Neonatal Leptin Treatment Reverses Developmental Programming
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《内分泌学杂志》
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 and 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.), Auckland 1020, New Zealand
Institute of Biochemistry (A.G.), Food Science and Nutrition, the Hebrew University of Jerusalem, Rehovot 76100, Israel
Abstract
An adverse prenatal environment may induce long-term metabolic consequences, in particular obesity and insulin resistance. Although the mechanisms are unclear, this programming has generally been considered an irreversible change in developmental trajectory. Adult offspring of rats subjected to undernutrition during pregnancy develop obesity, hyperinsulinemia, and hyperleptinemia, especially in the presence of a high-fat diet. Reduced locomotor activity and hyperphagia contribute to the increased fat mass. Using this model of maternal undernutrition, we investigated the effects of neonatal leptin treatment on the metabolic phenotype of adult female offspring. Leptin treatment (rec-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, and normalized caloric intake, locomotor activity, body weight, fat mass, and fasting plasma glucose, insulin, and leptin concentrations in programmed offspring in adult life in contrast to saline-treated offspring of undernourished mothers who developed all these features on a high-fat diet. Neonatal leptin had no demonstrable effects on the adult offspring of normally fed mothers. This study suggests that developmental metabolic programming is potentially reversible by an intervention late in the phase of developmental plasticity. The complete normalization of the programmed phenotype by neonatal leptin treatment implies that leptin has effects that reverse the prenatal adaptations resulting from relative fetal undernutrition.
Introduction
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, 6). Data from epidemiological as well as in vivo animal studies have given rise to the concept of developmental programming, whereby an unfavourable 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). These adaptations may later prove to be a disadvantage, however, when the pre- and postnatal environments are widely discrepant (2). Although the mechanisms underlying developmental programming are unclear, epigenetic modification of gene expression is increasingly implicated (3, 4). The cues inducing such long-term changes leading to such postnatal metabolic phenotypes may either be nutritional or involve the glucocorticoid system (5, 6, 7). The general premise underlying the concept of developmental programming is that although it is induced by the processes of developmental plasticity (8), once induced the organism has a reduced capacity to reverse the developmental trajectory chosen, making developmental programming irreversible in a particular nutritional range (8).
Using one experimental approach to induce developmental programming, we have previously shown that maternal undernutrition during pregnancy results in the offspring developing obesity, hyperinsulinemia, and hyperleptinemia in adult life, particularly when fed a high-fat diet (9). Inactivity and hyperphagia contribute to the obesity phenotype in the adult offspring of undernourished mothers (10, 11).
The field of metabolic physiology has been profoundly altered by the discovery of the adipokines (12, 13). The initial view was that leptin was an anti-obesity hormone, preventing the storage of excess adipose tissue by feeding back to the hypothalamus to reduce food intake and increase energy expenditure (14, 15). In most humans with obesity, however, systemic leptin levels are elevated, in keeping with a state of leptin resistance (16, 17). Although a chronic elevation in leptin may not prevent weight gain, a sudden decrease in leptin coordinates adaptations aimed at protecting body weight during fasting. Restoring circulating leptin levels in fasting mice normalizes all of the neuroendocrine responses elicited by fasting (18).
Recent data suggest that leptin may have a broader range of actions, particularly during growth and development (19). 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 (20). Leptin mRNA is expressed in the placenta, and the human leptin gene has a placental-specific upstream enhancer (21). Breast milk also contains significant amounts of leptin (22), 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 are associated with rapid postnatal weight gain in small-for-gestational-age infants (23). The temporal coexpression of the long isoform of the leptin receptor and its ligand in mesenchymal tissues during fetal development (19) raises the possibility that leptin may act as a paracrine or autocrine factor during fetal life.
