timed - pregnant ( second parity ) female sprague - dawley rats ( charles river , kingston , ny ) were received on gestation day 2 .
animals were individually housed in conventional tub cages with ad libitum access to food and water in an environmentally controlled room maintained on a 12-h light / dark cycle with light onset at 0600 .
all animal procedures were approved by the animal care and use committee of the johns hopkins university school of medicine .
pregnant rats were assigned to two diet groups : standard chow diet ( harlan teklad 2018 , 17% kcal from fat ; n = 21 ) or high - fat diet ( research diets , d12492 , 60% kcal from fat , n = 21 ) .
all dams remained on their respective diets from gestation day 2 throughout gestation and lactation .
beginning on gestation day 14 , 10 dams from each diet group ( n = 10 , standard chow  stress group and high - fat  stress group ) were subjected to an 8-day schedule of variable stress ( table 1 ) ( 20 ) . each stressor was applied during the light cycle unless noted otherwise .
we selected a variable stress paradigm for our studies to prevent the animals from habituating to the stress , as has been documented using predictable stressors such as repeated restraint stress ( 29 ) .
stress was restricted to the 3rd week of gestation because the neural circuits that regulate the hpa axis and energy homeostasis , including the hypothalamus , cortex , and hippocampus , are rapidly developing during this period ( 30 ) .
schedule of variable stress during gestation the remaining control dams were exposed to normal animal room husbandry practices in the animal facility ( n = 11 , standard chow  control group and high - fat  control group ) .
tail blood samples were collected from all dams on the morning of gestation days 14 ( prestress ) and 21 ( poststress ) for measurement of basal plasma corticosterone , leptin , and insulin . the day a litter was found
was designated postnatal day 0 . on postnatal day 1 , litters were culled to 10 pups each ( 5 male and 5 female ) .
pups were weaned on postnatal day 21 and housed in groups of two to three by sex and treatment group .
half of each group ( n = 45 pups ) were weaned onto either standard chow or high - fat diet . on postnatal day 22 and postnatal day 70 , rats were food - deprived overnight for 16 h with only water available .
a baseline blood sample was taken via a small tail nick for determination of plasma insulin .
baseline fasted blood glucose was determined at the same time by a handheld glucose meter ( freestyle ; therasense , alameda , ca ) .
an oral gavage of glucose ( 2.0 g / kg body wt , 20% glucose in sterile water solution ) was then administered .
blood samples were collected at 15 , 30 , 45 , 60 , and 120 min after glucose gavage to determine plasma insulin levels .
male pups ( n = 1 per litter ) were killed on postnatal day 1 , 7 , 14 , 21 , and 90 by decapitation .
blood was collected into a heparinized microcentrifuge tube , centrifuged at 4c to collect plasma , and stored at 80c for hormone analysis .
fat pads ( dorsosubcutaneous , inguinal , and retroperitoneal ) were unilaterally dissected and weighed . the entire subscapular brown adipose tissue fat pad was removed and weighed .
plasma hormone concentrations were determined by commercially available radioimmunoassay kits for corticosterone ( for rats and mice ; mp biomedicals , solon , oh ) and for leptin and insulin ( both for rat ; millipore , billerca , ma ) .
inter- and intra - assay variability for each assay , respectively , were as follows : corticosterone , 6.57.1% and 4.410.3% ; leptin , 3.05.7% and 2.04.6% ; and insulin , 8.59.4% and 1.44.6% .
data were analyzed by statistica 7.0 ( systat , tulsa , ok ) by anova , repeated - measures anova , or student 's t tests for independent samples as appropriate .
beginning on gestation day 14 , 10 dams from each diet group ( n = 10 , standard chow  stress group and high - fat  stress group ) were subjected to an 8-day schedule of variable stress ( table 1 ) ( 20 ) . each stressor was applied during the light cycle unless noted otherwise .
we selected a variable stress paradigm for our studies to prevent the animals from habituating to the stress , as has been documented using predictable stressors such as repeated restraint stress ( 29 ) .
stress was restricted to the 3rd week of gestation because the neural circuits that regulate the hpa axis and energy homeostasis , including the hypothalamus , cortex , and hippocampus , are rapidly developing during this period ( 30 ) .
schedule of variable stress during gestation the remaining control dams were exposed to normal animal room husbandry practices in the animal facility ( n = 11 , standard chow  control group and high - fat  control group ) .
