas morphology, significantly reduced (P 0.05) in male offspring from and fat and hypothalamus real-time PCR, data were analyzed restricted mothers compared with control male offspring using a mixed-model of SAS with offspring nested in the mother. when on a standard diet (Fig. 1, B–E). This effect was Offspring IPGTT, kidney glomeruli number, and liver real-time attenuated by leptin supplementation to food-restricted PCR data were analyzed by ANOVA using a GLM of SAS. Post mothers. After the HFD, there were no differences in hoc analysis was performed using Tukey’s test. For NAFLD as- sessment, median scores were compared individually by off- weight or body composition among male offspring from spring and by average per mother using a Mann-Whitney rank the different maternal treatment groups (Fig. 1, F–I), all sum test with SigmaPlot (Systat Inc., San Jose CA). For compar- of which had significantly (P Ͻ 0.0001) higher body fat

TABLE 1. Pregnancy measurements

Measurement Control Restricted Leptin Maternal factors Number plug positive 5 8 8 Number live litters 5 4 4 Starting weight (dpc 1.5) 26.7 Ϯ 0.7 27.0 Ϯ 0.6 26.9 Ϯ 0.6 End treatment weight (dpc 11.5) 29.6 Ϯ 0.6a 24.3 Ϯ 1.2b 26.2 Ϯ 0.7b Litter size 9.3 Ϯ 0.9 10.3 Ϯ 0.3 9.0 Ϯ 0.5 Measurements at time the animals were killed Sex ratio (percent male) 54.2 Ϯ 0.1 49.1 Ϯ 0.1 51.9 Ϯ 0.5 Preweaning weights (per pup) Day 8 6.3 Ϯ 0.3 5.6 Ϯ 0.6 6.0 Ϯ 0.1 Day 15 9.8 Ϯ 0.6 9.4 Ϯ 0.4 10.3 Ϯ 0.4 Day 22 14.0 Ϯ 0.5 13.3 Ϯ 0.3 14.5 Ϯ 0.3 Numbers with different superscripts are significantly different (P Ͻ 0.05); n ϭ 5 control, four restricted, and four restricted, leptin treated. 4560 Pennington et al. Effect of Leptin on Fetal Programming Endocrinology, September 2012, 153(9):4556–4567

mothers (Fig. 2A). Notably, several members of the IGF family were al- tered. Igf1r (IGF-I receptor) was up- regulated (P Ͻ 0.05) in male offspring from restricted mothers compared with males from control mothers. Igf1 and Igf2 were not different (P ϭ 0.12) in restricted male offspring compared with restricted, leptin-treated and con- trol male offspring. Igfbp1 (IGF bind-

ing protein 1) was significantly de- Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 creased (P Ͻ 0.05) in male offspring from restricted, leptin-treated mothers compared with male offspring from re- stricted mothers but not different (P ϭ 0.10) from control male offspring, whereas Igfbp2 and Igfbp3 were not different among treatment groups. There were no differences in expres- sion of Ppara, Pparg, Ppargc1a, leptin, its receptor, or fatty acid binding pro- teins Fabp1 and Fabp7. Gene expres- sion of 18s rRNA was significantly de- creased (P Ͻ 0.05) in male offspring from restricted mothers compared with male offspring from control and re- stricted, leptin-treated mothers. The expression of the same genes was ex- amined in sc (data not shown) and vis- ceral fat from male offspring, and there were no differences among maternal treatment groups (Fig. 2C). Unlike in male offspring, significant weight differences were observed in fe- male offspring, with females born to re- FIG. 1. Offspring postweaning weights and body composition by treatment. A, Male offspring weights were not different between treatment groups before or after the HFD. J, stricted, leptin-treated mothers being Female offspring from restricted, leptin-treated mothers tended (#, P Ͻ 0.10) to be higher significantly heavier (Fig. 1J). Begin- from postweaning wk 13–19 compared with female control and restricted offspring. After ning at 14 wk after weaning, on a stan- the HFD, female restricted, leptin-treated offspring were significantly heavier (*, P Ͻ 0.05) compared with control and restricted female offspring. Representative MRI images and dard diet, restricted, leptin-treated fe- graphical representation of male offspring body fat percentage at 18 wk after weaning prior male offspring had numerically but to the HFD (B–E), male offspring body fat percentage at 25 wk after weaning and 5 wk on not significantly increased (P Ͻ 0.1) the HFD (F–I), and female offspring body fat percentage at 25 wk after weaning and 5 wk on weights compared with control and re- the HFD (K–N). E, I, and N, Data are represented as mean Ϯ SEM, and columns with different superscripts are significantly different. For comparisons among treatments, n ϭ 5 control, stricted female offspring. This was ex- four restricted, and four restricted, leptin-treated for both males and females. acerbated by the HFD. Female re- stricted, leptin-treated offspring body percentage (31.47 Ϯ 0.01) than on a standard diet weights were significantly different (P Ͻ 0.05) beginning (25.41 Ϯ 0.01). at 20 wk after weaning until the animals were killed (Fig. Real-time PCR gene expression analysis of male off- 1J). The percent body fat of female restricted, leptin- spring liver after the HFD revealed that several genes in- treated offspring was significantly increased (P Ͻ 0.05) volved in regulating body composition were up-regulated compared with female restricted offspring, whereas the by maternal food restriction, and these effects were atten- body fat percentage of female control offspring was inter- uated in male offspring from restricted, leptin-treated mediate after the HFD (Fig. 1, K–N). No differences were Endocrinology, September 2012, 153(9):4556–4567 endo.endojournals.org 4561

