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Endocrinology Vol. 139, No. 9 3704-3711
Copyright © 1998 by The Endocrine Society

ARTICLES

Increased Fat Intake during Lactation Modifies Hypothalamic-Pituitary-Adrenal Responsiveness in Developing Rat Pups: A Possible Role for Leptin1

Geneviève Trottier, Kristine G. Koski, Thierry Brun2, Donna J. Toufexis, Denis Richard and Claire-Dominique Walker

Department of Psychiatry, Douglas Hospital Research Center, and School of Dietetics and Human Nutrition (K.G.K.), McGill University, and the Department of Nutrition, University of Montréal (T.B.), Montréal, Canada; and the Department of Physiology, Laval University (D.R.), Québec, Canada H4H 1R3

Address all correspondence and requests for reprints to: Claire-Dominique Walker, Ph.D., Department of Psychiatry, McGill Univer-sity, Douglas Hospital Research Center, 6875 Lasalle Boulevard, Verdun, Québec, Canada H4H 1R3. E-mail: waldom{at}douglas.mcgill.ca


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
High fat feeding reportedly enhances hypothalamus-pituitary-adrenal (HPA) responses to stress in adult rats. The present study tested whether elevated fat intake during suckling could have short and/or long lasting consequences on HPA regulation in the offspring. Mothers were fed either a control (C; 5% fat) or high fat (HF; 20% fat) diet during the last week of gestation and throughout lactation. After weaning (day 21), pups from C and HF mothers were fed a chow diet. Offspring from both C- and HF-fed mothers were tested for ACTH and corticosterone responses to stress on postnatal days 10 and 35. We found that HF feeding produced higher lipid levels in the milk of HF compared with C lactating rat dams and that offspring of these mothers had significantly increased retroperitoneal fat pad weight and relative adipose mass on day 21 as well as elevated plasma leptin levels on days 10 and 21 of age. After weaning, pups from the HF mothers had lower plasma leptin levels than those from C mothers. Maternal dietary fat affected HPA responsiveness in the offspring in an age-related manner. Neonatal pups (day 10) from the HF mothers exhibited a reduction in the ACTH and corticosterone responses to ether stress. However, in 35-day-old offspring from HF-fed dams, stress-induced ACTH secretion was increased compared with that in pups from the C-fed mothers. These results demonstrate that maternal diet and increased fat intake through the milk are important regulators of HPA responsiveness in neonates and prepubertal rats. During neonatal life, the blunted stress responsiveness seen with elevated fat intake and the resulting high leptin levels might protect the pups from excessive HPA activation. After removal of the maternal dietary influence and reduced leptin levels, enhanced ACTH stress responses are observed as in adult rats fed a HF diet. Because of the inverse relationship between plasma levels of leptin and HPA responses in pups, the possibility exists that the effects of the HF diet on stress responsiveness are mediated by changes in leptin exposure during development.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
IT IS NOW understood that complex interactions exist among nutrient ingestion, metabolic parameters, and neuroendocrine systems regulating homeostasis (1). In particular, the relationship between fat intake and hypothalamic-pituitary-adrenal (HPA) function is underscored by several studies showing that elevated fat intake in adult rats results in increased plasma concentrations of FFA (2) as well as elevations in basal levels of corticosterone and stress-induced ACTH and corticosterone secretion (2, 3, 4). In fact, other studies have shown a direct stimulatory effect of fatty acids on ACTH and corticosterone secretion (5, 6). Consequently, it was postulated that elevated consumption of fat in the adult rat functions as a chronic stressor to the HPA axis (2). However, considering that lipids are the main nutrient in maternal milk, neonatal physiology may be better adapted to high levels of dietary fat (7). For instance, it was found that fat ingestion possesses opioid-like antinociceptive and calming properties in the neonate, thereby reducing reactions to stress (8). Interestingly, research has indicated that changes in dietary fat content act to modify milk lipid levels in the lactating rat (9, 10). A high energy diet fed throughout lactation was found to increase milk lipid levels and metabolizable energy content compared with those in rats fed normal laboratory rat chow (9). Although early postnatal effects of increased fat intake on the HPA axis are not currently known, it was demonstrated that regulation of corticosterone-binding globulin (CBG) by fatty acids is considerably different in the 15-day-old rat compared with that in the adult. Treatments to elevate plasma FFA result in decreased corticosterone binding to CBG and liver glucocorticoid receptors in the neonate, whereas they increase CBG binding in the adult rat (11). This modulation of glucocorticoid systems by nutritional factors could be physiologically relevant at a time when many developmental processes are under glucocorticoid control.