Studies of developmental programming suggest that both central and peripheral mechanisms are involved (11, 24, 25, 26), and we hypothesized that leptin might affect developmental programming by virtue of either its central or peripheral effects. Hypothalamic arcuate nucleus projections that regulate body weight mature during the first 2 wk after birth in rodents (27). 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 (27). Leptin may also influence the normal proliferation of pancreatic -cells that occurs in the neonatal period. Pancreatic -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 (28). In addition, leptin provides a functional link with insulin to form the adipoinsular axis (29, 30, 31) and plays an important role in the control of -cell function in vivo via inhibition of insulin secretion and reduction of insulin transcript levels (32, 33, 34) It is possible that maintaining a critical leptin level during development allows the normal maturation of tissues involved in metabolic homeostasis and that a period of relative hypoleptinemia may induce some of the metabolic adaptations that underlie developmental programming. The aim of the present study was to establish whether neonatal leptin treatment can alleviate postnatal obesity and the associated metabolic sequelae that occur in the offspring of undernourished mothers.
Materials and Methods
Study design
A previously developed maternal undernutrition model of fetal programming was used in this study (6). 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. Animals were assigned to one of two nutritional groups: 1) undernutrition (30% of ad libitum) of a standard diet throughout gestation (UN group) and 2) standard diet ad libitum 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 assure 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, female 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 libitum feeding until weaning. At weaning, saline- or leptin-treated AD and UN offspring were weight matched and placed on either standard rat chow or a high-fat diet [HF; Research Diets Inc. (New Brunswick, NJ) no. 12451, 45% kcal as fat]. At postnatal d 170, rats were fasted overnight and killed by halothane anesthesia followed by decapitation. Blood was collected into heparinized vacutainers 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.
Measurements
Body composition was assessed using dual-energy x-ray absorptiometry (DEXA) (Hologic, Waltham, MA). 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 ED50 was 0.31 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). Voluntary locomotor activity was assessed at d 165 in trials of 20 min duration after three habituation trials using Optimax behavioral testing apparatus (Columbus Instruments, Columbus, OH) as described previously (10). Statistical analyses were carried out using SigmaStat (Jandel Scientific, San Rafael, CA) and StatView (SAS Institute Inc., Cary, NC) statistical packages. Differences between groups were determined by three-way ANOVA (prenatal nutrition, treatment, and postnatal diet as factors) followed by Bonferonni post hoc analysis, and data are shown as mean ± SEM.
Results
As reported previously (9), maternal undernutrition resulted in fetal growth retardation reflected by significantly decreased birth weight in the offspring from UN dams (UN, 4.02 ± 0.03 g; AD, 6.13 ± 0.04 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 resulted in a transient reduction in pup weight gain in both AD and UN pups (Fig. 1), although the response was more acute and more marked in the UN group (mean relative weight loss over treatment period: AD, 3.8 ± 0.5%; UN, 14.8 ± 1.2%; P < 0.0001) (mean relative weight loss in first 72 h after treatment: AD, 3.2 + 1.9%; UN, 22.9 + 1.7%; P < 0.0001).
Hypercaloric nutrition from after weaning to the end of the study significantly (P < 0.0001) increased body weight gain when compared with chow-fed animals. There was a marked amplification of the diet-induced weight gain in the UN group (programming x diet interaction, P < 0.0001). Neonatal leptin treatment resulted in a normalization of diet-induced body weight gain in UN animals to match that of AD animals (Fig. 2). Body weight gain in AD animals on the high-fat diet was not different between those animals that had received saline or leptin in the neonatal period. However, body length (nose to anus) remained significantly shorter in all UN treatment groups (P < 0.001).
Total fat mass as assessed by whole-body DEXA scanning showed that the postnatal high-fat diet significantly increased fat mass in both HF groups (P < 0.0001) (Table 1). However, UN saline-treated HF animals had increased total body fat compared with AD saline-treated HF animals with the increased adiposity normalized after neonatal leptin treatment (programming x diet interaction, P < 0.05) (Fig. 3). There was no difference in total fat mass between AD and UN animals on the chow diet. A diet x treatment interaction was present whereby neonatal leptin treatment had the overall effect of reducing total body fat mass in animals on the high-fat diet (diet x treatment interaction, P < 0.05). Neonatal leptin treatment had no significant effect on total fat mass in AD animals in adult life, and reductions in fat mass induced by leptin were specific to UN animals (programming x treatment interaction, P < 0.0001). A significant programming x diet x treatment interaction reflects the specificity of leptin treatment to UN animals in the presence of a high-fat diet with no effects of neonatal leptin exposure in AD animals (P < 0.001) (Table 1).