spring, only those born to restricted, leptin-treated mothers were insulin re- sistant, whereas all male offspring were insulin resistant, based on serum insu- lin levels and impaired glucose toler- ance (Fig. 3). In males, the maternal treatment group had no effect on insu- lin concentrations, which were uni- formly high (Fig. 3A), nor did it affect glucose tolerance, which was impaired

in all male offspring (Fig. 3B). Female Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 offspring from restricted, leptin- treated mothers had significantly in- creased (P Ͻ 0.05) serum insulin con- centrations compared with control and restricted female offspring (Fig. 3A). Female offspring from restricted, leptin-treated mothers also had nu- merically higher, but not significantly different, area under the curve after an IPGTT compared with female off- spring from control and restricted mothers (Fig. 3C). Pancreas morphology followed the same patterns as insulin levels and glu- cose tolerance. The ␣- and ␤-cell num- ber, ␤-cell mass, and ␤-to␣-cell ratio were not different among maternal treatment groups in male offspring (Fig. 3, D–F and J–M). Female off- spring from restricted (n ϭ 4) and re- stricted, leptin-treated (n ϭ 4) mothers had numerically higher but not signifi- ␤ ϭ FIG. 2. Real-time PCR analysis of expression of key metabolic genes in male (left panel, A–D) cantly different -cell number (P and female (right panel, E–H) offspring. A, B, E, and F, Liver. C, D, G, and H, Visceral fat. Data 0.08) and ␤-cell mass (P ϭ 0.13) com- are represented as mean fold change relative to control group mean. Error bars represent pared with control (n ϭ 5) female off- range of fold changes based on SEM of ⌬⌬Ct. *, Significant differences in ⌬Ct among ␣ treatment groups (ANOVA, P Ͻ 0.05); n ϭ 5 control, four restricted, and four restricted, spring (Fig. 3, J—K). The -cell number leptin treated for both males and females. and ␤-to␣-cell ratio were not different among female offspring groups (Fig. 3, observed in the expression of the genes involved in body G–M). composition examined by real-time PCR in liver, sc fat, Several adaptations were found in the gene expression and visceral fat (Fig. 2, E and G). of glucose metabolism regulators in the livers of male off- At the time the animals were killed, serum leptin levels spring from restricted mothers (Fig. 2B). Despite the ab- were not different among treatment groups in male or sence of a significant difference in glucose tolerance, male female offspring but were elevated across all groups (Table offspring of restricted mothers had gene expression 2). No differences were found among groups in heart changes indicative of enhanced insulin sensitivity. Gck weight, kidney glomeruli number, or serum triglyceride (glucokinase), a key enzyme in glycogen synthesis and gly- levels in male or female offspring (Table 2). colysis, was significantly increased (P Ͻ 0.05) in males from restricted mothers compared with controls but not Diabetic assessment different between male offspring of restricted, leptin- Insulin resistance and type 2 diabetes are consequences treated mothers and controls (P ϭ 0.08). Insr (insulin re- of metabolic syndrome. After the HFD, among female off- ceptor) was also significantly increased (P Ͻ 0.05) in re- 4562 Pennington et al. Effect of Leptin on Fetal Programming Endocrinology, September 2012, 153(9):4556–4567