In addition to FFA secretion, leptin derived from fat stores represents another possible regulator of HPA function. Leptin was shown to decrease plasma levels of corticosterone in obese ob/ob mice (12), to inhibit CRH release in response to hypoglycemia (13) and food restriction (14), and to potentiate CRH release under basal conditions (15). In fact, an inverse relationship between leptin secretion and HPA function has been recently suggested in humans (16, 17) and neonatal mice (18). Furthermore, it is known that leptin levels increase after ingestion of a high fat diet (19), thus providing a potential additional factor linking high fat intake to changes in adrenocortical function.

We hypothesized that if changes in maternal diet result in changes in milk lipid levels, then dietary manipulation may allow us to study the effects of elevated fat intake on stress responsiveness and development of the HPA axis in neonatal rat pups without introducing invasive procedures such as gastric fistula or artificial rearing. Experiments were designed to test the hypothesis that elevated fat intake during development significantly altered HPA responses to stress in the offspring and that part of this effect could be mediated by changing lipid metabolism (20), fat deposition (21), and circulating leptin concentrations (22, 23).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Pregnant Sprague-Dawley females (Charles River, St. Constant, Canada) were received on days 15–16 of gestation and kept in our animal facility under constant conditions of humidity (70–80%), temperature (22–25 C), and light (12-h light, 12-h dark cycle, with lights on at 0600 h). They were housed individually in clear plastic cages, with food and water available ad libitum. From the time of arrival, females were assigned to one of two groups, receiving either a control (C) or a high fat (HF) diet. The day of parturition was set as day 0, and litters were culled on day 1 to five males and five females per mother. Pups were kept with their mothers until weaning on day 21. Thereafter, weaned rats were housed three per cage and fed a normal Purina chow diet (Ralston-Purina, St. Louis, MO). Body weights of dams and pups (collective litter weight) as well as food intake of mothers were recorded every morning. Individual daily data for body weight and food intake of lactating dams were averaged over 4-day intervals for analysis (lactation days 1–4, 5–8, 9–12, 13–16, and 17–20). All protocols were approved by the animal care committee at McGill University.

Diets
Dams were fed one of two experimental powdered diets (custom-made by Harlan Teklad, Madison, WI) for the last week of gestation and throughout lactation (Table 1Go). The C diet was formulated to contain 5% fat, 15% protein, and 60% carbohydrate (CHO), whereas the HF diet contained 20% fat, 15% protein, and 53.5% CHO. Diets were stored at 4 C and kept no longer than 3 months. Food intake, measured in grams, was converted to energy intake (kilocalories) using the energy content of each diet (1 Cal = 4.186 kJ). Metabolizable energy content was calculated for each separate ingredient from the proportions of pure carbohydrate, fat, and protein using Atwater factors. The global metabolizable energy content of each diet was obtained from the sum of its ingredients. Calculated values were 3.45 Cal/g (14.44 kJ/g) and 4.26 Cal/g (17.83 kJ/g), respectively, for C and HF diets. Purina rat chow, fed after weaning, contained 4.5% fat, 52.1% CHO, and 22.5% protein. Total metabolizable energy for rat chow was 3.34 Cal/g (13.98 kJ/g).


