INTRODUCTION

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BOTH MATERNAL OBESITY and diabetes during gestation appear to promote the development of obesity and diabetes in the progeny (2, 24-26, 28). Because both obesity and diabetes can be genetically acquired, there has always been a question about the role of genetic background vs. the adverse metabolic effects associated with gestational diabetes and obesity in influencing outcome (24). Because this is difficult to resolve in human beings, various animal models have been used to address this issue. In rats, administration of insulin in high doses during the third trimester produces obesity in the offspring that is associated with abnormalities of brain noradrenergic pathways (9, 11-13). Direct injection of insulin into the ventrobasal hypothalamus in the immediate postnatal period also promotes obesity associated with abnormal glucose tolerance in later life (27). Offspring of dams fed a high-fat diet during pregnancy and lactation became obese and had abnormal glucose metabolism as adults (7). Such studies suggest that gestational abnormalities in diet or insulin levels may have adverse effects on offspring. However, the design of such studies leaves issues unanswered related to genetic background and the effects of pre- vs. postnatal environment on outcome in the progeny.

We recently derived two substrains of the Sprague-Dawley rats that were bred for their propensity to resist (DR) or develop diet-induced obesity (DIO) when fed a diet relatively high in energy, fat, and sucrose content (HE diet) (19). Although outbred Sprague-Dawley rats have virtually identical carcass fat and glucose metabolism when fed Chow (16, 18), selectively bred DIO rats have increased carcass fat and mild glucose intolerance at 2-3 mo of age compared with DR rats, even when fed Chow from weaning (19). Nevertheless, exposure to an HE diet for just 2 wk produces frank obesity and glucose intolerance in the selected DIO rats, whereas DR rats are largely unaffected by this diet (19). These substrains allow us to predict with certainty the phenotype of offspring of like matings. Thus we have used these animals to address the issue of how genetic background interacts with gestational obesity and hyperinsulinemia to affect the development of obesity and glucose intolerance in the offspring.

	METHODS

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Dams and breeding. The breeding pairs for this experiment were derived from our two colonies of rats bred selectively for their propensity to develop DIO or DR, respectively (19). These colonies were originally derived from outbred Sprague-Dawley rats (Charles River Laboratories). Rats were kept at 23-24°C on a 12:12-h light-dark cycle. One month before breeding, 18 DIO and 27 DR females were divided into one of five groups of nine dams each: 1) DR Chow dams were continued on Purina Rat Chow that contains 3.30 kcal/g with 23.4% as protein, 4.5% as fat, and 72.1% as carbohydrate, which is primarily in the form of complex polysaccharide (20); 2) DR HE diet dams were fed a HE diet composed of 8% corn oil, 44% sweetened condensed milk, and 48% Purina Rat Chow (Research Diets; this diet contains 4.47 kcal/g with 21% of metabolizable energy content as protein, 31% as fat, and 48% as carbohydrate, 50% of which is sucrose; Ref. 20); 3) DR Ensure dams were given HE diet ad libitum and, in addition, were given access to Ensure (Ross Products), which is a liquid diet containing 1.06 kcal/ml with 14% of the metabolizable energy content as protein, 22% as fat, and 64% as carbohydrate (DR rats overeat and become obese on this diet; Ref. 21); 4) DIO Chow dams were fed Chow; and 5) DIO HE diet rats were fed the HE diet. After 1 mo on their respective diets, all dams underwent tail bleeding for plasma glucose, insulin, and leptin levels and were then mated with males of the same genotype. Of these matings, 26 of 27 DR dams were successfully impregnated, whereas only 8 of 18 DIO dams were successfully impregnated. Dams were kept on their respective diets through gestation and weaning. A second blood sample was drawn on the 2nd wk of gestation. At birth, litter sizes ranged from 4 to 15 pups, with means in the five groups ranging from 10 to 14 without any significant differences in litter sizes among the groups. All litters were culled to 4-10 pups; i.e., random pups from litters larger than 10 were discarded. Two weeks into the lactation period a final weighing and blood drawing was carried out in the dams.