TABLE 2. Offspring measurements

Males Females

Control Restricted Leptin Control Restricted Leptin Heart weight (mg) 228 Ϯ 6 227 Ϯ 6 220 Ϯ 6 166 Ϯ 6 166 Ϯ 4 174 Ϯ 6 Kidney, n 195 Ϯ 11 166 Ϯ 17 178 Ϯ 19 154 Ϯ 13 150 Ϯ 19 152 Ϯ 6 Leptin (ng/ml) 29.2 Ϯ 2.5 32.1 Ϯ 6.5 29.1 Ϯ 1.9 28.5 Ϯ 3.4 37.6 Ϯ 8.2 43.9 Ϯ 7.9 Triglycerides (mg/ml) 2.3 Ϯ 0.1 2.3 Ϯ 0.1 2.4 Ϯ 0.2 2.8 Ϯ 0.2 2.6 Ϯ 0.2 2.6 Ϯ 0.1 Bone biomechanics analysis Length (mm) 16.4 Ϯ 0.2 16.1 Ϯ 0.1 16.2 Ϯ 0.2 16.3 Ϯ 0.2 16.0 Ϯ 0.2 15.9 Ϯ 0.2 Marrow diameter (mm) 0.67 Ϯ 0.02a 0.59 Ϯ 0.02b 0.58 Ϯ 0.03b 0.50 Ϯ 0.03 0.51 Ϯ 0.03 0.47 Ϯ 0.03

Cortical bone width (mm) 0.59 Ϯ 0.01 0.63 Ϯ 0.03 0.61 Ϯ 0.03 0.60 Ϯ 0.03 0.59 Ϯ 0.02 0.61 Ϯ 0.02 Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 Polar moment of area (mm4) 1.09 Ϯ 0.04 1.09 Ϯ 0.08 1.02 Ϯ 0.12 0.81 Ϯ 0.07 0.78 Ϯ 0.06 0.77 Ϯ 0.04 Maximum torque (Nmm) 46.4 Ϯ 3.0 49.5 Ϯ 3.3 48.6 Ϯ 3.3 39.0 Ϯ 1.8 42.5 Ϯ 2.0 40.3 Ϯ 2.0 Measured torsional stiffness 63.4 Ϯ 5.2 53.1 Ϯ 5.8 63.2 Ϯ 5.8 56.6 Ϯ 4.2 54.0 Ϯ 4.4 55.5 Ϯ 3.9 (Nmm/radian) Material shear modulus (N/mm2) 405.1 Ϯ 22.5 352.1 Ϯ 55.6 437.2 Ϯ 15.0 496.8 Ϯ 36.5 499.3 Ϯ 66.9 505.4 Ϯ 38.2 Tensile strength (N/mm2) 41.9 Ϯ 3.5 45.7 Ϯ 3.9 47.3 Ϯ 3.9 43.7 Ϯ 4.0 49.2 Ϯ 4.4 46.7 Ϯ 2.3 Energy to failure (U) 9.5 Ϯ 1.3a 11.9 Ϯ 1.5b 11.4 Ϯ 0.5b 6.9 Ϯ 0.5 8.5 Ϯ 0.6 8.1 Ϯ 0.6 Numbers with different superscripts are significantly different (P Ͻ 0.05); n ϭ 5 control, four restricted, and four restricted, leptin treated for both males and females. stricted male offspring compared with controls. Akt2 between all male (n ϭ 54) and female (n ϭ 44) offspring, (thymoma viral protooncogene 2), Nr3c1 (nuclear recep- regardless of treatment group, and males showed a greater tor subfamily 3, group C, member 1, glucocorticoid re- severity of NAFLD overall (P ϭ 0.008, Fig. 4I). ceptor), and Irs1 (insulin receptor substrate 1) gene ex- pression was not different among the treatment groups. Brain and behavior There was no effect of maternal treatment group on the The hypothalamus is a central site in the body that expression of genes related to glucose metabolism in male controls feeding behavior and energy homeostasis. Lep- offspring visceral fat (Fig. 2D). In females, no differences tin signaling in the hypothalamus via leptin receptors were found in liver or visceral fat gene expression (Figure acts on orexigenic and anorexigenic neuropeptides to 2, F and H) among the treatment groups. Overall, after the control satiety (41). Analysis of Lepra and Leprb in the HFD, male offspring were significantly (P Ͻ 0.05) more hypothalamus revealed that male offspring from re- insulin resistant and glucose intolerant compared with fe- stricted and restricted, leptin-treated mothers tended male offspring (Fig. 3, A–C). Male offspring (n ϭ 54) (P ϭ 0.06) to have decreased Lepra but not Leprb gene produced significantly (P Ͻ 0.05) more insulin (44.8 Ϯ 9.1 expression compared with control male offspring (Fig. ng/ml) than female offspring (n ϭ 44, 13.7 Ϯ 6.6 ng/ml). 5A). No differences in Lepra or Leprb were found Males (n ϭ 13) also had significantly (P Ͻ 0.05) higher among female offspring (Fig. 5B). area under the curve (36845 Ϯ 2352) on the IPGTT vs. The hypothalamic gene expression of orexigenic neu- female offspring (n ϭ 25, 29480 Ϯ 1951). The pancreas ropeptides, Agrp and Npy, and anorexigenic neuropep- morphology assessment revealed that male offspring (n ϭ tides, Cart and Pomc, were also examined in male and 42) had significantly decreased (P Ͻ 0.0001) ␣-cell num- female offspring (Fig. 5, A and B). Pomc trended (P ϭ bers and significantly increased (P Ͻ 0.0001) ␤-cell num- 0.06) higher in male offspring from restricted, leptin- bers, ␤-cell mass, and ␤-to␣-cell ratio compared with treated mothers compared with control and restricted female offspring (n ϭ 40) (Fig. 3, D–M). male offspring. Agrp, Npy, and Cart did not differ among maternal treatments in male or female offspring. NAFLD assessment Locomotor activity as measured by distance traveled in Another common feature of metabolic syndrome is 1 h was similar among the treatment groups in male and NAFLD. The pathological analysis of offspring livers re- female offspring (Supplemental Table 2). Additionally, vealed that the severity of NAFLD did not differ between anxiety level was not affected by maternal treatment in male offspring (Fig. 4C and Supplemental Figs. 1 and 2). either male or female offspring (Supplemental Table 2). All male offspring showed signs of NAFLD. No significant differences in the severity of NAFLD were observed Offspring bone biomechanics among treatment groups in female offspring (Fig. 4F). The Male offspring from food-restricted mothers had stron- distributions of total scores for NAFLD were compared ger bones than controls, and this effect was not attenuated Endocrinology, September 2012, 153(9):4556–4567 endo.endojournals.org 4563 Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021