tail blood samples were collected from all dams on the morning of gestation days 14 ( prestress ) and 21 ( poststress ) for measurement of basal plasma corticosterone , leptin , and insulin . the day a litter was found
was designated postnatal day 0 . on postnatal day 1 , litters were culled to 10 pups each ( 5 male and 5 female ) .
pups were weaned on postnatal day 21 and housed in groups of two to three by sex and treatment group .
half of each group ( n = 45 pups ) were weaned onto either standard chow or high - fat diet .
on postnatal day 22 and postnatal day 70 , rats were food - deprived overnight for 16 h with only water available .
a baseline blood sample was taken via a small tail nick for determination of plasma insulin .
baseline fasted blood glucose was determined at the same time by a handheld glucose meter ( freestyle ; therasense , alameda , ca ) .
an oral gavage of glucose ( 2.0 g / kg body wt , 20% glucose in sterile water solution ) was then administered .
blood samples were collected at 15 , 30 , 45 , 60 , and 120 min after glucose gavage to determine plasma insulin levels .
male pups ( n = 1 per litter ) were killed on postnatal day 1 , 7 , 14 , 21 , and 90 by decapitation .
blood was collected into a heparinized microcentrifuge tube , centrifuged at 4c to collect plasma , and stored at 80c for hormone analysis .
fat pads ( dorsosubcutaneous , inguinal , and retroperitoneal ) were unilaterally dissected and weighed . the entire subscapular brown adipose tissue fat pad was removed and weighed .
plasma hormone concentrations were determined by commercially available radioimmunoassay kits for corticosterone ( for rats and mice ; mp biomedicals , solon , oh ) and for leptin and insulin ( both for rat ; millipore , billerca , ma ) .
inter- and intra - assay variability for each assay , respectively , were as follows : corticosterone , 6.57.1% and 4.410.3% ; leptin , 3.05.7% and 2.04.6% ; and insulin , 8.59.4% and 1.44.6% .
data were analyzed by statistica 7.0 ( systat , tulsa , ok ) by anova , repeated - measures anova , or student 's t tests for independent samples as appropriate .
there were no significant differences in maternal body weight among the groups before stress exposure ( table 2 ) .
however , compared with beginning body weight on gestation day 2 , dams maintained on high - fat diet gained significantly more weight during the first 2 weeks of gestation compared with dams fed standard chow diet ( p < 0.05 ) .
the greater weight gain in high - fat dams may be attributable to significantly higher caloric intake of dams fed high - fat diet during gestation days 214 ( p < 0.05 ) .
there were no significant differences in plasma corticosterone among the groups before stress . maternal body weight , food intake , and endocrine measures during gestation * p < 0.05 vs. standard chow  control group ; p < 0.05 vs. standard chow  control group and standard chow  stress group .
stress during the last week of gestation ( gestation days 1421 ) resulted in less body weight gain in stress groups ( table 2 ) .
post hoc analysis revealed that dams in the high - fat  stress group gained significantly less body weight compared with standard chow  control group . however , body weight on the final day of gestation ( gestation day 21 ) was not different among the groups .
caloric intake was not affected by stress ( gestation days 1521 ) . to assess the effect of stress and consumption of high - fat diet , we measured plasma corticosterone , leptin , and insulin on gestation day 21 . at this time point ,
plasma corticosterone was significantly higher in the variable stress groups compared with nonstressed controls ( p < 0.05 ) , indicating that exposure to variable stress resulted in hpa axis activation and increased circulating glucocorticoid levels .
there was an overall effect of diet on plasma leptin levels after 20 days of high - fat diet feeding ( p < 0.05 ) .
leptin was significantly elevated in the high - fat  control group compared with standard chow 
fed groups . because leptin is secreted by adipocytes and is highly correlated to adiposity , these data suggest that even though they were not significantly heavier , the high - fat diet dams had greater body adiposity compared with the standard chow  fed dams .
blood glucose ( data not shown ) and plasma insulin were not different among the groups , suggesting that high - fat feeding did not result in symptoms of gestational diabetes in high - fat  fed dams . among the groups , there were no significant differences in litter size ( standard chow 
stress , 15.3  0.9 ; high - fat  control , 14.6  0.9 ; and high - fat  stress , 14.5  0.7 ) or male - to - female ratios ( standard chow 
stress , 0.48  0.03 ; high - fat  control , 0.45  0.04 ; and high - fat  stress , 0.50  0.03 ) .