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  		Table 1. Detailed composition of maternal diets

 
Milk composition
Milk samples were collected from dams on days 4, 9, 14, and 19 of lactation. All dams were milked between 0900–1200 h to avoid diurnal variation in milk composition (24). After separation from the pups for 30 min, dams were anesthetized with sodium brietal (Eli Lilly Co., Toronto, Canada) and injected ip with 1.5 IU oxytocin (Sigma Chemical Co., St. Louis, MO) to stimulate milk flow. Milking was initiated 5 min after oxytocin injection, and approximately 1 ml milk was collected into a Pasteur pipette by gentle hand stripping. Pups were kept in their home cage on a warming pad for the duration of the procedure, and separation from the mother never exceeded 120 min. Milk samples were aliquoted and kept at -80 C until assayed. Milk protein levels were measured by modified Lowry assay, total carbohydrates were measured by the orcinol-sulfuric acid method, and total lipids were determined using the sulfuric acid-vanillin reaction as described previously (25). Nonesterified fatty acids (NEFA) concentrations in the milk were determined using a colorimetric assay kit from Wako Chemicals (Richmond, VA).

Tissue collection and carcass analysis
All data for dams (carcass composition, fat pad weight, plasma leptin levels, hepatic enzyme levels) were collected on lactation day 21. The weight of the fresh right retroperitoneal fat pad was recorded in dams and pups (postnatal days 10, 21, and 35) from the two diet groups. The weight of the fat pad was converted to a ratio by correcting each individual value for body weight at the time of death. Animal carcasses from dams and pups (postnatal days 21 and 35) were emptied of intestinal content and frozen at -20 C for carcass composition analysis as described previously (26). Proportions of fat and protein were determined on dried homogenates from whole carcasses using adiabatic bomb calorimetry for energy content and the Kjeldahl procedure for total nitrogen (converted to protein content by dividing by 0.16, based on the assumption that proteins contain an average of 16% nitrogen).

Hepatic enzyme levels
Liver samples were frozen immediately upon collection in isopentane (-50 C) and were kept at -80 C until assayed for enzyme levels. For determination of enzyme levels, liver tissue homogenate proteins (25 µg) were resolved on 5% SDS-polyacrylamide gels and electrotransferred to nitrocellulose membranes. Biotin-bound carboxylase enzymes [pyruvate carboxylase (PC) and acetyl coenzyme A (ACC) carboxylase isozymes] were measured by direct streptavidin blotting (27). To this end, nitrocellulose membranes were incubated for 18 h at 4 C with streptavidin-biotinylated horseradish peroxidase complex (dilution 1:3000). Carboxylase enzymes were revealed using enhanced chemiluminescence reagents (Amersham, Arlington Heights, IL). Total proteins were determined by the Coomassie blue method (Bio-Rad, Richmond, CA). Results are expressed in arbitrary optical density units and are the mean ± SEM of two or three determinations per group.

Stress testing in pre- and postweaning rats
All testing was performed between 1000 and 1400 h. Ten-day-old pups from the two diet groups were separated from their mother for 20 min before being exposed to ether vapors for 3 min (28). Animals were killed 5, 15, 30, or 60 min after the onset of the stressor. Controls were killed immediately upon removal from the home cage. Both male and female pups were used in these experiments, because we consistently failed in several previous studies to observe a sexual dimorphism in the magnitude of the stress responses in preweaning pups. Trunk blood was collected in tubes containing EDTA, and plasma was kept frozen (-20 C) until assayed for ACTH, corticosterone, and leptin concentrations. On postnatal days 33–35, weaned male rats were implanted with a SILASTIC brand cannula (Dow Corning Corp., Midland, MI) inserted in the right jugular vein under metofane anesthesia (Methoxyflurane, Janssen Pharmaceutical, North York, Canada). All rats were individually housed post surgery. Two days later (postnatal days 35–37), rats were subjected to 15 min of forced swim stress in a Plexiglass cylinder containing water at 30 C (29). Blood samples (250 µl) were taken before (0 min) and 5, 15, 60, and 120 min after the onset of stress. Blood volume was replaced with a 0.9% saline solution containing 0.5% heparin (5000 IU/100 ml). Plasma was stored at -20 C until assayed.

