Litter Size Variation in Hypothalamic Gene Expression Determines Adult Metabolic Phenotype in Brandt's Voles (Lasiopodomys brandtii)

Background Early postnatal environments may have long-term and potentially irreversible consequences on hypothalamic neurons involved in energy homeostasis. Litter size is an important life history trait and negatively correlated with milk intake in small mammals, and thus has been regarded as a naturally varying feature of the early developmental environment. Here we investigated the long-term effects of litter size on metabolic phenotype and hypothalamic neuropeptide mRNA expression involved in the regulation of energy homeostasis, using the offspring reared from large (10–12) and small (3–4) litter sizes, of Brandt's voles (Lasiopodomys brandtii), a rodent species from Inner Mongolia grassland in China. Methodology/Principal Findings Hypothalamic leptin signaling and neuropeptides were measured by Real-Time PCR. We showed that offspring reared from small litters were heavier at weaning and also in adulthood than offspring from large litters, accompanied by increased food intake during development. There were no significant differences in serum leptin levels or leptin receptor (OB-Rb) mRNA in the hypothalamus at weaning or in adulthood, however, hypothalamic suppressor of cytokine signaling 3 (SOCS3) mRNA in adulthood increased in small litters compared to that in large litters. As a result, the agouti-related peptide (AgRP) mRNA increased in the offspring from small litters. Conclusions/Significance These findings support our hypothesis that natural litter size has a permanent effect on offspring metabolic phenotype and hypothalamic neuropeptide expression, and suggest central leptin resistance and the resultant increase in AgRP expression may be a fundamental mechanism underlying hyperphagia and the increased risk of overweight in pups of small litters. Thus, we conclude that litter size may be an important and central determinant of metabolic fitness in adulthood.


Introduction
Early-life environmental influences on the adult metabolic phenotype are of interest both scientifically and clinically, as it relates to the risk factors contributing to the obesity epidemic [1]. Epidemiological and experimental studies show a linkage between low birth weight and increased obesity [2], which implies the importance of intrauterine environment in remodeling adult phenotypes. In contrast, rapid growth during lactation also increases obesity risk. For instance, maternal high-fat diet during lactation can induce offspring insulin resistance and obesity in adulthood [3]. Adult rats [4,5] and mice [6,7] previously subjected to early postnatal overnutrition in small litters are hyperphagic, hyperleptinemic and differ in emotional behavior from control litters. These observations from either maternal high-fat diet or litter size manipulation underscore the critical importance of early postnatal nutritional environment in ''programming'' the long-term regulation of energy homeostasis [8,9].
Litter size is an important life history trait [10], which is correlated negatively with postnatal growth [11,12] and thus may determine an individual's reproductive success, longevity or other fitness-correlated traits [13]. Despite its importance in evolution, few studies have been made to investigate the physiology and central mechanisms contributing to the longterm effect of litter size on adult metabolic phenotype. We utilized seasonal breeding Brandt's voles (Lasiopodomys brandtii), which have a mean litter size of seven (litter size varies from 2 to 14) [14]. In a previous study, we found that the pup mass at birth was not related to litter size, however, the offspring raised in litter size of four were 18% heavier than those from a litter size of ten at peak lactation in the voles [15]. Therefore, the offspring raised in different natural litter sizes are an appropriate model to study consequences of nutritional variations during the critical postnatal period on the regulation of adult energy homeostasis.
The mediobasal hypothalamus is the site of energy homeostasis, and can exquisitely sense and integrate peripheral metabolic cues to coordinate peripheral metabolism [16]. Adipose-tissue derived leptin represents one important metabolic signal which acts on the hypothalamus. In response to feeding, leptin is secreted from adipose tissue and engages leptin receptors (OB-Rb) in various hypothalamic regions, leading to activation of the JAK2-STAT3 pathway and increased metabolic rate and decreased food intake via a reduction in orexigenic neuropeptide Y (NPY) and agoutirelated peptide (AgRP), and increase expression of anorexigenic proopiomelanocortin (POMC) and cocaine-and amphetamineregulated transcript (CART) [17,18,19]. Meanwhile, the JAK2-STAT3 pathway stimulates transcription of suppressor of cytokine signaling 3 (SOCS3), a negative regulator of leptin signaling following Ob-Rb activation [20]. The decreased activation of leptin signaling and the increase in hypothalamic SOCS3 expression have been clearly related with leptin resistance in obesity [21,22].
In the present study, we investigated the long-term effects of natural litter sizes on offspring metabolic phenotype and biomarkers, such as food intake, resting metabolic rate (RMR), nonshivering thermogenesis (NST), uncoupling protein 1 (UCP1) in brown adipose tissue (BAT), body compositions, serum leptin and tri-iodothyronine (T3) and thyroxine (T4) levels in adulthood. In addition, we analyzed gene expression of OB-Rb, SOCS3, and orexigenic and anorexigenic neuropeptides in hypothalamus from young and adult voles raised in large and small litters. We hypothesized that postnatal litter size would permanently influence offspring metabolic phenotype and hypothalamic neuropeptide expression. We predicted that offspring reared from small litters would exhibit hyperphagia, excessive weight gain and hyperleptinemia in adulthood as compared to the counterparts from large litters, and that these phenotypes in small litters would be related to greater expression in hyperthalamic orexigenic neuropeptides.

