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Endocrinology, doi:10.1210/en.2007-0981
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Endocrinology Vol. 149, No. 4 1906-1913
Copyright © 2008 by The Endocrine Society

The Effect of Neonatal Leptin Treatment on Postnatal Weight Gain in Male Rats Is Dependent on Maternal Nutritional Status during Pregnancy

Mark H. Vickers, Peter D. Gluckman, Alice H. Coveny, Paul L. Hofman, Wayne S. Cutfield, Arieh Gertler, Bernhard H. Breier and Mark Harris

Liggins Institute (M.H.V., P.D.G., A.H.C., P.L.H., W.S.C., B.H.B., M.H.), University of Auckland, Auckland 1023, New Zealand; National Research Centre for Growth and Development (M.H.V., P.D.G., A.H.C., P.L.H., W.S.C., B.H.B., M.H.), Auckland, New Zealand; and Institute of Biochemistry, Food Science and Nutrition (A.G.), the Hebrew University of Jerusalem, Jerusalem 76100, Israel

Address all correspondence and requests for reprints to: Dr. Mark Vickers, Liggins Institute, University of Auckland, Auckland 1023, New Zealand. E-mail: m.vickers{at}auckland.ac.nz.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
An adverse prenatal environment may induce long-term metabolic consequences, in particular obesity, hyperleptinemia, insulin resistance, and type 2 diabetes. Although the mechanisms are unclear, this "programming" has generally been considered an irreversible change in developmental trajectory. Adult offspring of rats subjected to undernutrition (UN) during pregnancy develop obesity, hyperinsulinemia, and hyperleptinemia, especially in the presence of a high-fat diet. Using this model of maternal UN, we have recently shown that neonatal leptin treatment in females reverses the postnatal sequelae induced by developmental programming. To examine possible gender-related effects of neonatal leptin treatment, the present study investigated the effect of neonatal leptin treatment on the metabolic phenotype of adult male offspring. Leptin treatment (recombinant rat leptin, 2.5 µg/g·d, sc) from postnatal d 3–13 resulted in a transient slowing of neonatal weight gain, particularly in programmed offspring. Neonatal leptin treatment of male offspring from normally nourished mothers caused an increase in diet-induced weight gain and related metabolic sequelae, including hyperinsulinemia and increased total body adiposity compared with saline-treated controls. This occurred without an increase in caloric intake. These effects were specific to offspring of normal pregnancies and were not observed in offspring of mothers after UN during pregnancy. In the latter, neonatal leptin treatment conferred protection against the development of the programmed phenotype, particularly in those fed the chow diet postnatally. These data further reinforce the importance of leptin in determining long-term energy homeostasis, and suggest that leptin’s effects are modulated by gender and both prenatal and postnatal nutritional status.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
AN ADVERSE intrauterine environment is associated with long-term metabolic consequences, in particular obesity, insulin resistance, and type 2 diabetes (1, 2, 3, 4, 5). Data from epidemiological as well as in vivo animal studies have contributed to the concept of developmental programming, whereby an unfavorable prenatal environment is believed to trigger adaptations that improve fetal survival or prepare the fetus in expectation of a particular range of environments postnatally (1). However, these adaptations (predictive adaptive responses) may later prove to be a disadvantage when the prenatal and postnatal environments are widely discrepant (2). The general premise underlying the concept of developmental programming is that, whereas it relies on plasticity, once induced the organism has a limited capacity to reverse the developmental trajectory chosen (6). Using one experimental approach to induce developmental programming, we have previously shown that maternal undernutrition (UN) during pregnancy results in the offspring developing obesity, hyperinsulinemia, and leptin resistance in adult life, particularly when fed a high-fat (HF) diet postnatally (7). Inactivity and hyperphagia contribute to the obesity phenotype in the adult offspring of undernourished mothers (8, 9).