Pups and diet manipulations. After weaning, all male pups were separated from their dams and placed in group cages (2-4/cage) according to the diet groups of their dams. There were 24 rats per DR group and 12 rats per DIO group. They were fed Chow and water ad libitum and weighed weekly. At 13 wk of age, they were placed in individual cages, and their intake of powdered Chow was monitored for 3 wk. At 16 wk of age, half of the rats in each group (DR = 12/group, DIO = 6/group) were then killed by decapitation with removal and weighing of the retroperitoneal adipose depots and collection of trunk blood for plasma glucose, insulin, and leptin. The remaining rats in each group (DR = 12/group, DIO = 6/group) had tail blood taken for plasma glucose, insulin, and leptin levels and were then placed on HE diet with their intake was monitored for an additional 4 wk. At the end of this period, rats were killed by decapitation, their retroperitoneal, perirenal, and epididymal adipose depots were removed and weighed, and their trunk blood was collected for plasma glucose, insulin, and leptin levels.

Assays of glucose, insulin, and leptin. Samples of trunk blood were collected into heparinized tubes, and plasma was removed for assay. Glucose was assayed by automated glucose oxidase method (Beckman), and both insulin and leptin were analyzed by RIA (Linco) using antibodies to authentic rat insulin and leptin, respectively.

Statistics. One-way ANOVA was used for single-point measures of total food intake, terminal body and fat depot weights, and plasma glucose, insulin, and leptin levels. Log transformation of plasma insulin and leptin data was carried out for additional comparison by ANOVA, although this did not produce additional significant intergroup differences. Repeated-measures ANOVA was used to compare intake and weight gain measures across time. When significant intergroup differences were found by ANOVA (P  0.05), post hoc comparisons were carried out by Scheffé's analysis.

	RESULTS

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Dam weight gain and plasma hormone levels. As in previous studies of these selected rats (19), DIO dams were 28% heavier than DR dams of the same age before they were begun on HE diet (Table 1). After 1 mo, DR dams fed the HE diet gained the same amount of body weight as Chow-fed DR dams. On the other hand, DR Ensure dams became as obese as the DIO HE diet dams. Their weight gain was thus more than threefold greater than DR Chow and HE diet-fed dams. The DIO HE diet dams gained 56% more weight than DIO Chow-fed dams even though their absolute body weights did not differ significantly. Plasma insulin levels in DR Ensure, DIO Chow, and DIO HE diet dams were comparable and were 2.5-fold greater than those in DR Chow and DR HE diet dams. Leptin levels showed a similar pattern, although levels in DR Ensure, DIO Chow, and DIO HE diet dams were six- to eightfold higher than those in DR Chow and HE diet rats. Glucose levels did not differ significantly among the groups at this time.

Weight gain and metabolic parameters in offspring. By the 3rd postweaning wk, DIO progeny became heavier than DR progeny (Fig. 1). This weight difference between DIO and DR rats persisted throughout the remainder of the 16 wk on Chow, regardless of the diet of their respective dams [F(48,1,092) = 24.31; P = 0.0001]. By the 13th wk on Chow, the two groups of DIO rats were 30% heavier and had gained 25% more weight than any of the three groups of DR rats (Table 2). Over the last 3 wk on Chow, DIO rats consumed 32% more energy than DR rats [Fig. 2, Table 2; F(8,144) = 2.94; P = 0.005]. At this point, body weights and weight gains did not differ significantly among the Chow-fed offspring of the three groups of DR dams. In the subgroup of rats killed at this time, retroperitoneal adipose pad weights were also comparable among these groups, although the DR Ensure offspring tended to have heavier pads than the others. Although the diet of dams had no effect on body weight in their progeny, the offspring of DIO HE diet dams had 42% heavier retroperitoneal adipose pads than those of DIO Chow-fed dams and 180-280% heavier than all DR rats. Despite comparable insulin levels, glucose levels were 5% higher in offspring of DIO HE diet than DIO Chow dams.