Hormone measurements
Plasma ACTH levels were measured by specific RIA as described previously (30). Total plasma corticosterone levels were determined by RIA using a kit from ICN Biomedicals (Costa Mesa, CA). The limit of detection was 15.6 pg/ml for ACTH and 0.2 µg/dl for corticosterone. Inter- and intraassay variabilities for ACTH were 26% and 8%, respectively. For corticosterone, interassay variability was 12%, and intraassay variability was 3%. Plasma leptin levels were measured on basal samples by specific RIA for rat leptin using a kit from Linco Research (St. Charles, MO). The limit of detection for leptin was 0.5 ng/ml.

Statistical analysis
Data distributions were tested for normality, and all variables complied, so no transformations were required. Data from repeated trials were pooled. Results were analyzed by ANOVA using the appropriate design (for repeated measures, when applicable). Nested statistics were considered when the outcome of maternal treatment was measured in the pups and for variables measured in group-housed animals. They were used in certain instances, but could not be performed on repeated measures designs. Significant interactions were tested by F tests for simple main effect, and pairwise comparisons were performed using Tukey’s honestly significant difference test. The level of significance chosen was P < 0.05. Values are expressed as the group mean ± SEM.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Effect of HF feeding on body weight and food intake in nursing rat dams and pups
Data for body weight and food intake of lactating dams were analyzed with repeated measures ANOVA after individual data were averaged over 4-day intervals. In both groups of nursing mothers (C and HF fed), food intake significantly increased over the course of lactation (main effect of time, P < 0.0001). However, dietary treatment did not influence either food or energy intake of dams (data not shown). Feeding a HF diet had no significant effect on body weight of dams or on pup growth during the suckling period (data not shown). Statistical analysis performed on daily pup weight revealed a significant effect of age (P < 0.001), but no effect of the maternal diet.

Changes in milk composition with HF feeding
Total protein and carbohydrate levels in the milk were not affected by the dam’s diet and did not vary significantly across milking days (data not shown). In contrast, there was a significant influence of maternal HF diet on total milk lipid concentrations as well as a significant effect of milking day (Fig. 1Go, top). Milk lipid levels were higher in mothers fed the HF diet throughout lactation than in those given the C diet (P = 0.0051). In both groups, milk lipid levels decreased at the end of the lactation period (lower on days 14 and 19 compared with those on days 4 and 9; P < 0.01). Levels of NEFA in the milk did not vary in response to maternal dietary treatment (Fig. 1Go, bottom). There was a significant effect of the day of lactation in both groups, with milk NEFA levels higher on day 19 than on days 4, 9, and 14 of lactation (P < 0.001). The interaction factor (diet x days of lactation) was not significant.



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  		Figure 1. Total lipid levels (milligrams per ml; top) and NEFAs (milliequivalents per liter; bottom) in the milk of HF and C dams as a function of lactational age. Values are the mean ± SEM. Main effects of diet (**, P = 0.0051) and day of lactation (a vs. b, P < 0.0001) are seen for total lipids. Milk NEFA levels were not affected by maternal diet, and NEFA levels were significantly higher on day 19 than at other milking sessions (a vs. b, P < 0.001).

 
Effect of HF feeding on retroperitoneal fat pad weight, carcass composition, and leptin levels in dams and pups
In dams on day 21 of lactation, we observed significant increases in fat pad to body weight ratio (C, 2.70 ± 0.58 mg/g; HF, 5.17 ± 0.73 mg/g; P = 0.0381) and relative adipose mass (percent fat: C, 5.86 ± 0.54%; HF, 8.91 ± 0.42%; P = 0.0014) as a function of experimental diet. However, the proportion of body weight as protein was not significantly different at the end of the lactation period in dams fed C vs. HF diets (percent protein: C, 20.58 ± 0.26%; HF, 21.6 ± 0.26%; P > 0.05). Maternal plasma leptin levels measured on day 21 of lactation did not change significantly as a function of the diet (C, 2.04 ± 0.24 ng/ml; HF, 2.59 ± 0.28 ng/ml; P > 0.05).