Leptin, the product of the ob gene, is known to play a major role in the regulation of metabolism and neuroendocrine functions in several mammalian species (10, 11). Initially, leptin was viewed as an antiobesity hormone, preventing the storage of excess adipose tissue by feeding back to the hypothalamus to reduce food intake and increase energy expenditure (11, 12). However, in most humans with obesity, systemic leptin levels are elevated, in keeping with a state of leptin resistance (13, 14). Leptin is now reported to have a broad range of actions, particularly during growth and development (15). Serum levels of leptin vary dramatically during intrauterine and early postnatal life, with a 5- to 10-fold increase in leptin occurring between postnatal d 4 and 10 in female mice (16). Breast milk also contains significant amounts of leptin (17), and it may contribute to circulating levels in the neonate. Although cord blood leptin levels tend to reflect neonatal fat mass, low cord blood leptin levels correlate with rapid postnatal weight gain in small for gestational age infants (18). The temporal coexpression of the long isoform of the leptin receptor (OBRb) and its ligand in mesenchymal tissues during fetal development (15) raises the possibility that leptin may act as a paracrine or autocrine factor during fetal development. Because circulating leptin levels in neonates vary according to maternal diet, leptin can, therefore, be viewed as a critical link between environmental and maternal factors and the developing physiology of the infant (19).

Hypothalamic arcuate nuclear projections that regulate body weight mature during the first 2 wk after birth in rodents (20). Work by Bouret et al. (20) has shown that in leptin-deficient (ob/ob) mice, these projections remain immature and that treatment with leptin has a neurotrophic effect on these hypothalamic projections, but only during the neonatal period. Leptin may also influence the normal proliferation of pancreatic β-cells that occurs in the neonatal period. Pancreatic β-cells express the long form of the leptin receptor, and leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis and increasing islet cell proliferation (21). In addition, leptin provides a functional link with insulin to form the adipoinsular axis (22, 23, 24), and plays an important role in the control of β-cell function in vivo via inhibition of insulin secretion and reduction of insulin transcript levels (25, 26, 27). It has also been reported that leptin can modulate the hormonal response to stress in young rats either by a direct effect on the hypothalamic-pituitary-adrenal axis or indirectly through changing some aspects of maternal behavior (28, 29).

We have recently shown that neonatal leptin treatment to female offspring can reverse the postnatal phenotype induced by development programming. Therefore, it is possible that neonatal leptin levels play a critical role in establishing physiological pathways that regulate energy balance throughout postnatal life. Given that there are important gender-based differences in the regulation and action of leptin in humans (30) and in the rat (31, 32), the aim of the present study was to establish whether neonatal leptin treatment could alleviate postnatal obesity and the associated metabolic sequelae that occur in male offspring after developmental programming.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Study design
A previously developed maternal UN model of developmental programming was used in this study (7, 33), and the leptin treatment protocol is identical to that described for females (34). Virgin Wistar rats (age 100 ± 5 d) were time mated using a rat estrous cycle monitor to assess the stage of estrous of the animals before introducing the male. After confirmation of mating, rats were housed individually in standard rat cages with free access to water. All rats were kept in the same room with a constant temperature maintained at 25 C and a 12-h light, 12-h dark cycle. The experimental design is outlined in Fig. 1Go. Animals were assigned to one of two nutritional groups: 1) UN [30% of ad libitum (AD)] of a standard diet throughout gestation (UN group); and 2) standard diet AD throughout gestation (AD group). Food intake and maternal weights were recorded daily until the end of pregnancy. After birth, pups were weighed, and litter size was adjusted to eight pups per litter to ensure adequate and standardized nutrition until weaning. Pups from undernourished mothers were cross fostered onto dams that had received AD feeding throughout pregnancy. At postnatal d 3, male AD and UN pups were randomized to receive either saline or recombinant rat leptin (2.5 µg/g·d) for 10 d by sc injection (n = 16 per group). During and after treatment, all animals were maintained on AD feeding until weaning. At weaning, saline or leptin-treated AD and UN male offspring were weight matched within the maternal group and placed on either standard rat chow or a HF diet (45% kcal as fat, D12451; Research Diets, Inc., New Brunswick, NJ). At postnatal d 110, rats were fasted overnight and killed by halothane anesthesia, followed by decapitation. Blood was collected into heparinized Vacutainers (BD, Franklin Lakes, NJ) and stored on ice until centrifugation and removal of supernatant for analysis. All animal work was approved by the Animal Ethics Committee of the University of Auckland.