there was an overall effect of maternal stress resulting in higher birth weight of both males ( p < 0.05 ) and females ( p < 0.05 ) . by postnatal day 21 , there were significant effects of both maternal diet and stress on body weight of male and female pups ( fig .
fat pad ( retroperitoneal and subcutaneous pads ) weight as a percentage of body weight was reliably higher in both male ( p < 0.01 ) and female ( p < 0.001 ) pups from dams that were fed high - fat diet during gestation and lactation ( fig .
thus , these data suggest that the increase in body weight on postnatal day 21 in pups from dams on high - fat diet is attributable , at least in part , to increased adiposity .
subscapular brown adipose tissue was not different among the groups ( data not shown ) .
a and b : body weight and fat as a percentage of body weight ( inset graphs ) of offspring through postnatal day 21 .
a : male offspring in each litter were weighed on postnatal days 1 , 7 , 14 , and 21 .
b : female offspring in each litter were weighed on postnatal days 1 , 7 , 14 , and 21 .
fat as a percentage of body weight was determined on postnatal day 21 ( inset graphs ) .
groups shown are : standard chow  control ( chow - con ; n = 11 litters ) , standard chow  stress ( chow - stress ; n = 10 litters ) , high - fat  control ( hf - con ; n = 11 litters ) , and high - fat  stress ( hf - stress ; n = 10 litters ) . * main effect of stress , p < 0.05 vs. control group ; main effect of high - fat diet , p < 0.05 vs. standard chow ; main effect of high - fat diet and stress , p < 0.05 .
c - c , standard chow  control ; c - s , standard chow  stress group ; h - c , high - fat  control group ; h - s , high - fat  stress group .
plasma leptin was elevated at birth in male pups from dams in the high - fat  stress group ( p < 0.05 ) ( fig .
, maternal high - fat diet resulted in significantly higher plasma leptin levels compared with pups of standard chow  fed dams , and this effect persisted throughout the preweaning period ( p < 0.05 ) .
pups from dams in the high - fat  stress group had significantly greater blood glucose levels on postnatal days 7 and 14 , and by postnatal day 21 , both high - fat  control and high - fat  stress groups had elevated blood glucose compared with the standard chow  control group ( p < 0.05 ) ( fig .
there was no difference in plasma insulin from postnatal day 1 through 14 ; however , by postnatal day 21 , insulin was significantly greater in pups from high - fat diet  fed dams ( high - fat  control group , p < 0.01 ; high - fat  stress group , p < 0.05 ) ( fig . 2c ) .
plasma corticosterone was significantly lower on postnatal day 1 in pups from stressed dams ( p < 0.05 ) , regardless of maternal diet , but there were no differences among the groups for the remainder of the preweaning period ( fig .
groups shown are : standard chow  control ( chow - con ; n = 11 ) , standard chow  stress ( chow - stress ; n = 10 ) , high - fat  control ( hf - con ; n = 11 ) , and high - fat  stress ( hf - stress ; n = 10 ) . * p < 0.05 vs. standard chow 
 control groups ; p < 0.05 vs. standard chow  control and standard chow  stress groups ; p < 0.05 vs. standard chow  control group . at weaning ,
there were no significant differences between males and females within each group in any of the measures associated with the gtt ; therefore , the data have been combined in fig .
, there was an overall effect of high - fat diet in elevating baseline blood glucose ( time 0 ) .
although there was no difference in blood glucose at 15 min , it remained elevated in the high - fat  control and high - fat  stress pups at 30 , 45 , 60 , and 120 min .
the glucose area under the curve ( auc ) was higher in high - fat diet pups compared with standard chow pups , indicating that pups from dams fed high - fat diet cleared the glucose load slower than pups from dams fed standard chow diet ( p < 0.001 ) ( fig .
there was a main effect of maternal high - fat diet on insulin secretion in response to the glucose load . at 30 , 45 , 60 , and 120
min , plasma insulin remained higher in both high - fat  control ( p < 0.05 ) and high - fat  stress ( p < 0.01 ) pups compared with standard chow pups .
insulin auc was higher in both high - fat  control ( p < 0.05 ) and high - fat  stress ( p < 0.01 ) pups compared with pups from standard chow dams ( fig .