We observed significant effects of the maternal diets on fat pad weight, carcass content, and leptin levels in the offspring. Although retroperitoneal fat pad to body weight ratio (expressed in milligrams per g) were not different in 10-day-old pups from the two dietary treatment groups, at the time of weaning (day 21) male offspring from the HF mothers had greater retroperitoneal fat pad/body weight ratios than their control counterparts (P = 0.0027). These differences had disappeared by day 35, after 2 weeks of normal rat chow feeding (Fig. 2Go).



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  		Figure 2. Retroperitoneal fat pad to body weight ratio in 10-, 21-, and 35-day-old offspring from mothers fed C and HF diets. Values are the mean ± SEM of 6–10 determinations/group. There were significant main effects of age (P < 0.0001) and diet x age (P = 0.0053). **, P < 0.01, HF compared with controls on postnatal day 21.

 
The proportion of body weight as fat and protein in weanling pups was altered by maternal diet (Fig. 3Go). There were significant main effects for maternal diet and age, and a significant age x maternal diet interaction for both parameters (P < 0.01 for all main effects). In 21-day-old pups, relative adipose mass was higher in HF pups than in the C animals (P < 0.0001), whereas the opposite effect was observed on protein stores that were lower in HF than in C pups (P < 0.0001). These diet-induced differences disappeared by postnatal day 35 after 2 weeks of rat chow feeding; no influence of maternal diet could be observed on either percent fat or percent protein at that age. In all pups, the proportion of fat was significantly higher on day 21 than on day 35 (P < 0.0001). As for carcass protein, it was higher on day 35 than on day 21 in offspring from HF dams (P < 0.0001), but did not vary with age in animals from control mothers.



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  		Figure 3. Proportion of the carcass as fat (top) and protein (bottom) in 21-day-old and 35-day-old offspring from HF and C mothers. Values are the mean ± SEM of 6–10 determinations/group. There were significant main effects of maternal diet, age, and diet x age for both relative adipose and protein mass (P < 0.01 for all main effects). ***, P < 0.0001, HF compared with C on postnatal day 21.

 
Plasma leptin concentrations were significantly increased by HF feeding on postnatal days 10 (P < 0.001) and 21 (P < 0.05) compared with those during the C diet (Fig. 4Go). On day 35, plasma leptin levels from both C and HF pups were low compared with those in preweaning pups. In contrast to day 10 and 21 pups, weaned pups from the HF-fed mothers now exhibited lower plasma leptin concentrations than pups from C mothers (C, 1.03 ± 0.06 ng/ml; HF, 0.84 ± 0.09; n = 11–13 animals/group; P = 0.058). There was a significant effect of age (P < 0.001) and treatment (P < 0.001) as well as a significant age x treatment interaction (P < 0.001).



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  		Figure 4. Plasma leptin levels in 10-, 21-, and 35-day-old pups from HF and C mothers. Values are the mean ± SEM of 5–13 determinations/group. The dashed line represents average values for adult rats (lactating females from the same experiment). Absolute values for the day 35 group are given in Results. There were significant main effects of maternal diet (P < 0.0001), age (P < 0.001), and diet x age (P < 0.001). ***, P < 0.001; **, P < 0.01 (HF compared with C at the same age).

 
Effect of HF feeding on hepatic levels of ACC and PC in dams and pups
To verify that liver metabolism of the fatty acids was not altered by prior maternal dietary treatment in weaned pups, we compared hepatic levels of ACC (260-kDa biotin-containing isoform) in pups coming from the HF and C litters. In 21-day-old pups, ACC levels were barely detectable (data not shown). In 35-day-old males, after 2 weeks of rat chow feeding, there were no differences in ACC levels between dietary groups (Fig. 5Go, bottom; C, 0.43 ± 0.12; HF, 0.43 ± 0.13 arbitrary optical density units; n = 3/dietary group). In contrast, direct dietary influence on the levels of ACC were observed in lactating mothers on day 21 of lactation. Absolute values of ACC were lower in HF dams than in dams receiving the C diet (Fig. 5Go, top; C, 0.67 ± 0.28; HF, 0.46 ± 0.12 arbitrary optical density units; n = 3/dietary group). There were no differences in hepatic levels of PC (120-kDa protein) in any of the groups studied.



