Objective
We determined the potential programming effects of maternal obesity and high-fat (HF) diet during pregnancy and/or lactation on offspring metabolic syndrome.
Study Design
A rat model of maternal obesity was created using an HF diet prior to and throughout pregnancy and lactation. At birth, pups were cross-fostered, thereby generating 4 paradigms of maternal diets during pregnancy/lactation: (1) control (Con) diet during pregnancy and lactation (Con/Con), (2) HF during pregnancy and lactation (HF/HF), (3) HF during pregnancy alone (HF/Con), and (4) HF during lactation alone (Con/HF).
Results
Maternal phenotype during pregnancy and the end of lactation evidenced markedly elevated body fat and plasma corticosterone levels in HF dams. In the offspring, the maternal HF diet during pregnancy alone programmed increased offspring adiposity, although with normal body weight, whereas the maternal HF diet during lactation increased both body weight and adiposity. Metabolic disturbances, particularly that of hyperglycemia, were apparent in all groups exposed to the maternal HF diet (during pregnancy and/or lactation), although differences were apparent in the manifestation of insulin resistant vs insulin-deficient phenotypes. Elevated systolic blood pressure was manifest in all groups, implying that exposure to an obese/HF environment is disadvantageous for offspring health, regardless of pregnancy or lactation periods. Nonetheless, the underlying mechanism may differ because offspring that experienced in utero HF exposure had increased corticosterone levels.
Conclusion
Maternal obesity/HF diet has a marked impact on offspring body composition and the risk of metabolic syndrome was dependent on the period of exposure during pregnancy and/or lactation.
An epidemic of metabolic syndrome is well recognized within the United States. Currently 65% of adult Americans are overweight and 30% obese, although a marked increase in obesity is apparent from childhood through adolescence. Notably, prepregnancy obesity prevalence continues to increase as well. Among women presenting for prenatal care, the incidence of obesity has doubled since 1980. Not only do women begin pregnancy at a higher body mass index, but women also gain excess gestational weight. Thus, clinicians caring for pregnant women are increasingly caring for women who are overweight or obese.
It is well established in human studies and animal models that maternal nutritional factors during pregnancy may have marked effects on fetal growth and ultimately influence the offspring’s predisposition to obesity. Studies that have examined the developmental origins of adult obesity have demonstrated that maternal obesity, weight gain during pregnancy, and/or the presence of gestational diabetes are each associated with an increased risk of the offspring becoming obese during childhood and/or as adults.
Recent human studies have demonstrated that among these factors, maternal prepregnancy weight may be the most predictive of offspring obesity. Animal studies have begun to examine the mechanisms of fetal programming, with evidence that maternal obesity and a Western high-fat (HF) diet program fetal adipose tissue to promote increased adipogenesis and hypothalamic neural pathways to promote appetite as compared with satiety.
Breast-feeding has been strongly encouraged for postpartum women in the United States, with evidence suggesting significant benefits in newborn immune function, nutrition, maternal weight loss, and maternal-newborn bonding. However, it is recognized that maternal breast milk reflects in part, the maternal diet, and maternal obesity and the HF Western diet may contribute to higher fat content in breast milk. Thus, maternal obesity may affect breast milk–induced newborn programming, independent of the maternal obesity effects on fetal programming.
In view of the potential programming effects of maternal obesity during both pregnancy and lactation, we sought to examine the effects of each of these periods on programmed metabolic syndrome in rats. We hypothesized that offspring exposed to maternal obesity during both pregnancy and lactation would be more obese and exhibit a greater degree of metabolic abnormalities.
Materials and Methods
Maternal diet and studies
A rat model of maternal obesity was created using a HF diet prior to and through pregnancy and lactation was utilized. Studies were approved by the Animal Research Committee of Harbor-UCLA Medical Research and Education Institute and were in accordance with the American Association for Accreditation of Laboratory Care and National Institutes of Health guidelines.