, these data suggest that maternal high - fat diet resulted in offspring that cleared glucose more slowly and required greater insulin to do so . a and b : glucose tolerance test ( 2.0
g / kg , oral gavage ) for male and female pups on postnatal day 23 .
groups shown are : standard chow  control ( chow - con ; n = 8) , standard chow  stress ( chow - stress ; n = 8) , high - fat  control ( hf - con ; n = 8) , and high - fat  stress ( hf - stress ; n = 8) .
p < 0.05 standard chow  stress , high - fat  control , and high - fat  stress vs. standard chow  control groups ; p < 0.05 high - fat  control and high - fat  stress vs. standard chow  control and standard chow 
c - c , standard chow  control ; c - s , standard chow  stress ; h - c , high - fat  control ; h - s , high - fat  stress . on postnatal day 21 , half of the male and female offspring were weaned onto standard chow diet and half on to high - fat diet . when pups were weaned onto standard chow diet , there were no longer any significant differences in body weight among the groups in either males or females at postnatal day 70 ( fig .
body composition was not different among males , but there was a significant increase in percent body fat in high - fat  stress females compared with the standard chow  control group ( p < 0.05 ) ( fig .
plasma insulin after an overnight fast was elevated in the high - fat  stress offspring ( p < 0.05 ) , but there were no significant differences among the groups in plasma leptin or fasted blood glucose ( table 3 ) . when adult male offspring ( postnatal day 70 ) were again challenged with a gtt
5a ) . offspring from high - fat  fed dams had higher fasting insulin and showed a prolonged elevation of insulin at 60 and 120 min , although there was no overall effect on insulin auc ( fig .
5c ) . however , among females , the high - fat  stress group cleared glucose slower and had greater glucose auc compared with standard chow  control and standard chow  stress groups ( p < 0.05 ) ( fig .
6a ) . the standard chow  stress group had lower insulin auc compared with the standard chow  control group , suggesting that this group may be more efficient in clearing the glucose load ( fig .
d : body weight and fat as a percentage of body weight for adult male ( a ) and female ( b ) offspring .
( d ) is expressed as the weight of dorsosubcutaneous , inguinal , and retroperitoneal fat pads as a percentage of body weight .
males weaned on standard chow ( chow wean ) are : standard chow  control ( chow - con ; n = 6 ) , standard chow  stress ( chow - stress ; n = 4 ) , high - fat  control ( hf - con ; n = 4 ) , and high - fat  stress ( hf - stress ; n = 4 ) .
males weaned on high - fat diet ( hf wean ) are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) .
females weaned on standard chow are : standard chow  control ( n = 5 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 4 ) , and high - fat  stress ( n = 4 ) .
females weaned on high - fat diet are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) . *
p < 0.05 vs. weaned on standard chow ; p < 0.05 vs. standard chow  control group .
endocrine measures in adult male offspring * p < 0.05 vs. standard chow  control group ; p < 0.05 vs. corresponding weaned on standard chow .
blood glucose ( a and b ) and plasma insulin ( c and d ) were determined for 2 h after oral administration of glucose for male offspring weaned onto standard chow ( a and c ) or high - fat diet ( b and d ) .
males weaned on standard chow are : standard chow  control ( chow - con ; n = 6 ) , standard chow  stress ( chow - stress ; n = 4 ) , high - fat  control ( hf - con ; n = 4 ) , and high - fat  stress ( hf - stress ; n = 4 ) .
males weaned on high - fat are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) . * high - fat diet main effect , p < 0.05 ; stress main effect , p < 0.05 ; high - fat diet and stress interaction , p < 0.05 . a  d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method . * p < 0.05 vs. corresponding standard chow  weaned group ; p < 0.05 vs. standard chow  control and standard chow  stress groups ; p < 0.05 vs. standard chow  control and high - fat  control groups .
c - c , standard chow  control ; c - s , standard chow  stress ; h - c , high - fat  control ; h - s , high - fat  stress . a 
blood glucose ( a and b ) and plasma insulin ( c and d ) were determined for 2 h after oral administration of glucose for female offspring weaned onto standard chow ( a and c ) or high - fat diet ( b and d ) .
females weaned onto standard chow are : standard chow  control ( chow - con ; n = 5 ) , standard chow  stress ( chow - stress : n = 4 ) , high - fat  control ( hf - con ; n = 4 ) , and high - fat  stress ( hf - stress ; n = 4 ) .
females weaned onto high - fat are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) . * high - fat diet main effect , p < 0.05 ; stress main effect , p < 0.05 . a 
d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method . * p < 0.05 vs. corresponding standard chow wean group ; p < 0.05 vs. standard chow  control and standard chow  stress groups ; p < 0.05 vs. standard chow  control and high - fat  control groups .
c - c , standard chow  control ; c - s , standard chow 
stress ; h - c , high - fat  control ; h - s , high - fat  stress .
when male and female pups were weaned onto a high - fat diet on postnatal day 21 , those pups that were from dams in the high - fat diet or stress groups or combination high - fat  stress group gained more weight compared with pups from the standard chow 
the increase in body weight could be attributed to significantly greater subcutaneous and retroperitoneal fat pad weights ( p < 0.05 ) ( fig .