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  		Figure 5. Representative duplicate of streptavidin-biotinylated horseradish peroxidase blot showing protein levels for ACC and PC from liver tissue homogenates. In the top panel, ACC and PC protein levels of dams (day 21 of lactation), after 4 weeks of feeding the C or HF diet are shown. On the bottom panel, enzyme levels are also shown for 35-day-old offsprings from HF or C mothers fed rat chow for 2 weeks after weaning.

 
Effect of maternal diet on stress responsiveness in 10-day-old neonates
Plasma ACTH and corticosterone responses to ether stress in 10-day-old pups are depicted in Fig. 6Go. Maternal diet had a significant effect on plasma ACTH levels. Overall ANOVA analysis for repeated measures across time showed significant main effects for maternal diet (P = 0.0139) and time poststress (P < 0.0001), and a significant interaction between the two factors (P = 0.0227). Peak ACTH stress responses at the 5 min point were significantly lower in HF compared with C pups (P = 0.0002). For corticosterone secretion, we also observed significant main effects for maternal diet (P = 0.0064) and time (P < 0.0001), and a significant interaction (P = 0.0273). Plasma corticosterone levels 60 min poststress were significantly lower in HF pups compared with controls (P < 0.0001), indicating a more rapid return to baseline values in these animals. Basal levels of ACTH or corticosterone (time zero) were not different between diet groups.



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  		Figure 6. Plasma ACTH (top) and corticosterone (CORT; bottom) responses to 3-min exposition to ether vapor in 10-day-old offspring from dams fed C and HF diets. The gray box along the x-axis represents the duration of the stressor (initiated at time zero). Values are the mean ± SEM of 15–16 determinations/group, pooled from 2 experimental trials. Significant main effects of maternal diet, time, and diet x time for both ACTH and CORT (P < 0.05 for all main effects). ***, P < 0.0001; *, P < 0.05 (HF compared with controls at the same time point).

 
Effect of maternal diet on stress responsiveness in postweaning 35-day-old offspring
Two weeks after weaning to a normal rat chow diet, the influence of maternal diet on stress-induced plasma ACTH secretion was still observed in 35-day-old offspring (Fig. 7Go, top). Main effects for maternal diet (P = 0.0031), time (P < 0.0001), and maternal diet x time interaction (P = 0.001) were significant (by repeated measures ANOVA). Plasma ACTH levels were significantly higher in pups from HF mothers compared with those from C mothers at 5 and 15 min after the onset of stress (P = 0.0225 and P = 0.0032, respectively). There were no differences in basal levels of ACTH in response to maternal diet. There were also no significant differences in either basal or stress-induced corticosterone levels between groups (Fig. 7Go, bottom).



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  		Figure 7. Plasma ACTH (top) and corticosterone (CORT; bottom) response to 15-min forced swim stress in 35-day-old offspring from HF and C dams. The gray box along the x-axis represents the duration of the stressor (initiated at time zero). Values are the mean ± SEM of 9–12 determinations/group, pooled from two experimental trials. Significant main effects of maternal diet (P = 0.0476) and time poststress (P < 0.0001) for ACTH levels. There were no differences in CORT levels between groups (main effect of time; P < 0.0001). ***, P < 0.0001; **, P < 0.01 (HF compared with controls at the same time point).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In these studies, we found that one of the critical consequences of increased dietary fat intake in nursing mothers was the increase in milk lipid content, which resulted in a higher fat intake in pups of the HF-fed mothers. This, in turn, had profound consequences on metabolic and neuroendocrine parameters in the offspring. Elevations in fat intake during suckling, a period during which maternal milk is the sole source of nutrient intake, influenced HPA function in the pups in an age-related manner. Indeed, we showed that 10-day-old pups fed higher levels of fat in maternal milk have elevated circulating leptin levels associated with a decreased amplitude in their ACTH response to ether stress and a faster return to baseline corticosterone values. An interesting parallel can be drawn with earlier studies (8, 31, 32), indicating that ingestion of sugars or fat (corn oil) in 10-day-old rats reduces separation distress and pain threshold. Others (33, 34) hypothesized that lack of nutrient intake during maternal separation was in part, but not solely, responsible for increases in basal corticosterone levels and adrenocortical responses to stress. Based on these findings and our own data, we suggest that nutrients and hormones present in maternal milk (lipids, leptin, opioids, etc.) may signal the adrenocortical system that food is available, and thus, extreme responses to stress are not required. This restriction of unnecessary hormonal and behavioral responses to stressful stimuli undoubtedly promotes energy conservation in very young animals.