Figure 1
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  		FIG. 1. Study design. Dams were fed either standard chow AD throughout pregnancy (AD) or at 30% of AD (UN). At postnatal d 3, AD and UN litters were randomized to receive either saline or recombinant rat leptin (2.5ug/g·d, sc) until d 13. All dams were fed AD throughout lactation. At weaning (d 21), offspring were weight matched within treatment groups and placed on either a standard chow or a HF diet fed AD for the remainder of the study (d 110).

 
Measurements
Body composition, bone mineral content (BMC), and bone mineral density (BMD) were quantified at d 100 by dual-energy x-ray absorptiometry (DEXA) using a dedicated small animal software package (Lunar Hologic, DPX Small Animal Software, Waltham, MA). Precision of measurements for body composition and bone parameters is less than 1.8% and less than 1.5%, respectively, based on repeated measurements and phantom bone analysis. Food intake was measured over a 5-d period before the end of the trial. Plasma leptin was measured by RIA as described previously (35). The ED-50 was 0.34 ng/ml, and the intraassay coefficient of variation was less than 10% (all samples measured within a single assay). Fasting plasma insulin was measured using a rat insulin ELISA (no. 10-1124-10; Mercodia, Uppsala, Sweden). C peptide was measured using a rat C-peptide RIA (RCP-21K; LINCO Research Inc., St. Charles, MO). Fasting plasma glucose was measured using a YSI Glucose Analyzer (model 2300; Yellow Springs Instrument Co., Yellow Springs, OH). Statistical analyses were performed using SigmaStat (Jandel Scientific, San Rafael, CA) and StatView (SAS Institute Inc., Cary, NC) statistical packages. Differences between groups were determined by three-way factorial ANOVA (maternal nutrition, treatment, and postnatal diet as factors), followed by Bonferroni post hoc analysis, and data are shown as mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Maternal UN
Maternal UN resulted in fetal growth retardation reflected by significantly decreased birth weight in the offspring from UN dams (UN 4.12 ± 0.1 g, AD 6.68 ± 0.1 g; P < 0.0001). Litter size was not different between the two groups (AD 12.3 ± 1.8, UN 11.9 ± 2.0).

Neonatal leptin treatment
Neonatal leptin treatment resulted in a transient reduction in pup weight gain in both AD and UN pups, although the response was more acute and more marked in the UN group. Absolute body weights were identical within treatment group at the start of leptin treatment (AD saline 8.55 ± 0.2 g, AD leptin 8.57 ± 0.2 g, UN saline 5.95 ± 0.3 g, UN leptin 5.97 ± 0.2 g; UN effect P < 0.0001). Absolute body weight gains were significantly reduced after 72-h leptin treatment in UN animals compared with AD animals (AD saline 13.70 ± 0.2 g, AD leptin 13.45 ± 0.3 g, UN saline 9.54 ± 0.3 g, UN leptin 8.20 ± 0.2 g; P < 0.0001 for leptin treatment effect) (Fig. 2Go). The mean relative weight loss over the treatment period was significantly greater in UN compared with AD offspring (total for leptin treatment period, AD 3.32 ± 0.38%, UN 11.68 ± 0.78%, P < 0.0001; mean relative weight loss in first 72 h after treatment, AD 1.86 + 0.4%, UN 16.31 + 3.1%, P < 0.0001).


Figure 2
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  		FIG. 2. Change in absolute body weights in leptin-treated AD and UN neonates compared with saline-treated animals. Male UN offspring exhibited an acute response to leptin treatment compared with offspring born to AD mothers. P < 0.05 for effect of leptin, P < 0.05 for effect of programming, programming x leptin treatment interaction P < 0.05. Data are mean ± SEM (n = 8 per group).

 
Postnatal weight gain
Body weights at postnatal d 110 were significantly reduced in UN offspring compared with AD offspring (P < 0.05; Table 1Go and Fig. 3Go). AD and UN animals fed the HF diet were significantly heavier than those fed the chow diet (P < 0.0001). There was a programming x treatment interaction in which body weights were significantly reduced in UN offspring treated with leptin as neonates compared with saline-treated UN offspring (Fig. 3Go). Conversely, in AD animals fed the HF diet, there was a significant increase in body weight in leptin-treated offspring compared with saline-treated offspring. Of note, body weights of AD animals on the chow diet were not different after either neonatal saline or leptin treatment.