Sprague Dawley rats (Charles River Laboratories, Inc, Hollister, CA) were housed in a facility with constant temperature and humidity and on a controlled 12 hour light–12 hour dark cycle. Beginning as weanlings, female rats were fed a HF (60% k/cal fat; Purified Diet 58Y1, New Brunswick, NJ; n = 16) or control (Con; 10% k/cal fat, Purified Diet 58Y2; n = 16) diet. The nutrient composition is given in Table 1 . At 11 weeks of age, the rats were mated and continued on their respective diets during pregnancy and lactation.
| Variable | Purified diet 58Y2, 10% k/cal fat | Purified diet 58Y1, 60% k/cal fat |
|---|---|---|
| Nutrients, % | ||
| Carbohydrates | 67.4 | 25.9 |
| Protein | 17.3 | 23.1 |
| Fat | 4.3 | 34.9 |
| Lard | 1.9 | 31.7 |
| Soybean oil | 2.4 | 3.2 |
| Fat type, % | ||
| Saturated | 25 | 37 |
| Monounsaturated | 35 | 46 |
| Polyunsaturated | 40 | 17 |
Maternal body weights and their food intake were recorded daily. In addition, maternal blood was obtained via a tail bleed at term (gestational age embryonic day [e] 20) and at the end of lactation (21 days postpartum) for glucose, lipid, and hormonal analysis (details are given in the following text). The food was removed 1 hour prior to the blood sampling. Furthermore, at the end of lactation, dams underwent a noninvasive dual-energy x-ray absorptiometry (DEXA) scan (details are given in the following text) for evaluating percentage body fat and lean body mass.
The offspring
At birth, pups were culled to 8 per litter (4 males and 4 females) to normalize rearing and were cross-fostered, thereby generating 4 paradigms of maternal diets during pregnancy/lactation: (1) Con diet during pregnancy and lactation (Con/Con), (2), HF during pregnancy and lactation (HF/HF), (3) HF during pregnancy alone (HF/Con), and (4) HF during lactation alone (Con/HF). At 3 weeks of age, the offspring in each of the 4 groups were housed individually and 2 males and 2 females per litter were weaned to a normal-fat diet (10% k/cal).
Body weights and food intake
Each litter from the 4 groups was weighed weekly, and the weight of an individual pup was calculated from it (ie, litter weight per number of pups). The first weight was recorded at 1 day of age, and subsequent weights were taken at 7, 14, and 21 days of age. Thereafter the body weight and food intake were monitored weekly on an individual basis.
Body composition
At 3 and 24 weeks of age, offspring of both sexes underwent noninvasive DEXA scanning using the DEXA system with a software program for small animals (QDR 4500A; Hologic, Bedford, MA). An in vivo scan of whole-body composition was obtained, including the lean and fat tissue mass, the total mass, and the percentage body fat measurements.
Blood pressure
Blood pressure was determined in conscious 8 week old male and female offspring using a noninvasive tail-cuff sphygmomanometry (ML125 NIPB system; AD Instruments, Colorado Springs, CO) method. Several cuff sizes were used, depending on the weight of the animal. To circumvent the potential problem of restrain-induced stress, the animals were acclimatized for at least 1 week with placement in the restraint.
Glucose tolerance test
At 6 and 24 weeks of age, offspring of both sexes underwent a glucose tolerance test (GTT) as follows: after an overnight fast, D-glucose (1 mg/g body weight) was injected intraperitoneally in conscious rats. Blood was taken via a tail bleed prior to (time 0) and 15, 30, 60, 120, and 180 minutes after glucose injection.
Blood collection
One day after birth (approximately 24 hours after birth), the excess pups (4 pups per litter) from Con (n = 16) and HF (n = 16) groups were decapitated and blood was pooled from pups from the same litter. At ages 3 and 24 weeks, 1 male and 1 female from each litter (n = 8 per group) were fasted overnight, and blood was collected via cardiac puncture in heparinized tubes for plasma analysis.