Locomotor activity was significantly reduced in UN saline-treated animals on both chow and HF diets compared with AD saline-treated animals (P < 0.05) (Table 1). Leptin treatment did not alter locomotor activity in AD chow animals but increased locomotor activity in AD HF animals, UN chow animals and particularly in UN HF animals (Fig. 3). Caloric intake (kilocalories consumed per gram body weight) was significantly increased in UN animals compared with AD animals (P < 0.05) (Table 1). Caloric intake was reduced in all leptin treatment groups with intake in the UN leptin-treated HF group normalizing to that of AD animals (P < 0.05) (Fig. 3).
At postnatal d 170, fasting plasma leptin, C-peptide, and insulin concentrations were elevated in UN saline-treated HF animals compared with AD saline-treated HF animals (programming x postnatal diet interaction, P < 0.005) (Fig. 4). Neonatal leptin treatment normalized plasma leptin, C-peptide, and insulin in UN leptin-treated HF animals to match that of AD HF animals. In contrast, neonatal leptin treatment had no statistically significant effect on adult leptin, C-peptide, and insulin levels in AD animals fed chow, AD animals fed a high-fat diet, or UN animals fed chow. However, a trend toward an increase in plasma leptin, insulin, and C-peptide concentrations was noted in AD leptin-treated animals on both the chow and high-fat diets, but these did not reach statistical significance (P = 0.11, 0.08, and 0.07, respectively). Fasting plasma glucose concentrations were in the normal range for all treatment groups (data not shown).
Discussion
The present study demonstrates that all the measured metabolic consequences of maternal undernutrition were reversed by a period of neonatal leptin treatment in female rats. These effects of leptin to reverse metabolic programming were permanent and specific to the offspring of maternally undernourished animals, with neonatal leptin treatment having no significant effect on fat depots in animals born of normal birth weight. The observed findings are likely to result in part from a resetting of central and/or peripheral pathways that regulate energy homeostasis. Of interest, voluntary locomotor activity was significantly increased in the offspring of maternally undernourished animals given leptin compared with maternally undernourished offspring given saline.
Although the mechanisms underlying developmental programming are unclear, the process has been considered irreversible. The current findings indicate that, at least in the rat, there is an early postnatal window during which the process can be reversed. SGA (small for gestational age) children have been shown to have low cord blood and plasma leptin levels (36, 37) and a predisposition to develop the metabolic syndrome in adult life (38), although very little is known about the leptin surge in these cohorts. Our animal model closely resembles the increased weight gain and metabolic abnormalities seen in humans born of low birth weight, and thus we propose that the underlying mechanistic basis proposed in the present study will have parallels in the clinical setting. Defining the postnatal age limit after which a potential intervention would be ineffective and the underlying mechanisms will require further study. However, the continuing developmental plasticity of the hypothalamus (27), and the pancreas in the rodent (39), provides the opportunity to alter developmental programming. The timing of neonatal leptin treatment in the current study, from d 3–13, was an attempt to replicate the normal postnatal leptin surge (20).
Long-term effects of neonatal leptin treatment were restricted to the programmed animals. Energy homeostasis in the offspring of normally fed mothers was not significantly altered by neonatal leptin treatment. This specificity of leptin’s action argues in favor of neonatal leptin treatment correcting the mechanism of programming rather than having a general effect on metabolic determination. Additional work will be required to define the relationship between maternal undernutrition and leptin levels during fetal life and also in the immediate postnatal period. It is possible, however, that maternal undernutrition results in hypoleptinemia during a critical period of development and that this reduction in leptin is the cue that initiates the programming cascade. Gluckman and Hanson (2) have suggested that developmental programming is the consequence of a mismatch between the environment seen during the period of developmental plasticity and the mature environment. Maternal undernutrition has indicated to the fetus that the current and therefore future nutritional environments are likely to be poor, but the high-fat diet applied after weaning creates a severe metabolic mismatch. It is notable in the present study in females that the programmed phenotype is expressed only in the presence of a high-fat diet postnatally, whereas we have previously shown in the male that programming can manifest independently of postnatal nutrition (9). Irrespective of the mechanism that the fetus uses to read the nutritional cue, a high leptin level in the neonate might be interpreted as a high nutritional state (because leptin levels and fat mass generally correlate in the neonate), and given that there is still potential for plastic response, the organism readjusts from its earlier trajectory. Neonatal leptin caused a very different weight response in the pups of undernourished mothers. This suggests that sensitivity to leptin is affected by prenatal undernutrition and again might infer a central role for perinatal leptin levels having a key role in metabolic programming.