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  		TABLE 1. Phenotypical markers in male offspring at postnatal d 110 after treatment with either saline or leptin in the neonatal period

 

Figure 3
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  		FIG. 3. Body growth until postnatal d 110 of AD (left panel) and UN (right panel) offspring treated with either saline or leptin as neonates and fed either a chow or HF diet after weaning. P < 0.05 for effect of programming, P < 0.0001 for effect of diet, programming x treatment interaction P < 0.05. Data are mean ± SEM (n = 8 per group).

 
Nose-anus (NA) lengths were significantly reduced in UN offspring compared with AD offspring at postnatal d 110 (P < 0.0001; Table 1Go) and were significantly increased overall in HF fed animals (P < 0.005). AD and UN animals treated as neonates with leptin were significantly shorter as adults than those treated with saline (P < 0.005). A programming x diet interaction indicated that NA lengths were increased significantly more in HF vs. chow-fed UN animals compared with AD animals (P < 0.05).

Food intake
As reported by our group previously (7), food intake was slightly but significantly increased in UN offspring compared with AD offspring in adulthood (P < 0.05; Table 1Go). There was no significant effect of neonatal leptin treatment on food intake at postnatal d 110.

Fat mass
Total body fat as assessed by DEXA scanning was significantly increased in UN animals compared with AD animals treated with saline as neonates (P < 0.05) and was significantly increased in all HF fed animals (P < 0.0001; Fig. 4Go). A programming x leptin treatment interaction was present showing that AD offspring treated with leptin as neonates had increased total body fat as adults independent of postnatal diet. Conversely, neonatal leptin treatment to UN offspring resulted in a decreased fat mass in chow-fed UN animals as adults compared with saline-treated UN chow-fed controls, and there were no differences in body fat between saline and leptin-treated HF fed animals. The excised retroperitoneal fat depot data were consistent with the DEXA data. UN saline-treated offspring had significantly greater white adipose tissue (WAT) as quantified in retroperitoneal fat depots compared with AD saline-treated offspring (P < 0.05), which were further increased after HF feeding (P < 0.0001). There was a significant programming x leptin treatment interaction with neonatal leptin treatment increasing WAT retroperitoneal fat pad mass in AD but not UN offspring as adults (P < 0.05).


Figure 4
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  		FIG. 4. Total body fat as quantified by DEXA scanning. Effect of programming P < 0.05, effect of postnatal diet P < 0.0001, programming x leptin treatment interaction P < 0.02. Data are mean ± SEM (n = 8 per group). ADLC, AD leptin chow; ADLHF, AD leptin HF; ADSC, AD saline chow; ADSHF, AD saline HF; UNLC, UN leptin chow; UNLHF, UN leptin HF; UNSC, UN saline chow; UNSHF, UN saline HF.

 
Leptin
Fasting plasma leptin concentrations were significantly increased in UN saline-treated offspring compared with AD saline-treated offspring as adults (P < 0.05; Fig. 5AGo) and were significantly increased after postnatal HF feeding (P < 0.0001). There were no statistically significant interactions between any of the treatment groups.


Figure 5
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  		FIG. 5. A, Fasting plasma leptin concentrations at d 110 in AD and UN male offspring on either a chow or HF diet after neonatal saline or leptin treatment. Effect of programming P < 0.05, effect of postnatal diet P < 0.0001, no interactions. B, Fasting plasma insulin concentrations. Effect of programming P < 0.05, effect of postnatal diet P < 0.0001, programming x leptin treatment interaction P < 0.05. C, Fasting plasma C-peptide concentrations. Effect of programming P < 0.05, effect of postnatal diet P < 0.0001, programming x leptin treatment interaction P < 0.005. Data are mean ± SEM (n = 8 per group). ADLC, AD leptin chow; ADLHF, AD leptin HF; ADSC, AD saline chow; ADSHF, AD saline HF; UNLC, UN leptin chow; UNLHF, UN leptin HF; UNSC, UN saline chow; UNSHF, UN saline HF.