Ethics Statement
All experimental protocols were reviewed and approved by the Animal Care and Use Committee of Institute of Zoology, the Chinese Academy of Sciences. The institute does not issue a number to any animal study, but each study requires the permit to use animals from the ethical committee. The animal facility must be licensed by the experimental animal committee of Beijing, and all staff, fellows and students must receive appropriate training before performing animal studies.

Animals
Brandt's voles were the offspring of our laboratory breeding colony founded by field-captured animals. After weaning (21 days of age), voles were housed as same gender sibling pairs in plastic cages (3065620 cm) and maintained in temperature (2361uC) and humidity-controlled rooms under a 16:8 h light/dark photoperiod with lights on at 04:00 h. All animals were provided standard rabbit pellet chow (KeAo Feed Co., Beijing) and water ad libitum.
At 3-4 months of age, virgin female voles were housed individually and acclimated for 2 weeks and then were paired with males for 4 days to allow mating. On the day of parturition, the dams with 10-12 pups (regarded as large) and 3-4 pups (regarded as small) were selected to compare the effect of litter size, since we found previously that there was a difference in pup body mass only between these two groups [15]. Four out of twelve dams in large litters and five out of seventeen dams in small litters killed some of their pups (5 pups were dead in either group). At weaning, one pup from every litter was sacrificed to collect tissues (without regard to gender), and another two (one male and one female) were housed individually until 13 weeks. During lactation, all the pups from one nest were weighed every 3 days. After weaning, body mass and food intake were recorded weekly. Food intake was determined for three consecutive days and the remains were collected after the 3-day test. RMR and NST were measured at 12 weeks of age. We compared the effects of litter size and gender in adulthood; therefore four groups (LM, male from large litter, n = 12; SM, male from small litter, n = 9; LF, female from large litter, n = 13; SF, female from small litter, n = 10) were used in this study.
All the animals were sacrificed by CO 2 overdose between 09:00 h and 11:00 h at 13 weeks of age. Trunk blood was collected and centrifuged at 4000 rpm for 30 min at 4uC and serum stored at 280uC until assayed. The whole brains were rapidly removed and placed on dry ice for slow freezing. A slice of brain tissue was cut between the optic chiasm and the mammillary bodies, and the hypothalamus was dissected by one horizontal cut immediately below the anterior commissure and sagittal cuts through the edge of the septum and perihypothalamic sulcus as previously described [23]. The hypothalamus was frozen in liquid nitrogen immediately and stored at 280uC until subsequent analysis. The interscapular brown adipose tissue (iBAT) was immediately and carefully dissected, weighed and stored at 280uC until assayed.

Metabolic trials
RMR and NST were measured by using an open-circuit respirometer (FOXBOX, Sable Systems International Inc., Las Vegas, NV, USA). To avoid possible effects of circadian rhythm interfering with the group effects, two groups of animals were measured in an alternating manner between 08:00 h and 17:00 h. RMR was assessed from the rate of O 2 consumption and CO 2 production at 30uC (within their thermal neutral zone) (constanttemperature incubator; model LRH-250; Yiheng Co., Shanghai, CHN). A vole was placed in a chamber (2006130685 mm, volume 1.4 L) for 2 h. The flow rate of incurrent and excurrent air (dried with anhydrous CaSO 4 ; W. A. Hammond Drierite Co., USA) was approximately 300-400?ml?min -1 and 100 ?ml?min -1 , respectively. The baseline of oxygen and carbon dioxide concentration were measured before and after each test. Oxygen consumption was recorded at intervals of 10 s. RMR was estimated from the stable lowest consecutive rate of oxygen consumption over 5 min.
The voles stayed in the chamber for another 1 h for NST measurement. Maximum NST was induced by a subcutaneous injection of norepinephrine (NE) at 2561uC and mass-dependent dosage of NE (Shanghai Harvest Pharmaceutical Co. LTD) was calculated according to Heldmaier [24] and the recommended dosage in Brandt's voles [25]. NST was calculated from the stable highest consecutive rate of oxygen consumption over 5 min. The rate of oxygen consumption was calculated according to the equation.