The concomitant reduction in caloric intake and increased locomotor activity in programmed animals treated with neonatal leptin may reflect a direct effect of leptin on the hypothalamus. Leptin-deficient animals have reduced neural projections from the arcuate nucleus to a number of other hypothalamic nuclei involved in energy homeostasis (27, 40). These projections can be normalized by exogenous leptin treatment but only if leptin is given during the neonatal period (27). It has also been shown that fetal hypothalamic development can be modified by maternal nutrition, and weanling offspring of rat dams fed a low-protein diet during pregnancy are reported to have malformed hypothalamic nuclei (24).
As well as having a central hypothalamic effect, neonatal leptin treatment may have reversed developmental programming by modifying postnatal pancreatic organogenesis. Fetal malnutrition causes a reduction in -cell number and pancreatic islet size in rat pups by suppressing islet cell proliferation (41) and increasing -cell apoptosis (25). Pancreatic -cells express the long form of the leptin receptor, and leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis (28). Work in our laboratory has described a dysregulation of the feedback loop between insulin and leptin in the pancreatic islet in adult offspring after maternal undernutrition (11). The normalization of insulin and C-peptide levels in adult programmed animals given neonatal leptin may be primary but could alternatively be secondary to the reduction in fat mass in these animals.
The results of this study indicate that developmental adaptations during fetal life can be reversed by interventions in the neonatal period, at least in an altricial species, and that alterations in perinatal leptin levels may play a crucial role in determining the occurrence of long-term metabolic sequelae. Given the implications of these findings, it will be important to determine leptin’s ontogeny in infants born prematurely or small for gestational age.
Footnotes
This work was funded by the Health Research Council of New Zealand and the National Research Centre for Growth and Development.
Abbreviation: DEXA, Dual-energy x-ray absorptiometry.
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Institute of Biochemistry (A.G.), Food Science and Nutrition, the Hebrew University of Jerusalem, Rehovot 76100, Israel
Abstract
An adverse prenatal environment may induce long-term metabolic consequences, in particular obesity and insulin resistance. Although the mechanisms are unclear, this programming has generally been considered an irreversible change in developmental trajectory. Adult offspring of rats subjected to undernutrition during pregnancy develop obesity, hyperinsulinemia, and hyperleptinemia, especially in the presence of a high-fat diet. Reduced locomotor activity and hyperphagia contribute to the increased fat mass. Using this model of maternal undernutrition, we investigated the effects of neonatal leptin treatment on the metabolic phenotype of adult female offspring. Leptin treatment (rec-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, and normalized caloric intake, locomotor activity, body weight, fat mass, and fasting plasma glucose, insulin, and leptin concentrations in programmed offspring in adult life in contrast to saline-treated offspring of undernourished mothers who developed all these features on a high-fat diet. Neonatal leptin had no demonstrable effects on the adult offspring of normally fed mothers. This study suggests that developmental metabolic programming is potentially reversible by an intervention late in the phase of developmental plasticity. The complete normalization of the programmed phenotype by neonatal leptin treatment implies that leptin has effects that reverse the prenatal adaptations resulting from relative fetal undernutrition.
Introduction
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, 6). Data from epidemiological as well as in vivo animal studies have given rise to the concept of developmental programming, whereby an unfavourable 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). These adaptations may later prove to be a disadvantage, however, when the pre- and postnatal environments are widely discrepant (2). Although the mechanisms underlying developmental programming are unclear, epigenetic modification of gene expression is increasingly implicated (3, 4). The cues inducing such long-term changes leading to such postnatal metabolic phenotypes may either be nutritional or involve the glucocorticoid system (5, 6, 7). The general premise underlying the concept of developmental programming is that although it is induced by the processes of developmental plasticity (8), once induced the organism has a reduced capacity to reverse the developmental trajectory chosen, making developmental programming irreversible in a particular nutritional range (8).