Plasma analysis
Plasma insulin, leptin, and coritcosterone levels were measured using rat-specific commercial radioimmunoassay kits (insulin and leptin radioimmunoassay kit; LINCO Research Inc, St Charles, MO; coritcosterone radioimmunoassay kit; Diagnostic Systems Laboratories, Inc, Webster, TX). Plasma lipid levels were measured using reagents from Raichem, Inc (San Diego, CA) and run on an automated Cobas-Mira chemistry analyzer (Roche Diagnostic Systems Inc, Sommerville, NJ). Plasma triglyceride (catalog no. 80008) and cholesterol (catalog no. 80015) concentrations were analyzed using Raichem enzymatic reagents (with control serum level 1 [no. 83082] and control serum level 2 [no. 83083]). Blood glucose was determined using a Hemocue B-glucose analyzer (HemoCue Inc, Mission Viejo, CA).
Statistical analysis
For all offspring studies at 3, 6, 8, and 24 weeks of age, 8 males and 8 females from 8 litters were studied per group. Differences between Con and the experimental groups were compared using an unpaired Student t test (1 day old neonate), repeated measures of analysis of variance (body weight and food intake), or analysis of variance with Dunnett’s post hoc tests (body composition and plasma hormones/metabolites). At ages 1 day and 3 weeks, combined data for males and females are shown because no sex differences were evident. However, at the age 24 weeks, sex differences justified analyzing the data according to sex. Values are expressed as means ± SE.
Results
Maternal dams
Pregnancy
Maternal body weight was increased at the initiation of pregnancy as per the experimental model. Both HF and Con dams gained nearly identical amounts of weight during the pregnancy ( Figure 1 , A). However, there were marked differences immediately following delivery because Con dams experienced a significantly greater weight loss from pregnancy day e20 to postnatal day one (100 ± vs 40 g; Figure 1 , B). Among the 4 lactation groups, 3 of the groups demonstrated a similar maternal weight change during lactation (Con/Con, Con/HF, and HF/HF) with a slight increase in maternal weight through postnatal day (p) 10-12 and a slight decrease in maternal weight at the completion of lactation (p20). The HF dams nursing Con pups evidenced a continued decrease in maternal weight throughout the lactation period.
When assessed by the percentage of carbohydrate, fat, and protein intake, HF dams consumed a greater percentage of kilocalories via fat and a lower percentage by carbohydrate during both pregnancy and lactation, with nearly identical levels of protein intake as Con dams ( Figure 2 ). The total food intake was similar among HF and Con dams during pregnancy. However, food intake was significantly greater during the terminal portion of lactation among HF dams nursing HF pups and HF dams nursing Con pups ( Figure 3 ).
In conjunction with maternal obesity and a HF maternal diet, the plasma profile of pregnant dams at gestation day e21 included increased plasma cholesterol and coritcosterone, although HF and Con dams had similar plasma leptin, plasma triglyceride, and blood glucose levels ( Table 2 ).
| Variable | Con | HF |
|---|---|---|
| Plasma triglycerides, mg/dL | 164 ± 13 | 196 ± 16 |
| Plasma cholesterol, mg/dL | 110 ± 5 | 128 ± 3 a |
| Blood glucose, mg/dL | 57 ± 3 | 63 ± 5 |
| Plasma leptin, ng/mL | 3.3 ± 0.3 | 4.3 ± 0.5 |
| Plasma corticosterone, ng/mL | 266 ± 34 | 776 ± 64 a |
Lactation
At the completion of lactation, there were marked differences among the dams, dependent on the offspring being nursed ( Table 3 ). HF/HF dams demonstrated markedly increased percentage body fat and reduced lean body mass as compared with all groups. Notably, the HF/Con dams’ body fat was significantly less than the HF/HF, consistent with the loss of maternal body weight among HF/Con dams during lactation. Plasma leptin levels reflected the percentage body fat with elevated levels in the HF/HF group. Plasma cholesterol levels, which were elevated among HF dams at the completion of pregnancy, demonstrated a reduction in HF/HF dams as well as HF/Con dams, coinciding with lower levels of plasma triglycerides. Blood glucose levels were similar among all groups, although HF/HF and HF/Con dams demonstrated elevated levels of plasma insulin. Notably, Con/HF dams had markedly elevated levels of plasma corticosterone at the completion of lactation.