 
Insulin and glucose
Fasting plasma insulin concentrations were significantly increased in UN animals compared with AD animals treated with saline as neonates (P < 0.05). Neonatal leptin treatment in AD HF fed offspring resulted in a significant increase in plasma insulin concentrations compared with saline-treated AD offspring on the HF diet (P < 0.0001) (Fig. 5BGo). Plasma insulin concentrations in UN offspring fed the HF diet were not different between saline and leptin treatment groups. Neonatal leptin treatment in UN chow-fed offspring resulted in a significant decrease in plasma insulin concentrations compared with saline-treated UN offspring on the chow diet (P < 0.05). There was no significant effect of neonatal leptin treatment in AD chow-fed animals, and the HF diet did not significantly increase insulin concentrations in saline-treated AD animals compared with chow-fed AD controls. A programming x leptin treatment interaction indicated that leptin treatment had the overall effect of significantly increasing plasma insulin concentrations in AD but not UN animals (P < 0.05). There were no significant differences between any of the groups in fasting plasma glucose concentrations (Table 1Go), although there was a strong trend toward a decrease in plasma glucose concentrations in UN chow-fed offspring treated with leptin as neonates compared with saline-treated UN chow-fed animals (P = 0.056).

Insulin to glucose ratio
Fasting plasma insulin to glucose ratios [insulin (ng/ml) divided by fasting glucose (mmol/liter)] were significantly increased in UN offspring compared with AD offspring treated with saline as neonates (P < 0.05) and were further increased in animals fed the HF diet (P < 0.0001; Table 1Go). As observed with insulin, a programming x leptin treatment interaction was present in which the plasma insulin to glucose ratios were significantly increased in AD but not UN leptin-treated animals (P < 0.05). In animals fed the chow diet postnatally, neonatal leptin treatment resulted in a significant increase in insulin to glucose ratios in adult AD offspring but caused a significant decrease in insulin to glucose ratio concentrations in adult UN offspring treated with leptin as neonates compared with saline-treated controls (P < 0.05).

C peptide
As observed with plasma insulin, fasting plasma C-peptide concentrations were significantly increased in UN offspring compared with AD offspring treated with saline as neonates (P < 0.05).

Neonatal leptin treatment in AD HF fed offspring resulted in a significant increase in plasma C-peptide concentrations compared with saline-treated AD offspring on the HF diet (P < 0.0001) (Fig. 5CGo). Plasma C-peptide concentrations in UN offspring fed the HF diet were not different between saline and leptin treatment groups. Neonatal leptin treatment in UN chow-fed offspring resulted in a significant decrease in plasma C-peptide concentrations compared with saline-treated UN offspring on the chow diet (P < 0.001). There was no significant effect of neonatal leptin treatment in AD chow-fed animals, and the HF diet did not significantly increase C-peptide concentrations in saline-treated AD animals compared with chow-fed AD controls. A programming x leptin treatment interaction indicated that leptin treatment had the overall effect of significantly increasing plasma C-peptide concentrations in AD but not UN animals (P < 0.05).

Bone density and bone mass
BMC (g) was significantly reduced in UN offspring compared with AD offspring (P < 0.05; Table 1Go). BMC was significantly increased in all HF fed animals. A leptin treatment x postnatal diet interaction indicated that AD and UN chow-fed animals treated with leptin as neonates had decreased BMC compared with saline-treated animals, whereas HF fed animals treated with leptin as neonates had increased BMC compared with saline-treated animals. There were no significant effects of neonatal leptin treatment on total BMD (g/cm2) as adults. BMD was slightly but significantly increased in all HF fed animals (P < 0.005), and was not different between AD and UN animals.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In this study the long-term effects of neonatal leptin treatment on body composition and metabolism in male offspring were dependent upon prior developmental programming and postnatal nutrition. Neonatal leptin treatment promoted obesity in AD male offspring, particularly in the context of a HF diet, whereas in UN males it prevented induced-induced obesity, but only if the animals were fed a standard chow diet. Any protective effect was negated when the UN animals were fed a HF diet. These findings are in contrast to female pups in which neonatal leptin treatment in AD animals had no significant effect on body composition or metabolism, regardless of the postnatal diet, whereas neonatal leptin treatment protected UN females from becoming obese on both a HF and a standard chow diet (34).