The effect of elevated fat intake during the nursing period was carried over after weaning, because even after 2 weeks of normal rat chow feeding, HPA responses to stress were modified in pups from HF compared with C mothers. However, in contrast to results in preweanling pups, 35-day-old male offspring from HF dams had significantly greater ACTH responses to swim stress than control animals. This finding parallels studies in adult rats showing that elevations in plasma concentrations of FFA cause marked increases in circulating levels of both ACTH and corticosterone (2, 5). Thus, the lasting effect of maternal diet on HPA responsiveness in the offspring, which is observed after weaning to rat chow and normalization of carcass composition is opposite to that observed in suckling pups. Such age-dependent effects could be explained by an inverse relationship between leptin and HPA function, as suggested previously for adults (16, 17), or might involve other metabolic effectors, such as neuropeptide Y or galanin (1). In fact, our recent preliminary data support a direct inhibitory effect of chronic leptin injection on ACTH secretion in 10-day-old pups (35). Age-related effects are not unprecedented, as the effects of early environmental factors on responses to stress have been shown to vary considerably with age. For instance, pups exposed prenatally to ethanol show suppressed HPA responses to stress during the preweaning period and increased responsiveness to stressor from weaning until adult life. Interestingly, data from pairfed animals indicate that some of the actions of prenatal alcohol on offspring development and hormonal responsiveness may be mediated by nutritional factors (36).

In contrast to stress-induced secretion, we did not observe changes in basal levels of ACTH or corticosterone as a function of maternal diets. This is consistent with the fact that many postnatal manipulations affect mostly stress-induced hormone secretion (37, 38). Similarly, the absence of an increase in stress-induced corticosterone levels even when peak ACTH secretion is augmented in 35-day-old rats is not an unusual finding (39) and probably reflects the fact that adrenal production of corticosterone saturates at moderate levels of ACTH stimulation, in the range of what we observed in our studies (~200 pg/ml ACTH) (40, 41).

Although we did not determine milk levels of leptin in our experimental groups, increased maternal fat intake elevated plasma leptin concentrations in 10- and 21-day-old pups. Two weeks after weaning, plasma leptin levels were reduced and tended to be lower in pups from HF compared with C mothers. Plasma leptin levels in the adult are thought to be mainly regulated by body fat (42), and this might be true also for postweaning pups, as HF pups had smaller fat pads than C pups. However, during neonatal development, this regulation might be quite different, because we found a concordance among elevated leptin levels, elevated fat content of the carcass, and increased fat pad weight in 21-day-old pups, but not in 10-day-old pups. Because of the extremely low fat pad to body weight ratio in 10-day-old compared with 21-day-old pups, it is likely that in young pups, leptin levels are determined by either dietary factors (probably fat intake) that would stimulate endogenous leptin production and/or by leptin transfer via maternal milk (43). An earlier report has shown significant correlations between milk and plasma leptin levels in humans, and if this remains true for rodents, mothers receiving the HF diet might have slightly higher milk leptin levels than C-fed mothers. This could also explain why levels of leptin are elevated in preobese fa/fa pups without significant changes in adipose mass (44). We hypothesize that plasma levels of leptin in normal rat pups are elevated compared with those in weaned or adult animals because of the high fat content of maternal milk and possible leptin transfer through the milk.