Body composition analysis
After dissection of the hypothalamus and iBAT, the following organs and tissues, including the heart, lungs, liver, kidneys, spleen, gonad, stomach, small intestine, caecum, colon, together with subcutaneous fat, epididymal fat, mesenteric fat, epigonadal fat were extracted and weighed (61 mg). The organs and carcass with fat pads were then dried in an oven at 60uC to constant weight. Body fat extraction from dry grinded carcass was performed with a Soxhlet Fat Extraction System (Avanti 2050; FOSS, Hogänä s, Sweden) with petroleum ether.

Serum assays
Serum leptin levels were determined by radioimmunoassay (RIA) with the 125 I Multi-species Kit (Cat. No. XL-85K, Linco Research Inc.), which had been validated in Brandt's voles [26,27]. The lowest leptin level detected by this assay when using a 100 ml sample was 1.0 ng/ml. The intra-and inter-assay coefficients of variation were 3.6% and 8.7%, respectively.
Serum T3 and T4 were quantified using RIA kits (Institute of Chinese Atomic Energy, Beijing) according to the instructions and we have validated this kit for use in Brandt's voles previously [26]. Intra-and inter-assay coefficients of variation were 2.4% and 8.8% for the T3, and 4.3% and 7.6% for T4, respectively.

Measurement of UCP1, COX4 and SIRT1 content in iBAT
Total protein content in iBAT was determined by Folin phenol method with bovine serum albumin as standard [28]. Uncoupling protein 1 (UCP1) and cytochrome c oxidase 4 (COX4), and SIRT1 content in iBAT was measured by Western blotting [27,29]. Total iBAT protein (90 mg/lane) was separated in a discontinuous SDS-polyacylamide gel (12.5% running gel and 3% stacking gel for UCP1, COX4 and b-tubulin; 8% running gel and 3% stacking gel for SIRT1 and b-tubulin) and transferred onto PVDF membranes (Hybond-P; Amersham, Buckinghamshire, UK). After transfer, membranes were stained with Ponceau S to confirm equal loading and transfer. Membrane were then blocked in 5% milk in Tris-buffered saline-Tween for 1 h at room temperature and probed with the indicated antibodies overnight at 4uC. Following incubation with the appropriate horseradish peroxidase-conjugated secondary antibody for 1 h, the bands were visualized by chemiluminescence (Amersham Life Sciences, Little Chalfont, UK). Densitometry was performed using Quantity One (version 4.4.0) software (BioRad, Hercules, CA).
Real-time PCR for measurement of hypothalamic OB-Rb, SOCS3, NPY, AgRP, POMC and CART mRNA expression Real time PCR reactions were performed in a 12.5 mL total volume comprised of 6.25 mL 26SYBR Premix EX Taq TM master mix, 1 mL cDNA templates and 0.2 mmoL/L primers using the SYBR Green I qPCR kit (Cat. No. DRR041D, TaKaRa, Shiga, Japan) in the Mx3005P quantitative PCR system (Stratagene, La Jolla, CA, USA). Thermal cycling conditions were: 95uC for 10 s, 40 cycles of 95uC for 5 s, 60uC for 20 s, and 72uC for 20 s. Samples were run in duplicate and all runs were accompanied by the housekeeping gene b-actin. Species-specific primers were designed (Table 1) and verified effectively in Brandt's voles [30]. Standard curves were constructed for each gene via serial dilutions of cDNA (1 to 26-fold dilutions). Analysis of standard curves between target genes and b-actin showed that they had similar amplification efficiency, which ensures the validity of the comparative quantity method. The data derived from Mx3005P quantitative software were expressed as relative amounts, which were calculated by normalizing the amount of target gene to bactin mRNA levels. No amplification was detected in absence of template or in the no RT control.