FIG. 3. Diabetic assessment of male and female offspring. A, Serum insulin concentrations in male and female offspring. Glucose tolerance test curves in male (B) and female (C) offspring. D--F, Representative images of pancreas morphology in male control, restricted, and restricted, leptin- treated offspring, respectively. G–I, Representative images of pancreas morphology in female control, restricted, and restricted, leptin-treated offspring, respectively. Line draw around ␤-cell area represents the selected region of interest for ␤-cell mass index. Average ␤-cell number (J), ␤- cell mass (K), ␣-cell number (L), and ␤-to␣-cell ratio (M) compared among treatment groups in male and female offspring are shown. Insulin- positive ␤-cells are stained cyan and glucagon-positive ␣-cells are magenta. Data are represented as mean Ϯ SEM. *, Significant difference from control of same sex (P Ͻ 0.05). For comparisons among treatments, n ϭ 5 control, four restricted, and four restricted, leptin treated for both males and females. by maternal leptin supplementation. Bone marrow diam- males having a significantly increased (P Ͻ 0.005) energy eter was significantly decreased (P Ͻ 0.05) in male off- to failure compared with females. spring from restricted and restricted, leptin-treated moth- ers compared with control male offspring (Table 2). The bone energy to failure, or total energy the bone can absorb Discussion before fracturing, was significantly increased (P Ͻ 0.05) in male offspring from restricted and restricted, leptin- Effects of maternal food restriction on male treated mothers compared with controls (Table 2). There offspring was a trend (P ϭ 0.06) for energy to failure to be increased Based on previous studies in humans, rats, and sheep (7, in female offspring from restricted mothers compared 8, 12), we predicted that maternal food restriction during with control female offspring (Table 2). early pregnancy would result in higher incidence of met- Overall, males had stronger bones than females. Males abolic syndrome features (elevated body weight, body fat had a significantly larger (P Ͻ 0.0001) marrow diameter percentage, glucose intolerance and NAFLD) in male off- than females. Males also had significantly increased (P Ͻ spring and that this would be exacerbated by a HFD (6). 0.0001) polar moment of area and (P Ͻ 0.001) maximal If anything, maternal food restriction had the opposite torque compared with females. Females had a significantly effect. On a standard diet, male offspring from restricted greater (P Ͻ 0.005) femur material shear modulus com- mothers had a body weight similar to controls, with a pared with males. All of these differences account for lower body fat percentage, and thus a higher lean mass. 4564 Pennington et al. Effect of Leptin on Fetal Programming Endocrinology, September 2012, 153(9):4556–4567