The results observed for male offspring of normal AD pregnancies are in some agreement with those recently reported by Yura et al. (36), showing that treatment of control offspring with leptin in the neonatal period resulted in a modest increase in the risk for obesity as compared with saline-treated controls. Work by Toste (37) and de Oliveira Cravo (38) et al. has also shown that neonatal leptin administration in male pups from normal pregnancies results in obesity associated with reduced expression of hypothalamic leptin receptors and central leptin resistance. However, in all of these studies, only male offspring of normal control dams were treated with leptin. Therefore, the potential interaction between developmental programming and alterations in neonatal leptin levels was not addressed. In addition, gender-specific differences associated with neonatal leptin treatment were not investigated in these studies.

The effect of leptin treatment on neonatal weight gain in the AD and UN groups was fundamentally different, raising the possibility that UN pups are more sensitive to leptin in the neonatal period. In the AD leptin-treated pups, there was a moderate reduction in weight gain throughout the treatment period when compared with the saline-treated group. However, in the UN group, there was a rapid and significant effect on weight gain that lessened during the treatment period so that by d 13, the relative effect of leptin on weight gain was similar in the UN and AD groups. Hypothalamic levels of OBRb mRNA are very low to undetectable shortly after birth (postnatal d 4) and increase significantly between postnatal d 4 and 14 (39). It is possible that developmental programming may alter the ontogeny of hypothalamic leptin receptor expression, and partly explain the observed difference in leptin sensitivity between the AD and UN pups.

Although neonatal leptin treatment had an effect on body weight during the period of treatment, data confirming that this weight loss was a consequence of reduced milk intake were not available for this study. Daily ip injections of 1 mg/kg leptin from d 7–10 in C57BL/6J mice were previously reported to have no effect on milk intake or body weight (40), however, the dose used was 2.5 times less than in the current study. When 1 µg leptin was administered intracerebroventricularly to 17-d-old pups, it markedly increased oxygen consumption, and by 28 d intracerebroventricular leptin inhibited food intake (40). In addition, the observed differences in response to neonatal leptin treatment on weight gain may have been a direct result of differential effects of leptin on energy expenditure in AD and UN pups. Work by Stehling et al. (41) has shown that treatment with murine leptin from neonatal d 7–16 can reduce juvenile fat storage solely by increasing energy expenditure and independent of its effects on food intake. It has also been shown that, despite adult-like effects of leptin treatment on appetite-related neuropeptides proopiomelanocortin and neuropeptide Y expression in neonates, leptin does not regulate food intake during early development (42). It is of interest that the maternal food intake of leptin-treated pups in the current study (data not shown) was decreased during lactation, and this may have been a proxy for reduced energy demand on the mother.

Leptin sensitivity of adult offspring was not directly tested in the current study. The fact that leptin levels were positively correlated with fat mass and, therefore, different between groups but there was no intergroup difference in food intake suggests that there was a difference in hypothalamic leptin sensitivity across the groups. It has been shown that neonatal leptin treatment to normal male rats can lead to hypothalamic leptin resistance and increased body weight gain in adulthood (36, 37). We have also recently shown that offspring of mothers that were food restricted during pregnancy are leptin resistant in adulthood compared with control animals (43).

A number of studies have highlighted the complex relationship between gender and energy homeostasis. Serum leptin levels in humans are higher in females than males, and this is partly attributed to the greater sc fat depots in females. Moreover, sex steroids may not only modulate adipocyte leptin secretion, but also the sensitivity of the hypothalamus to leptin. Testosterone has an inhibitory effect on leptin secretion, whereas estrogen has a stimulatory effect. Recent studies suggest that male rats are more sensitive to the anorectic effects of insulin, whereas females are more responsive to alterations in serum leptin. Part of the increased leptin sensitivity in females appears to be due to estrogen-dependant effects in the arcuate nucleus of the hypothalamus (44). Male mice are also more susceptible to dietary induced weight gain than female mice (45). Together, these findings suggest that although serum leptin levels are higher in female rats, this does not necessarily indicate leptin resistance but, rather, an altered set point in relation to leptin’s effect on energy homeostasis.