Statistical analysis
Data were analyzed using SPSS 13.0 (SPSS, Chicago, IL, USA). Prior to all statistical analyses, data were examined for assumptions of normality of variance using the Kolmogorov-Smirnov tests. Non-normally distributed data underwent logarithm or arcsine square root transformation. The temporal changes in body mass and food intake were assessed by repeated-measures ANOVA, followed by LSD post-hoc test. Group differences in body mass and food intake were assessed by two-way ANOVA and ANCOVA respectively. Differences among groups in serum leptin, T3, T4 levels, the mRNA levels of BAT UCP1, hypothalamic Ob-Rb, SOCS3, NPY, AgRP, POMC and CART in adult were assessed by two-way ANOVA, followed by Tukey post-hoc test. Differences in body compositions were analyzed by two-way ANCOVA with body mass as a covariate. At weaning, the differences in UCP1 and hypothalamic gene expression were assessed by independent samples t-test. Pearson correlation analyses were used to detect possible associations of serum leptin levels with body fat mass and food intake. Data are expressed as mean6SE. Values of P,0.05 were considered statistically significant.
During weeks 6 and 8, voles from small litters ate more food than those from large litters (P.0.05). However, there were no effects of litter size or gender on food intake between weeks 10-13 (P.0.05).

Hypothalamic neuropeptide mRNA expression
There was no difference in hypothalamic OB-Rb, SOCS3, NPY, AgRP, POMC and CART mRNA expression between large and small litters at weaning (Table 2).

Body compositions
The data for organ mass are presented in Table 3. The offspring from small litters had a larger brain than those from large litters (F 1, 39 = 14.592, P,0.001). In addition, BAT mass (F 1, 39 = 12.016, P = 0.001) and dry stomach mass (F 1, 39 = 5.131, P,0.05) were higher in females as compared to males. However, there were no differences observed in other organs between either litter sizes or gender.

Discussion
In this study, we used a wild rodent model to examine the consequences of postnatal litter size on offspring growth and adult metabolic phenotype, and to investigate the central mechanisms contributing to the long-term effect of litter size on metabolic fitness. As observed in several other rodent species [12,31,32], voles from small litters showed more rapid growth per pup during postnatal development than those from large litters. We also found that increases in hypothalamic SOCS3 and AgRP expression were associated with higher food intake and body mass in voles raised in small compared to large litters. These findings demonstrate that litter size may program adult metabolic phenotype by permanently influencing central leptin sensitivity and hypothalamic neuropeptide expression.