more resistant to metabolic syndrome than controls. They had increased ex- pression of IGF pathway components, indicative of their potential for in- creased lean mass, particularly muscle (42), and had significantly increased bone strength compared with control offspring. The IGF signaling axis can be programmed by maternal undernutri- tion (43–45). Skeletal growth is also

regulated by the IGF signaling axis (46). Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 There was an up-regulation of expres- sion of multiple insulin signaling com- ponents including Gck and Insr in male offspring from restricted mothers com- pared with control male offspring. FIG. 4. NAFLD diagnosis of male and female offspring. Representative images of NAFLD in male control (A), restricted (C), leptin-treated (E), and female control (B), restricted (D), and There are a few potential explana- leptin-treated (F) offspring are shown. Histogram of distribution of NAFLD severity in male (G) tions for the unexpected effect of ma- and female (H) offspring is also shown. For comparisons among treatments, n ϭ 5 control, ternal food restriction on male off- four restricted, and four restricted, leptin-treated for both males and females. I, Comparison of mean NAFLD severity Ϯ SEM between male and female offspring. *, Significant difference spring. One may be a species difference, between sexes (P Ͻ 0.05), n ϭ 54 males and 44 females. particularly with regard to the precise timing of food restriction. Although it is After exposure to the HFD, all male offspring, regardless the period with the strongest evidence of maternal treatment, exhibited features of the metabolic across multiple species, there are no data specifically in syndrome: increase in weight, high body fat percentage, mice on food restriction during early pregnancy. Food glucose intolerance, and NAFLD. However, even after the restriction throughout pregnancy in mice and rats can pro- HFD, male offspring of restricted mothers appeared to be gram obesity after a HFD (19, 47). Although restriction throughout pregnancy has shown similar effects in rats and mice, there is a lack of effect seen in humans (48), suggesting different timing sensitivities. It may also be of note that food restriction in our study began on d 1.5, thus missing the periconceptual period, which may be crucial. Another possibility is that leptin levels were not actually reduced in the food-restricted mothers. Previous work from our laboratory demonstrated in detail that 50% food restric- tion from d 1.5–11.5 severely decreases maternal leptin con- centrations, (31). In this study, both control and restricted mothers were given a saline injection to control for handling stress in the leptin-treated mothers. When we measured lep- tin levels under these conditions, 2 h after the leptin injection, there was no difference in leptin concentrations in control and restricted mothers (Schlitt, J. M., and L. C. Schulz, un- published data). A direct comparison of leptin concentra- tions in a small number of restricted mothers who were or were not given saline injections suggests that it may have increased serum leptin levels (unpublished data). We do not know whether the injection effect lasted beyond 2 h. There is one important piece of evidence suggesting that the food- FIG. 5. Real-time PCR analysis of hypothalamic gene expression in restricted mothers were truly leptin deficient: leptin injection male (A) and female (B) offspring. Data are represented as mean fold reversed most of the effects of food restriction on offspring. change relative to control mean Ϯ range of fold change based on SEM of ⌬⌬Ct, n ϭ 5 control, four restricted, and four restricted, leptin Bone is also known to be affected by fetal programming treated for both males and females. (49). In one study, whole pregnancy protein restriction Endocrinology, September 2012, 153(9):4556–4567 endo.endojournals.org 4565 caused a decrease in offspring bone mass (50). This is an apparent with age alone (17, 55), whereas others have opposite outcome compared with our observations, with found that feeding a HFD is necessary to exacerbate pro- early-pregnancy nutrient restriction resulting in male off- gramming effects (19, 56). Our findings indicate that in spring having stronger bones. One possibility for these utero exposure to increased maternal leptin levels (hyper- differences is the timing of treatment; a shorter period of leptinemia), even during food restriction, can lead to fetal nutrient deprivation may allow offspring to compensate programming events in female offspring. These findings and thus develop stronger bones. Furthermore, although mimic fetal programming studies