4c and d ) , and this was associated with elevated plasma leptin levels in standard chow  stress and high - fat  control groups ( p < 0.05 ) ( table 3 ) . when male offspring were challenged with a gtt at postnatal day 70 after being weaned onto a high - fat diet , those from dams in the high - fat diet or stress groups or combination high - fat  stress group cleared glucose more slowly relative to male offspring from the same groups weaned onto standard chow diet ( fig .
5b ) ( p < 0.05 ) . overall , male offspring from the stress group required greater insulin to clear the glucose load , regardless of maternal diet , compared with those pups weaned onto a standard chow diet ( p < 0.05 ) ( fig .
5d ) . among female offspring that were weaned onto high - fat diet , the standard chow  control and standard chow  stress groups cleared glucose slower than high - fat  control and high - fat  stress groups ( p < 0.05 ) ( fig .
although the offspring weaned onto high - fat diet had greater insulin responses compared with those weaned onto standard chow , there was no significant difference among the high - fat  weaned groups in insulin auc ( fig .
there were no significant differences in maternal body weight among the groups before stress exposure ( table 2 ) .
however , compared with beginning body weight on gestation day 2 , dams maintained on high - fat diet gained significantly more weight during the first 2 weeks of gestation compared with dams fed standard chow diet ( p < 0.05 ) .
the greater weight gain in high - fat dams may be attributable to significantly higher caloric intake of dams fed high - fat diet during gestation days 214 ( p < 0.05 ) .
there were no significant differences in plasma corticosterone among the groups before stress . maternal body weight , food intake , and endocrine measures during gestation * p < 0.05 vs. standard chow  control group ; p < 0.05 vs. standard chow  control group and standard chow  stress group .
stress during the last week of gestation ( gestation days 1421 ) resulted in less body weight gain in stress groups ( table 2 ) .
post hoc analysis revealed that dams in the high - fat  stress group gained significantly less body weight compared with standard chow  control group . however , body weight on the final day of gestation ( gestation day 21 ) was not different among the groups .
caloric intake was not affected by stress ( gestation days 1521 ) . to assess the effect of stress and consumption of high - fat diet , we measured plasma corticosterone , leptin , and insulin on gestation day 21 . at this time point ,
plasma corticosterone was significantly higher in the variable stress groups compared with nonstressed controls ( p < 0.05 ) , indicating that exposure to variable stress resulted in hpa axis activation and increased circulating glucocorticoid levels .
there was an overall effect of diet on plasma leptin levels after 20 days of high - fat diet feeding ( p < 0.05 ) .
leptin was significantly elevated in the high - fat  control group compared with standard chow 
fed groups . because leptin is secreted by adipocytes and is highly correlated to adiposity , these data suggest that even though they were not significantly heavier , the high - fat diet dams had greater body adiposity compared with the standard chow  fed dams .
blood glucose ( data not shown ) and plasma insulin were not different among the groups , suggesting that high - fat feeding did not result in symptoms of gestational diabetes in high - fat  fed dams .
among the groups , there were no significant differences in litter size ( standard chow 
stress , 15.3  0.9 ; high - fat  control , 14.6  0.9 ; and high - fat  stress , 14.5  0.7 ) or male - to - female ratios ( standard chow 
stress , 0.48  0.03 ; high - fat  control , 0.45  0.04 ; and high - fat  stress , 0.50  0.03 ) .
there was an overall effect of maternal stress resulting in higher birth weight of both males ( p < 0.05 ) and females ( p < 0.05 ) . by postnatal day 21
, there were significant effects of both maternal diet and stress on body weight of male and female pups ( fig .
fat pad ( retroperitoneal and subcutaneous pads ) weight as a percentage of body weight was reliably higher in both male ( p < 0.01 ) and female ( p < 0.001 ) pups from dams that were fed high - fat diet during gestation and lactation ( fig . 1a and b , inset ) .
thus , these data suggest that the increase in body weight on postnatal day 21 in pups from dams on high - fat diet is attributable , at least in part , to increased adiposity .