View larger version (30K): [in this window] [in a new window]   Fig. 1.   Body weight gains from 4 wk of age in progeny of dams inbred for diet-induced obesity (DIO) or diet-resistant (DR) traits and fed Chow of high-energy (HE) diet for 4 wk before impregnation. Additional DR dams were fed HE diet + Ensure to make them obese and hyperinsulinemic. Groups of 24 DR and 12 DIO progeny per diet group of dam were fed Chow from 4 to 16 wk of age, and then half of each was killed for metabolic assessment. Remaining rats were fed HE diet for an additional 4 wk. There were significant intergroup × time differences among groups for 12 wk on Chow [F(48,1,092) = 24.32; P = 0.0001] and for the 4 wk on HE diet [F(12,126) = 19.77; P = 0.0001].

View larger version (32K): [in this window] [in a new window]   Fig. 2.   Energy intake for progeny of DIO dams fed Chow or HE diet and DR dams fed Chow, HE diet, or HE diet + Ensure. Intake of Chow was assessed from wk 13 to 16 in groups of 12 DIO and 24 DR rats per group and then on HE diet for 4 wk in groups of 6 DIO and 12 DR per group. There were significant intergroup × time differences among groups for 3 wk on Chow [F(8,144) = 2.94; P = 0.005] and 4 wk on HE diet [F(12,93) = 3.60; P = 0.0001].

	DISCUSSION

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Maternal obesity and disorders of glucose metabolism during pregnancy and lactation appear to predispose offspring to become obese and/or diabetic later in life (2, 24-26, 28). In humans, it has been difficult to separate genetic predisposition from maternal environment as a cause of this phenomenon (24). Here we show in rats that both factors must be present to promote the likelihood of offspring developing enhanced obesity with insulin resistance. Unlike previous studies (7, 15), the current one shows that exposure to these factors solely during gestation is sufficient to affect the offspring, since all lactating dams had resumed their prepartum weights and plasma leptin and insulin levels during the lactation period, but genotype was also a critical, independent factor in determining outcome. Even though DR Ensure dams were made obese and hyperinsulinemic to almost the level of the DIO rats during gestation, only the offspring of the DIO HE diet dams had more carcass fat (as indexed by retroperitoneal fat pad weights; Ref. 29) and higher plasma glucose levels than their respective Chow-fed controls. Four weeks of HE diet feeding produced even greater weight gain and carcass fat accretion in association with elevated plasma leptin levels in offspring of DIO HE diet-fed dams. The only effect of maternal obesity in DR progeny was the exclusive (among DR offspring) increase in plasma insulin levels seen in the DR Ensure offspring after 1 mo on HE diet.

Clearly, selectively bred DIO rats have a genetic predisposition toward greater fat storage and abnormal glucose metabolism, which appears to be secondary to increased metabolic efficiency (19). This phenomenon was seen here in both DIO dams and their offspring compared with DR rats, regardless of maternal diet. Chow-fed, selected adult DIO rats also can have nonfasting hyperglycemia and hyperinsulinemia compared with DR rats (19). Here Chow-fed DIO offspring all weighed more and had heavier fat depots and higher plasma leptin levels than DR offspring. All of these features suggest that the selected DIO genotype predisposes DIO rats to become more obese than DR rats, even when fed Chow from weaning (19). In addition, there was a selective effect of maternal obesity on the Chow-fed offspring of DIO HE diet-fed dams. They developed heavier fat pad weights and had higher nonfasting plasma glucose levels than all other comparable groups.

After an additional 4 wk on HE diet, all DIO rats remained hyperinsulinemic compared with DR rats. Here again the interaction of genotype with maternal obesity played an important role in the outcome of the progeny. Only in DIO rats did maternal obesity cause an increase in the obesity of the offspring. Thus progeny of DIO HE diet-fed dams gained more carcass weight and fat content and had higher leptin levels than those from Chow-fed DIO dams. Interestingly, maternal obesity had relatively little influence on glucose metabolism in the DIO offspring. On the other hand, the only effect of maternal obesity in DR progeny was a selective increase in nonfasting insulin levels after 4 wk on HE diet seen only in the offspring of DR dams fed HE diet. In addition, these DR offspring of Ensure dams showed a tendency toward heavier fat depots and higher insulin and leptin levels on Chow or after 4 wk on HE diet. Dietary fat and energy content had a pervasive effect on all of the rats, regardless of genotype. Exposure to the HE diet produced increases in retroperitoneal fat pad weights associated with higher nonfasting leptin and glucose levels in all groups. Thus genetic background predisposes rats to develop DIO and abnormal glucose metabolism, whereas maternal diet further enhances the predisposition to obesity selectively in DIO rats.