Using one experimental approach to induce developmental programming, we have previously shown that maternal undernutrition during pregnancy results in the offspring developing obesity, hyperinsulinemia, and hyperleptinemia in adult life, particularly when fed a high-fat diet (9). Inactivity and hyperphagia contribute to the obesity phenotype in the adult offspring of undernourished mothers (10, 11).
The field of metabolic physiology has been profoundly altered by the discovery of the adipokines (12, 13). The initial view was that leptin was an anti-obesity hormone, preventing the storage of excess adipose tissue by feeding back to the hypothalamus to reduce food intake and increase energy expenditure (14, 15). In most humans with obesity, however, systemic leptin levels are elevated, in keeping with a state of leptin resistance (16, 17). Although a chronic elevation in leptin may not prevent weight gain, a sudden decrease in leptin coordinates adaptations aimed at protecting body weight during fasting. Restoring circulating leptin levels in fasting mice normalizes all of the neuroendocrine responses elicited by fasting (18).
Recent data suggest that leptin may have a broader range of actions, particularly during growth and development (19). 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 (20). Leptin mRNA is expressed in the placenta, and the human leptin gene has a placental-specific upstream enhancer (21). Breast milk also contains significant amounts of leptin (22), 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 are associated with rapid postnatal weight gain in small-for-gestational-age infants (23). The temporal coexpression of the long isoform of the leptin receptor and its ligand in mesenchymal tissues during fetal development (19) raises the possibility that leptin may act as a paracrine or autocrine factor during fetal life.
Studies of developmental programming suggest that both central and peripheral mechanisms are involved (11, 24, 25, 26), and we hypothesized that leptin might affect developmental programming by virtue of either its central or peripheral effects. Hypothalamic arcuate nucleus projections that regulate body weight mature during the first 2 wk after birth in rodents (27). 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 (27). Leptin may also influence the normal proliferation of pancreatic -cells that occurs in the neonatal period. Pancreatic -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 (28). In addition, leptin provides a functional link with insulin to form the adipoinsular axis (29, 30, 31) and plays an important role in the control of -cell function in vivo via inhibition of insulin secretion and reduction of insulin transcript levels (32, 33, 34) It is possible that maintaining a critical leptin level during development allows the normal maturation of tissues involved in metabolic homeostasis and that a period of relative hypoleptinemia may induce some of the metabolic adaptations that underlie developmental programming. The aim of the present study was to establish whether neonatal leptin treatment can alleviate postnatal obesity and the associated metabolic sequelae that occur in the offspring of undernourished mothers.
Materials and Methods
Study design
A previously developed maternal undernutrition model of fetal programming was used in this study (6). 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. Animals were assigned to one of two nutritional groups: 1) undernutrition (30% of ad libitum) of a standard diet throughout gestation (UN group) and 2) standard diet ad libitum 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 assure 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, female 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 libitum feeding until weaning. At weaning, saline- or leptin-treated AD and UN offspring were weight matched and placed on either standard rat chow or a high-fat diet [HF; Research Diets Inc. (New Brunswick, NJ) no. 12451, 45% kcal as fat]. At postnatal d 170, rats were fasted overnight and killed by halothane anesthesia followed by decapitation. Blood was collected into heparinized vacutainers 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.
Measurements
Body composition was assessed using dual-energy x-ray absorptiometry (DEXA) (Hologic, Waltham, MA). 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 ED50 was 0.31 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). Voluntary locomotor activity was assessed at d 165 in trials of 20 min duration after three habituation trials using Optimax behavioral testing apparatus (Columbus Instruments, Columbus, OH) as described previously (10). Statistical analyses were carried out using SigmaStat (Jandel Scientific, San Rafael, CA) and StatView (SAS Institute Inc., Cary, NC) statistical packages. Differences between groups were determined by three-way ANOVA (prenatal nutrition, treatment, and postnatal diet as factors) followed by Bonferonni post hoc analysis, and data are shown as mean ± SEM.