subscapular brown adipose tissue was not different among the groups ( data not shown ) .
a and b : body weight and fat as a percentage of body weight ( inset graphs ) of offspring through postnatal day 21 . a : male offspring in each litter were weighed on postnatal days 1 , 7 , 14 , and 21 .
b : female offspring in each litter were weighed on postnatal days 1 , 7 , 14 , and 21 .
fat as a percentage of body weight was determined on postnatal day 21 ( inset graphs ) .
groups shown are : standard chow  control ( chow - con ; n = 11 litters ) , standard chow  stress ( chow - stress ; n = 10 litters ) , high - fat  control ( hf - con ; n = 11 litters ) , and high - fat  stress ( hf - stress ; n = 10 litters ) . * main effect of stress , p < 0.05 vs. control group ; main effect of high - fat diet , p < 0.05 vs. standard chow ; main effect of high - fat diet and stress , p < 0.05 .
c - c , standard chow  control ; c - s , standard chow  stress group ; h - c , high - fat  control group ; h - s , high - fat  stress group .
plasma leptin was elevated at birth in male pups from dams in the high - fat  stress group ( p < 0.05 ) ( fig .
, maternal high - fat diet resulted in significantly higher plasma leptin levels compared with pups of standard chow  fed dams , and this effect persisted throughout the preweaning period ( p < 0.05 ) .
pups from dams in the high - fat  stress group had significantly greater blood glucose levels on postnatal days 7 and 14 , and by postnatal day 21 , both high - fat  control and high - fat  stress groups had elevated blood glucose compared with the standard chow  control group ( p < 0.05 ) ( fig .
there was no difference in plasma insulin from postnatal day 1 through 14 ; however , by postnatal day 21 , insulin was significantly greater in pups from high - fat diet  fed dams ( high - fat  control group , p < 0.01 ; high - fat  stress group , p < 0.05 ) ( fig . 2c ) .
plasma corticosterone was significantly lower on postnatal day 1 in pups from stressed dams ( p < 0.05 ) , regardless of maternal diet , but there were no differences among the groups for the remainder of the preweaning period ( fig .
d : endocrine parameters in male offspring postnatal days ( pnd ) 121 . a : plasma leptin .
groups shown are : standard chow  control ( chow - con ; n = 11 ) , standard chow  stress ( chow - stress ; n = 10 ) , high - fat  control ( hf - con ; n = 11 ) , and high - fat  stress ( hf - stress ; n = 10 ) . * p < 0.05 vs. standard chow  control , standard chow  stress , and high - fat  control groups ; p < 0.05 vs. standard chow  control and standard chow  stress groups ; p < 0.05 vs. standard chow  control group . at weaning ,
there were no significant differences between males and females within each group in any of the measures associated with the gtt ; therefore , the data have been combined in fig .
, there was an overall effect of high - fat diet in elevating baseline blood glucose ( time 0 ) .
although there was no difference in blood glucose at 15 min , it remained elevated in the high - fat  control and high - fat  stress pups at 30 , 45 , 60 , and 120 min .
the glucose area under the curve ( auc ) was higher in high - fat diet pups compared with standard chow pups , indicating that pups from dams fed high - fat diet cleared the glucose load slower than pups from dams fed standard chow diet ( p < 0.001 ) ( fig .
there was a main effect of maternal high - fat diet on insulin secretion in response to the glucose load .
at 30 , 45 , 60 , and 120 min , plasma insulin remained higher in both high - fat  control ( p < 0.05 ) and high - fat  stress ( p < 0.01 ) pups compared with standard chow pups .
insulin auc was higher in both high - fat  control ( p < 0.05 ) and high - fat  stress ( p < 0.01 ) pups compared with pups from standard chow dams ( fig .
3b ) . together , these data suggest that maternal high - fat diet resulted in offspring that cleared glucose more slowly and required greater insulin to do so
. a and b : glucose tolerance test ( 2.0 g / kg , oral gavage ) for male and female pups on postnatal day 23 . a : blood glucose after oral administration of glucose .
b : plasma insulin after oral administration of glucose . the integrated auc was determined for glucose and insulin using the trapezoidal method .
groups shown are : standard chow  control ( chow - con ; n = 8) , standard chow  stress ( chow - stress ; n = 8) , high - fat  control ( hf - con ; n = 8) , and high - fat  stress ( hf - stress ; n = 8) . * p < 0.05 standard chow  stress , high - fat  control , and high - fat  stress vs. standard chow  control groups ; p < 0.05 high - fat  control and high - fat  stress vs. standard chow  control and standard chow  stress groups .