of maternal obesity in leptin affects bone mass in adulthood (for review, see Ref. which female offspring are obese and diabetic (9, 11). In 51), we found no effect of leptin during early pregnancy on contrast, maternal food restriction during early pregnancy adult bone strength. generally does not affect female offspring (6, 12) because Downloaded from https://academic.oup.com/endo/article/153/9/4556/2424252 by guest on 29 September 2021 Our second prediction was that effects of maternal food it did not in this study. Leptin levels are positively corre- restriction on male offspring would be reversed by leptin lated with BMI in nonpregnant women and in first-tri- treatment, and this did occur. Before the HFD, body com- mester pregnant women (27, 57). By giving leptin to re- position of male offspring of restricted, leptin-treated stricted mothers, we have isolated the effects of mothers was more similar to controls than restricted off- hyperleptinemia from other consequences of maternal spring. Similarly, effects of maternal food restriction on obesity. Thus, exposure to hyperleptinemia during gesta- the IGF system and glucose metabolism of male offspring tion is one mechanism by which obesity, especially in fe- after the HFD were partially or completely attenuated by male offspring, is perpetuated. the leptin treatment. The only exception was increased bone strength of restricted offspring, which was not at- Leptin may play a role in the sexual dimorphism in tenuated by leptin treatment. Thus, we conclude that d nutritional fetal programming 1.5-d11.5 maternal food restriction programs reduced The effects of maternal nutritional status on offspring susceptibility of male offspring to metabolic syndrome and that this is due, in part, to reduced maternal leptin. are known to be sexually dimorphic. Previous studies in animal models and humans indicate that male offspring HFD more severely impacted male offspring than are more susceptible to programming by maternal food female offspring restriction (7, 8, 12), whereas female offspring are more The data presented here clearly indicate that males, re- susceptible to fetal programming by maternal obesity (9– gardless of maternal treatment group, are more suscepti- 11). Our data indicate that male offspring are more af- ble to metabolic syndrome than females when on a HFD. fected by maternal hypoleptinemia and female offspring Overall, male offspring were diabetic at the time the an- by maternal hyperleptinemia. Only female mice born to imals were killed and developed more severe NAFLD com- restricted mothers with hyperleptinemia during the first pared with female offspring. This is consistent with sta- half of gestation were more likely to become obese and tistics indicating that men have a higher incidence of insulin resistant. Only male offspring born to food re- metabolic syndrome compared with women (3) and with stricted mothers were different from controls. The ability previous data in mice (52). Males also had stronger bones of leptin to reverse this effect of maternal food restriction compared with females, a phenomenon that has been pre- shows that it is due at least in part to hypoleptinemia. viously described (53, 54). Based on these observations, we conclude that differential responses to leptin contribute to the sexual dimorphism Fetal programming of metabolic syndrome seen in fetal programming events caused by under- and occurred in female offspring from restricted, overnutrition. Further studies are needed to examine the leptin-treated mothers mechanism through which leptin may act to affect fetal Female offspring from restricted, leptin-treated moth- programming events in a sex-specific manner. ers developed multiple signs and symptoms of metabolic syndrome, including obesity and insulin resistance, com- pared with control and restricted female offspring. Signif- Acknowledgments icant weight differences were detected only after HFD, but it is not clear whether the diet alone, increasing age, or a We gratefully acknowledge the support provided by Lixin Ma combination, caused the weights to diverge significantly, and Ming Yang and the Veterans Affairs Biomolecular Imaging and we did not conduct other measures in the absence of Center at the Harry S. Truman Veterans Affairs Hospital Uni- HFD. Some maternal nutritional programming studies versity of Missouri-Columbia, Dennis Miller and the University have shown that effects of fetal programming may become of Missouri Center for Translational Neuroscience Behavior 4566 Pennington et al. Effect of Leptin on Fetal Programming Endocrinology, September 2012, 153(9):4556–4567

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