| Variable | Con dams nursing Con pups | Con dams nursing HF pups | HF dams nursing HF pups | HF dams nursing Con pups |
|---|---|---|---|---|
| Body fat, % | 4.6 ± 0.4 | 5.6 ± 0.6 | 12.1 ± 1.2 a | 7.5 ± 0.8 a |
| Lean body mass, % | 92.9 ± 0.3 | 91.5 ± 0.6 | 85.4 ± 1.2 a | 90.0 ± 1.9 |
| Plasma triglycerides, mg/dL | 72 ± 9 | 60 ± 9 | 43 ± 6 a | 45 ± 3 a |
| Plasma cholesterol, mg/dL | 95 ± 7 | 91 ± 6 | 54 ± 5 a | 65 ± 2 a |
| Blood glucose, mg/dL | 57 ± 7 | 53 ± 6 | 68 ± 7 | 62 ± 5 |
| Plasma leptin, ng/mL | 1.6 ± 0.3 | 1.3 ± 0.2 | 2.1 ± 0.4 a | 1.6 ± 0.4 |
| Plasma insulin, ng/mL | 0.12 ± 0.02 | 0.16 ± 0.02 | 0.23 ± 0.05 a | 0.18 ± 0.02 a |
| Plasma corticosterone, ng/mL | 247 ± 47 | 588 ± 47 a | 414 ± 45 a | 397 ± 45 a |
The offspring
Growth
Both Con and HF newborns at day p1 had similar body weights (7.3 ± 0.1; 7.4 ± 0.2, respectively). During the nursing period, offspring weight gain diverged into a rapid weight gain group among HF/HF and Con/HF offspring, which was evident in both males and females by 3 weeks of age ( Figure 4 ). Females continued the divergent pattern through 30 weeks of age, whereas males exhibited 3 growth patterns as follows: HF/HF offspring demonstrated a continued acceleration of body weight gain through 30 weeks of age, Con/HF males demonstrated intermediate growth, and HF/Con demonstrating weight gain similar to Con/Con.
Food intake
Unlike the females, the food intake of male offspring paralleled weight gain, with HF/HF offspring demonstrating the greatest food intake from the end of lactation through 24 weeks of age. Con/HF offspring demonstrated intermediate food intake, slightly greater than Con offspring. Notably, HF/Con offspring demonstrated accelerated food intake through 18 weeks of age, after which food intake normalized to levels of the Con/Con offspring ( Figure 5 ).
Body composition
Consistent with the weight gain, both HF/HF and Con/HF offspring demonstrated significantly increased percentage body fat and a reduction in lean body mass at 3 weeks of age ( Figure 6 , A). At 24 weeks, all 3 groups exposed to an HF diet during pregnancy and/or lactation demonstrated markedly increased percentage body fat as compared with Con/Con ( Figure 6 , B). However, as described previously, only the HF/HF and Con/HF demonstrated significantly increased body mass. The HF/Con males and females had significantly reduced lean body mass as compared with respective Con/Con offspring, as measured by both grams and percentage body weight, whereas all 3 groups exposed to an HF diet during pregnancy and/or lactation demonstrated a reduced percentage lean body mass at 24 weeks.
Plasma profile
At 1 day of age, HF newborns had lower plasma triglyceride, cholesterol, leptin, and corticosterone levels, although similar plasma insulin levels as Con/Con ( Table 4 ).
| Variable | Con | HF |
|---|---|---|
| Body weight, g | 7.3 ± 0.1 | 7.4 ± 0.2 |
| Plasma triglyceride, mg/dL | 126 ± 9 | 92 ± 12 a |
| Plasma cholesterol, mg/d L | 95.7 ± 2.7 | 82.5 ± 3.3 a |
| Plasma leptin, ng/mL | 4.9 ± 0.9 | 2.1 ± 0.5 a |
| Plasma insulin, ng/mL | 0.52 ± 0.10 | 0.52 ± 0.20 |
| Plasma corticosterone, ng/mL | 80 ± 12 | 52 ± 8 a |
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