It is perhaps not surprising then that neonatal leptin treatment of male and female pups resulted in different long-term effects on energy homeostasis. The combined data suggest a complex interplay between programming, postnatal diet, and neonatal leptin treatment in male and female rats. UN females were more susceptible than UN males to dietary induced weight gain, as a result of increased caloric intake and reduced energy expenditure. Neonatal leptin treatment amplified the effects of a HF diet in AD males, but not AD females, however, this effect was independent of food intake and may reflect a lasting effect of neonatal leptin exposure on energy use.

NA lengths were significantly reduced in all AD and UN offspring treated with leptin as neonates. There is a body of literature that suggests that leptin can exert both direct stimulatory and indirect suppressive effects on bone formation via the hypothalamus (46). Leptin signaling in osteoblasts has not been clearly demonstrated, but intracerebroventricular infusion of leptin causes bone loss in leptin-deficient and wild-type mice. Takeda et al. (47) have identified neuronal networks required for leptin antiosteogenic function and demonstrated that the sympathetic nervous system is a negative regulator of bone formation. The suppressive effects are believed to be mediated by release of noradrenaline from the sympathetic nervous system. However, leptin-deficient ob/ob mice are shorter than wild-type mice, suggesting that an absence of circulating leptin has a negative effect on bone growth. Further research will be necessary to elucidate the mechanism by which neonatal leptin treatment results in a long term effect on skeletal growth.

The mechanisms underlying the observation of reversibility after neonatal leptin intervention in females are not yet fully understood. However, because the plasticity of systems that control energy homeostasis occurs during early developmental stages, the perinatal period is the most important time in which extrinsic factors may permanently alter metabolic set points (48). It has been suggested that neonatal leptin exposure can modulate the hormonal response to stress in young rats either by a direct effect on the hypothalamic-pituitary-adrenal axis or indirectly through changing some aspects of maternal behavior (28). In the present study, mothers were monitored daily during the lactation period for signs of behavioral differences, including suckling/grooming behavior and retrieval of displaced pups. Although not quantifiable, we observed no differences in maternal bonding or behavior toward offspring of leptin-treated animals compared with saline-treated controls.

Recent work has highlighted the plasticity of arcuate nuclear projections in the neonatal period and shown the importance of leptin as a signal for the development of hypothalamic circuits (20, 49). Hypothalamic arcuate nuclear projections that regulate body weight mature during the first 2 wk after birth in rodents (20, 50). In leptin-deficient (ob/ob) mice, these projections remain immature. Exogenous leptin has a neurotrophic effect on these hypothalamic projections, but only during the neonatal period (20). Although ob/ob mice administered leptin during this crucial neonatal period had no detectable leptin in their circulation as adults, their feeding behavior was closer to that of wild-type mice than untreated ob/ob littermates. This led to the conclusion that hypoleptinemia during a crucial period of development would predispose an animal to later obesity as a result of permanent structural abnormalities in the hypothalamic circuits that regulate energy homeostasis (51).

Inducing a supraphysiological leptin surge by administering leptin to control male pups may have relevance to models of neonatal overnutrition. One of the interesting observations from human epidemiological studies is that the relationship between birth weight and adult metabolic abnormalities is "U" shaped (52). Animal models of neonatal overnutrition have also demonstrated a link between excessive weight gain early in life and later metabolic complications (53). Neonatal leptin treatment to AD male offspring subsequently placed on a HF diet appears to have exacerbated the degree of leptin resistance in these animals. It is possible that the mechanism underlying the long-term effects of neonatal overnutrition on energy homeostasis may depend upon a period of hyperleptinemia during a critical stage of development.

In summary, the results of the present study further implicate the importance of leptin in the perinatal period and highlight that alterations in perinatal leptin levels play a crucial role in determining the occurrence of long-term metabolic sequelae. Further elucidation of the neonatal leptin surge and leptin receptor expression during this window of plasticity will bring us closer to defining the precise role of leptin in developmental programming.


   Footnotes
 
Disclosure Statement: The authors have nothing to declare.

First Published Online January 10, 2008

Abbreviations: AD, Ad libitum; BMC, bone mineral content; BMD, bone mineral density; DEXA, dual-energy x-ray absorptiometry; HF, high-fat; NA, nose-anus; UN, undernutrition; WAT, white adipose tissue.

Received July 17, 2007.

Accepted for publication December 28, 2007.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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