c - c , standard chow  control ; c - s , standard chow  stress ; h - c , high - fat  control ; h - s , high - fat  stress .
on postnatal day 21 , half of the male and female offspring were weaned onto standard chow diet and half on to high - fat diet . when pups were weaned onto standard chow diet
, there were no longer any significant differences in body weight among the groups in either males or females at postnatal day 70 ( fig .
body composition was not different among males , but there was a significant increase in percent body fat in high - fat  stress females compared with the standard chow  control group ( p < 0.05 ) ( fig .
plasma insulin after an overnight fast was elevated in the high - fat  stress offspring ( p < 0.05 ) , but there were no significant differences among the groups in plasma leptin or fasted blood glucose ( table 3 ) . when adult male offspring ( postnatal day 70 ) were again challenged with a gtt
5a ) . offspring from high - fat  fed dams had higher fasting insulin and showed a prolonged elevation of insulin at 60 and 120 min , although there was no overall effect on insulin auc ( fig .
5c ) . however , among females , the high - fat  stress group cleared glucose slower and had greater glucose auc compared with standard chow  control and standard chow  stress groups ( p < 0.05 ) ( fig
. 6a ) . the standard chow  stress group had lower insulin auc compared with the standard chow  control group , suggesting that this group may be more efficient in clearing the glucose load ( fig .
d : body weight and fat as a percentage of body weight for adult male ( a ) and female ( b ) offspring .
( d ) is expressed as the weight of dorsosubcutaneous , inguinal , and retroperitoneal fat pads as a percentage of body weight .
males weaned on standard chow ( chow wean ) are : standard chow  control ( chow - con ; n = 6 ) , standard chow  stress ( chow - stress ; n = 4 ) , high - fat  control ( hf - con ; n = 4 ) , and high - fat  stress ( hf - stress ; n = 4 ) .
males weaned on high - fat diet ( hf wean ) are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) . females weaned on standard chow are : standard chow  control ( n = 5 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 4 ) , and high - fat  stress ( n = 4 ) . females weaned on high - fat diet are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) . * p < 0.05 vs. weaned on standard chow ; p < 0.05 vs. standard chow  control group . endocrine measures in adult male offspring * p < 0.05 vs. standard chow  control group ; p < 0.05 vs. corresponding weaned on standard chow .
blood glucose ( a and b ) and plasma insulin ( c and d ) were determined for 2 h after oral administration of glucose for male offspring weaned onto standard chow ( a and c ) or high - fat diet ( b and d ) .
males weaned on standard chow are : standard chow  control ( chow - con ; n = 6 ) , standard chow  stress ( chow - stress ; n = 4 ) , high - fat  control ( hf - con ; n = 4 ) , and high - fat  stress ( hf - stress ; n = 4 ) .
males weaned on high - fat are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) . * high - fat diet main effect , p < 0.05 ; stress main effect , p < 0.05 ; high - fat diet and stress interaction , p < 0.05 . a  d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method . * p < 0.05 vs. corresponding standard chow  weaned group ; p < 0.05 vs. standard chow  control and standard chow  stress groups ; p < 0.05 vs. standard chow  control and high - fat  control groups .
c - c , standard chow  control ; c - s , standard chow 
stress ; h - c , high - fat  control ; h - s , high - fat  stress . a 
blood glucose ( a and b ) and plasma insulin ( c and d ) were determined for 2 h after oral administration of glucose for female offspring weaned onto standard chow ( a and c ) or high - fat diet ( b and d ) .
females weaned onto standard chow are : standard chow  control ( chow - con ; n = 5 ) , standard chow  stress ( chow - stress : n = 4 ) , high - fat  control ( hf - con ; n = 4 ) , and high - fat  stress ( hf - stress ; n = 4 ) .
females weaned onto high - fat are : standard chow  control ( n = 4 ) , standard chow  stress ( n = 4 ) , high - fat  control ( n = 5 ) , and high - fat  stress ( n = 4 ) . * high - fat diet main effect , p < 0.05 ; stress main effect , p < 0.05 .
d insets : the integrated auc was determined for glucose and insulin using the trapezoidal method .