An important feature of these studies was the documentation of the time of onset of the divergence of body weights between selected DIO and DR rats. Even when fed Chow from weaning, the selected DIO rats become more obese than weight-matched DR rats at 2-3 mo of age (19). This appears to be due to increased metabolic efficiency, although increased energy consumption also contributes to the excess deposition of carcass fat. Here weights of the DIO offspring diverged from those of the DR rats at 6 wk of age. Because no estimate of energy intake or carcass fat was made at this time, it is unknown whether differences in weight gain reflected excess fat deposition or energy intake, although both are likely. Interestingly, when outbred Sprague-Dawley rats are fed the HE diet from 4 wk of age, no bimodal distribution of weight gain occurs (23) as it does in outbred rats started on HE diet at 2-3 mo of age (22). All rats become equally as obese when started on this diet just after weaning (23). This discrepancy may have to do with the onset of sexual maturity between 4 and 6 wk, since, at least in male rats, the obesity-promoting effects on progeny of insulin injection into their dams during the third trimester can be prevented by castration (11).

The severe undernutrition suffered by human mothers and their offspring born during the post-World War II famine in Europe (3, 4) and the abnormal glucose metabolism in diabetic mothers (25, 26) both appear to predispose progeny to develop obesity and/or diabetes in later life. However, such human studies cannot separate out a contribution of genetic factors (24) and/or the selective survival of individuals who were most metabolically efficient. In rats, in which conditions can be better controlled, several issues still remain unresolved. Of the manipulations of maternal environment known to affect the onset of obesity and/or diabetes in the offspring, maternal undernutrition during either the first two trimesters (1, 8, 10, 14; but see 5) or injections of insulin in the third trimester (9, 11, 13) produce similar results in rats. Third-trimester insulin injections predispose male, but not female, offspring to become obese (9, 11, 13). Such injections should cause hyperphagia in the third trimester as occurs when dams are undernourished during the first two trimesters (1, 5). This hyperphagia might lead to maternal hyperinsulinemia, although this has not been documented. Regardless, the obesity in offspring of dams subjected to either undernutrition or insulin injection is associated with increased hypothalamic norepinephrine release (11) and apparent noradrenergic hyperinnervation of the hypothalamic paraventricular nucleus (11). This suggests that insulin could influence brain development so as to promote obesity in the progeny. This is supported by the finding that intrahypothalamic injections of insulin in the first 8 postnatal days produce impaired glucose tolerance in later life (27). Insulin thus appears to be a major candidate as a causative agent for such change in the offspring. Compared with insulin injections, neither corticosterone nor thyroxine given in the third trimester produce similar changes in the offspring (9). Because insulin is a neuronal growth factor (6), it is conceivable that exposure to high insulin levels during the development of the brain might alter the pattern of neural innervation and promote the later development of obesity and abnormal glucose metabolism.

In conclusion, obesity accompanied by hyperinsulinemia during gestation is associated with increased deposition of carcass adiposity in the progeny of selected rats that are already genetically predisposed to DIO. This effect appears to require exposure only during gestation. It is unclear whether other metabolic features, besides hyperinsulinemia, associated with maternal obesity might play a role here. However, given its known role as a trophic factor for neuronal growth and survival (6), exposure of the developing fetal brain to excess insulin levels may be an important determinant of abnormal brain development leading to obesity and glucose intolerance in later life. Insulin might also underlie changes in brain function associated with the sustained obesity that develops in adult outbred DIO rats after several months' exposure to HE diet (17).