Metabolic phenotype associated with different litter sizes
Consistent with studies in rats with natural [33] or manipulated litter sizes [34,35] or maternal overnutrition [36,37], Brandt's vole offspring from small litters were heavier at weaning, and remained heavier than those from large litters until the end of the experiment at 13 weeks of age, but there were no differences in peripheral and visceral fat pads between litter sizes. After adjusting for the effect of body mass at weaning, there was no difference observed in post-weaning body mass between different litter sizes. This suggests that the difference in adult body mass is totally determined by pre-weaning litter size. In addition, some studies suggest that maternal obesity interacts with post-weaning high-fatdiet consumption to cause greater adiposity [37]. However, the consequences resulting from the nutritional environment during lactation differ from the intrauterine environment. For example, intrauterine growth restriction may result in offspring catch-up growth during development and program obesity in adult [10,38]. However, if malnutrition was prolonged throughout lactation, adult body weight can be permanently reduced [10]. These findings support the ''thrifty phenotype hypothesis'' generated by Hales and Barker [39,40].
Although we did not measure locomotion, we found that higher energy intake contributed to the heavier body mass in the vole offspring from small litters. In a wide range of mammals, litter size is correlated negatively with milk intake and growth per pup during lactation [11,15]. Even during post-weaning development, offspring from small litters still ate more compared to those from large litters, which is similar to the study in litter size-manipulated rats [34]. In the present study, we did not find any differences in protein levels of UCP1 (a molecular marker of BAT thermogenesis), COX4 (reflecting mitochondrial oxidative capacities) [41] and SIRT1 (evolutionarily conserved NAD+ dependent deacetylase regulating transcriptional networks in various critical metabolic processes) [42] in BAT, and in RMR and NST of the whole animal level between litter sizes. Moreover, thyroid hormone, especially the ratio of serum T3/ T4, which is an important determinant of energy expenditure [43,44], was not affected by litter size. Therefore, these results suggest that natural litter size did not induce long-term changes in energy expenditure in the voles. In contrast, when rats are raised in reduced nursing litter size, they demonstrated a reduction in cold-induced adaptive thermogenesis compared to controls [45]. The diverse results may attribute to the different models of early postnatal nutritional environment. Dams with manipulated litter sizes would have different energy output from those with the same natural litter size, thus the extent of malnutrition or overnutrition of the offspring in these models are different.
The similar energy intake, but higher RMR and NST associated with higher BAT mass, more UCP1 and COX4 content in BAT, may contribute to the lower body mass in the females as compared with the male voles. However, there was no gender difference in metabolic phenotype affected by litter size. This is in agreement with rat studies with different litter sizes or maternal nutrition [33,36]. Thus, the effect of litter size on an animal's phenotype in adulthood is independent of gender.  Central leptin sensitivity and hypothalamic neuropeptide expression programmed by litter size In the present study, there was no difference in serum leptin levels at weaning or in adulthood between different litter sizes. This was also found in litter size-manipulated rats [46], whereas the rat offspring from undernutritioned dams throughout pregnancy showed hyperleptinemia in adulthood [47]. Similarly, this same phenomenon is observed in human studies with hyperleptinemia in infants from gestational undernutritioned or diabetic mothers [48]. Further, intracerebroventricular leptin administration to neonatal rats altered adult female phenotypes, including a reduction in body mass and food intake [49]. These studies suggest that perinatal leptin plays the critical role in programming adult metabolic phenotypes although serum leptin showed different responses to early nutritional environments.
Despite the similar levels in serum leptin and hypothalamic OB-Rb mRNA, we found that hypothalamic SOCS3 expression increased in the adult offspring from small litters, indicating central leptin resistance. Some recent studies in postnatal overfed rats and mice reported higher SOCS3 expression and lower STAT3 activity in adulthood [46,50], similar to our present result in voles. Additionally, our findings are supported by another study which showed that the offspring from enlarged litter sizes had enhanced leptin sensitivity and were protected from obesity [35]. Indeed, leptin resistance was functionally verified by the absence of a decrease in food intake and body mass in response to leptin injection as well as by a lower expression of the hypothalamic leptin receptor and an increased expression of SOCS3 in neonatal leptin-treated [51,52,53] and maternal leptin-treated rats [54]. Interestingly, the rat offspring of intrauterine growth restriction also demonstrated leptin resistance, indicated by suppressed leptininduced STAT phosphorylation [2]. These findings using diverse animal models imply the universality of both prenatal undernu-  trition and postnatal overnutrition resulting in impaired leptin signaling pathway and may explain the persistent hyperphagia and overweight observed in these models. We further analyzed mRNA expression of hypothalamic neuropeptides related to orexigenic and anorexigenic pathways at weaning and in adulthood. We found increased mRNA expression of orexigenic AgRP and NPY (especially AgRP), but no changes in anorexigenic POMC and CART in the vole offspring from small litters in adulthood. Hypothalamic orexigenic NPY and AgRP mRNA expression also increased in high-fat fed rats from reduced litter size [55]. Moreover, the findings were partly supported by studies in maternal high-fat offspring which showed increased NPY immunoreactivity in arcuate nucleus at day 1 [56] or NPY Y1 receptor mRNA in the periventricular nucleus of the hypothalamus in adulthood [37]. Other orexigenic peptides, such as galanin, enkephalin and dynorphin in the paraventricular nucleus and orexin and melanin-concentrating hormone in the lateral hypothalamus, were found to increase in the offspring of rat dams on a high-fat diet during pregnancy [57]. These rats also exhibited an increase in neurogenesis in the hypothalamus, ultimately with a great proportion of new neurons expressed orexigenic peptides. All these findings indicate that hypothalamic neuropeptide expression may be programmed by nutritional environment induced by litter size during a critical window of postnatal development.
Taken together, voles from small litters showed greater food intake and body mass than voles from large litters. Litter size did not affect adult serum leptin levels, but did have long-term effects on hypothalamic leptin sensitivity. In addition, increased AgRP expression was associated with hyperphagia in the offspring from small litters. These findings provide further evidence that litter size can permanently influence leptin sensitivity and hypothalamic  Table 3. Dry mass of organs but brain and BAT (wet mass) in adult male and female offspring from large and small litters. neuropeptides, resulting in bonafide changes in the adult metabolic phenotype. From a physiological point of view, this study also highlights the importance of litter size in evolution, and suggests that animals raised in natural litter sizes would not be susceptible to obesity unless subjected to artificial manipulation during the early postnatal period.