Maternal high fat feeding had significant effects on metabolic parameters in pups. At weaning age (day 21), we observed an increased retroperitoneal fat pad to body weight ratio and carcass fat in HF compared with C offspring. Higher carcass fat in pups was compensated for by lower percent protein in these animals. Interestingly, the increased fat deposition in pups was seen even in the face of elevated circulating leptin levels, which are normally decreasing fat pad depots in adults and neonates after exogenous administration (35). Fat pad/body weight ratio decreased markedly from 21 to 35 days of age, and the proportions of carcass fat and protein were no longer different between groups in 35-day-old pups, most likely reflecting exclusive feeding with rat chow after weaning. In addition to components of the diet, it is known that corticosterone can increase the proportion of carcass fat to lean body mass (45, 46). However, our results in 21-day-old rat pups showed differences in body fat without changes in basal corticosterone secretion, indicating that factors other than corticosterone were promoting fat deposition in pups from HF dams. A maternal dietary influence on hepatic lipogenesis of the pups was absent 2 weeks after weaning on rat chow, demonstrating that elevated fat intake during the neonatal period had no long term consequences on hepatic lipid metabolism and that levels of ACC are primarily regulated by proximal diet composition.

Lipid deposition was also altered in mothers receiving the experimental diets for 4 weeks. Retroperitoneal fat pad/body weight ratio and carcass fat percentage were elevated in HF-fed dams despite the absence of changes in caloric intake or body weight between HF- and C-fed mothers. Furthermore, hepatic levels of ACC, a marker of lipogenesis, were significantly reduced by HF feeding, in agreement with earlier studies showing that levels of ACC respond to changes in the proportion of dietary fat and carbohydrate (47). The source of dietary fat in our diets (half of which is soybean oil, providing important amounts of polyunsaturated fatty acids) may have played a role in decreasing ACC levels in HF dams, as polyunsaturated fatty acids are known to reduce ACC transcription (48). Protein levels of PC, a specific enzyme of gluconeogenesis measured as a control marker, were not affected by dietary manipulations. This suggests a selective accumulation of dietary fat in adipose tissue and a significant reduction of hepatic lipogenesis in mothers in the HF group.

The effects of diet on milk lipid content were not related to actual caloric intake in lactating dams, as they were similar in HF- and C-fed mothers. As opposed to total fat, we obtained no significant differences in nonesterified FFA concentrations in the milk with dietary manipulations, suggesting that milk triglyceride and FFA secretions respond to distinct control mechanisms.

In summary, we demonstrated that increased fat intake during lactation increased milk lipid content and produced opposite effects on the stress responses of the offspring during the neonatal and prepubertal periods. Because of the inverse relationship between plasma leptin levels and the magnitude of the stress response in both C and HF groups, we hypothesize that elevated leptin levels in pups from mothers receiving the high fat diet might contribute to the blunted stress responsiveness in these neonates. After weaning to normal rat chow, declining concentrations of leptin and/or elevated fatty acid secretion could enhance ACTH responses to stress in prepubertal rats from the HF fed mothers. We believe that our data provide evidence to understand how maternal influence is able to maintain low stress responsiveness during a critical period of neonatal development and how such a maternal effect can influence the HPA axis over the long term.


   Acknowledgments
 
We are grateful to Dominique Lavallée, Aghdas Zamani, Susan Anderson, and April Matsuno for much appreciated technical help, and to Dr. W. C. England for providing the ACTH antiserum. We also thank Dr. Barbara Woodside for helpful suggestions and insightful discussions.


   Footnotes
 
1 This work was supported by Grant 0GPO138199 from the National Science and Engineering Research Council of Canada (to C.D.W.) and a National Science and Engineering Research Council of Canada fellowship (to G.T.). This work was performed in partial fulfillment of a M.Sc. thesis (G.T.). Back

2 Recipient of a postdoctoral fellowship from the Swiss National Fund for Research (823A-046639). Back

Received February 6, 1998.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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