Results
As reported previously (9), maternal undernutrition resulted in fetal growth retardation reflected by significantly decreased birth weight in the offspring from UN dams (UN, 4.02 ± 0.03 g; AD, 6.13 ± 0.04 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 resulted in a transient reduction in pup weight gain in both AD and UN pups (Fig. 1), although the response was more acute and more marked in the UN group (mean relative weight loss over treatment period: AD, 3.8 ± 0.5%; UN, 14.8 ± 1.2%; P < 0.0001) (mean relative weight loss in first 72 h after treatment: AD, 3.2 + 1.9%; UN, 22.9 + 1.7%; P < 0.0001).
Hypercaloric nutrition from after weaning to the end of the study significantly (P < 0.0001) increased body weight gain when compared with chow-fed animals. There was a marked amplification of the diet-induced weight gain in the UN group (programming x diet interaction, P < 0.0001). Neonatal leptin treatment resulted in a normalization of diet-induced body weight gain in UN animals to match that of AD animals (Fig. 2). Body weight gain in AD animals on the high-fat diet was not different between those animals that had received saline or leptin in the neonatal period. However, body length (nose to anus) remained significantly shorter in all UN treatment groups (P < 0.001).
Total fat mass as assessed by whole-body DEXA scanning showed that the postnatal high-fat diet significantly increased fat mass in both HF groups (P < 0.0001) (Table 1). However, UN saline-treated HF animals had increased total body fat compared with AD saline-treated HF animals with the increased adiposity normalized after neonatal leptin treatment (programming x diet interaction, P < 0.05) (Fig. 3). There was no difference in total fat mass between AD and UN animals on the chow diet. A diet x treatment interaction was present whereby neonatal leptin treatment had the overall effect of reducing total body fat mass in animals on the high-fat diet (diet x treatment interaction, P < 0.05). Neonatal leptin treatment had no significant effect on total fat mass in AD animals in adult life, and reductions in fat mass induced by leptin were specific to UN animals (programming x treatment interaction, P < 0.0001). A significant programming x diet x treatment interaction reflects the specificity of leptin treatment to UN animals in the presence of a high-fat diet with no effects of neonatal leptin exposure in AD animals (P < 0.001) (Table 1).
Locomotor activity was significantly reduced in UN saline-treated animals on both chow and HF diets compared with AD saline-treated animals (P < 0.05) (Table 1). Leptin treatment did not alter locomotor activity in AD chow animals but increased locomotor activity in AD HF animals, UN chow animals and particularly in UN HF animals (Fig. 3). Caloric intake (kilocalories consumed per gram body weight) was significantly increased in UN animals compared with AD animals (P < 0.05) (Table 1). Caloric intake was reduced in all leptin treatment groups with intake in the UN leptin-treated HF group normalizing to that of AD animals (P < 0.05) (Fig. 3).
At postnatal d 170, fasting plasma leptin, C-peptide, and insulin concentrations were elevated in UN saline-treated HF animals compared with AD saline-treated HF animals (programming x postnatal diet interaction, P < 0.005) (Fig. 4). Neonatal leptin treatment normalized plasma leptin, C-peptide, and insulin in UN leptin-treated HF animals to match that of AD HF animals. In contrast, neonatal leptin treatment had no statistically significant effect on adult leptin, C-peptide, and insulin levels in AD animals fed chow, AD animals fed a high-fat diet, or UN animals fed chow. However, a trend toward an increase in plasma leptin, insulin, and C-peptide concentrations was noted in AD leptin-treated animals on both the chow and high-fat diets, but these did not reach statistical significance (P = 0.11, 0.08, and 0.07, respectively). Fasting plasma glucose concentrations were in the normal range for all treatment groups (data not shown).
Discussion
The present study demonstrates that all the measured metabolic consequences of maternal undernutrition were reversed by a period of neonatal leptin treatment in female rats. These effects of leptin to reverse metabolic programming were permanent and specific to the offspring of maternally undernourished animals, with neonatal leptin treatment having no significant effect on fat depots in animals born of normal birth weight. The observed findings are likely to result in part from a resetting of central and/or peripheral pathways that regulate energy homeostasis. Of interest, voluntary locomotor activity was significantly increased in the offspring of maternally undernourished animals given leptin compared with maternally undernourished offspring given saline.
Although the mechanisms underlying developmental programming are unclear, the process has been considered irreversible. The current findings indicate that, at least in the rat, there is an early postnatal window during which the process can be reversed. SGA (small for gestational age) children have been shown to have low cord blood and plasma leptin levels (36, 37) and a predisposition to develop the metabolic syndrome in adult life (38), although very little is known about the leptin surge in these cohorts. Our animal model closely resembles the increased weight gain and metabolic abnormalities seen in humans born of low birth weight, and thus we propose that the underlying mechanistic basis proposed in the present study will have parallels in the clinical setting. Defining the postnatal age limit after which a potential intervention would be ineffective and the underlying mechanisms will require further study. However, the continuing developmental plasticity of the hypothalamus (27), and the pancreas in the rodent (39), provides the opportunity to alter developmental programming. The timing of neonatal leptin treatment in the current study, from d 3–13, was an attempt to replicate the normal postnatal leptin surge (20).
Long-term effects of neonatal leptin treatment were restricted to the programmed animals. Energy homeostasis in the offspring of normally fed mothers was not significantly altered by neonatal leptin treatment. This specificity of leptin’s action argues in favor of neonatal leptin treatment correcting the mechanism of programming rather than having a general effect on metabolic determination. Additional work will be required to define the relationship between maternal undernutrition and leptin levels during fetal life and also in the immediate postnatal period. It is possible, however, that maternal undernutrition results in hypoleptinemia during a critical period of development and that this reduction in leptin is the cue that initiates the programming cascade. Gluckman and Hanson (2) have suggested that developmental programming is the consequence of a mismatch between the environment seen during the period of developmental plasticity and the mature environment. Maternal undernutrition has indicated to the fetus that the current and therefore future nutritional environments are likely to be poor, but the high-fat diet applied after weaning creates a severe metabolic mismatch. It is notable in the present study in females that the programmed phenotype is expressed only in the presence of a high-fat diet postnatally, whereas we have previously shown in the male that programming can manifest independently of postnatal nutrition (9). Irrespective of the mechanism that the fetus uses to read the nutritional cue, a high leptin level in the neonate might be interpreted as a high nutritional state (because leptin levels and fat mass generally correlate in the neonate), and given that there is still potential for plastic response, the organism readjusts from its earlier trajectory. Neonatal leptin caused a very different weight response in the pups of undernourished mothers. This suggests that sensitivity to leptin is affected by prenatal undernutrition and again might infer a central role for perinatal leptin levels having a key role in metabolic programming.
The concomitant reduction in caloric intake and increased locomotor activity in programmed animals treated with neonatal leptin may reflect a direct effect of leptin on the hypothalamus. Leptin-deficient animals have reduced neural projections from the arcuate nucleus to a number of other hypothalamic nuclei involved in energy homeostasis (27, 40). These projections can be normalized by exogenous leptin treatment but only if leptin is given during the neonatal period (27). It has also been shown that fetal hypothalamic development can be modified by maternal nutrition, and weanling offspring of rat dams fed a low-protein diet during pregnancy are reported to have malformed hypothalamic nuclei (24).
As well as having a central hypothalamic effect, neonatal leptin treatment may have reversed developmental programming by modifying postnatal pancreatic organogenesis. Fetal malnutrition causes a reduction in -cell number and pancreatic islet size in rat pups by suppressing islet cell proliferation (41) and increasing -cell apoptosis (25). Pancreatic -cells express the long form of the leptin receptor, and leptin increases the viability of isolated rat pancreatic islets by suppressing apoptosis (28). Work in our laboratory has described a dysregulation of the feedback loop between insulin and leptin in the pancreatic islet in adult offspring after maternal undernutrition (11). The normalization of insulin and C-peptide levels in adult programmed animals given neonatal leptin may be primary but could alternatively be secondary to the reduction in fat mass in these animals.
The results of this study indicate that developmental adaptations during fetal life can be reversed by interventions in the neonatal period, at least in an altricial species, and that alterations in perinatal leptin levels may play a crucial role in determining the occurrence of long-term metabolic sequelae. Given the implications of these findings, it will be important to determine leptin’s ontogeny in infants born prematurely or small for gestational age.
Footnotes
This work was funded by the Health Research Council of New Zealand and the National Research Centre for Growth and Development.
Abbreviation: DEXA, Dual-energy x-ray absorptiometry.
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