< 0.05 vs. corresponding standard chow wean group ; p < 0.05 vs. standard chow  control and standard chow  stress groups ; p < 0.05 vs. standard chow  control and high - fat  control groups .
c - c , standard chow  control ; c - s , standard chow  stress ; h - c , high - fat  control ; h - s , high - fat  stress .
when male and female pups were weaned onto a high - fat diet on postnatal day 21 , those pups that were from dams in the high - fat diet or stress groups or combination high - fat  stress group gained more weight compared with pups from the standard chow 
the increase in body weight could be attributed to significantly greater subcutaneous and retroperitoneal fat pad weights ( p < 0.05 ) ( fig .
4c and d ) , and this was associated with elevated plasma leptin levels in standard chow  stress and high - fat  control groups ( p < 0.05 ) ( table 3 ) . when male offspring were challenged with a gtt at postnatal day 70 after being weaned onto a high - fat diet , those from dams in the high - fat diet or stress groups or combination high - fat  stress group cleared glucose more slowly relative to male offspring from the same groups weaned onto standard chow diet ( fig .
5b ) ( p < 0.05 ) . overall , male offspring from the stress group required greater insulin to clear the glucose load , regardless of maternal diet , compared with those pups weaned onto a standard chow diet ( p < 0.05 ) ( fig .
5d ) . among female offspring that were weaned onto high - fat diet , the standard chow  control and standard chow 
stress groups cleared glucose slower than high - fat  control and high - fat  stress groups ( p < 0.05 ) ( fig .
although the offspring weaned onto high - fat diet had greater insulin responses compared with those weaned onto standard chow , there was no significant difference among the high - fat  weaned groups in insulin auc ( fig .
the intrauterine environment is critical to fetal development , and perturbations of this environment can have significant short- and long - term consequences on the offspring .
a growing body of epidemiological data demonstrates that obesity and other metabolic disease may have developmental origins , and determination of mechanisms contributing to those conditions may lead to treatment strategies and early interventions to prevent these disorders .
we hypothesized that exposure to prenatal stress or maternal high - fat diet in utero would predispose offspring to develop obesity and that weaning onto a high - fat diet would exacerbate this effect .
our data suggest that exposure to maternal prenatal stress or high - fat diet feeding results in offspring that gain more weight and have greater adiposity than controls .
consistent with their body composition , offspring of stressed or high - fat diet  fed dams are hyperleptinemic and hyperinsulinemic at weaning .
males and females are similarly affected by maternal prenatal stress and high - fat diet , resulting in impaired glucose tolerance at weaning .
although weaning on standard chow diet appears to normalize early obesity , the combination of maternal prenatal stress and high - fat diet continued to have some effects on glucose tolerance in both male and female offspring . weaning onto high - fat diet resulted in obesity and impaired glucose tolerance in offspring exposed to prenatal stress , high - fat diet , or both .
together , the data suggest that maternal prenatal stress or high - fat diet alters susceptibility of offspring to diet - induced obesity and its metabolic consequences .
although high - fat diet  fed dams were not heavier , their plasma leptin levels were elevated on gestation day 21 , suggesting that they had greater adiposity .
the offspring that were born to dams on high - fat diet were heavier at birth , remained heavier throughout the suckling period , and had impaired glucose tolerance at weaning , suggesting that they were developing insulin resistance .
prior studies that have attempted to identify the effects of prolonged maternal high - fat diet consumption have been difficult to interpret because of concurrence of high - fat diet exposure and maternal obesity ( 31 ) .
maternal obesity is usually associated with other comorbid conditions , such as gestational diabetes , that also have a significant influence on the phenotype of the offspring independent of dietary fat consumption during gestation . indeed ,
offspring of rodents with gestational diabetes show increased adiposity , impaired pancreatic function , impaired glucose tolerance , and altered hypothalamic development ( 3234 ) . for these reasons ,
the dams in our study were provided with high - fat diet only during gestation and lactation . although dams in the current study were hyperleptinemic , indicating that they had greater adiposity after 3 weeks on high - fat diet
, there were no differences in plasma concentrations of insulin or glucose , suggesting that they had not developed signs of gestational diabetes .
it is unclear from this experiment whether maternal high - fat diet consumption during gestation or lactation had a greater influence because high - fat dams remained on high - fat diet throughout these periods .
future studies with cross - fostered control groups will be required to make this distinction .
baseline corticosterone was elevated in dams in the stress group , suggesting that exposure to variable stress during the 3rd week of gestation was effective . maternal prenatal stress in our study resulted in higher birth weights in both male and female offspring , and this occurred in the absence of significant differences in litter size or male - to - female ratios . by postnatal day 21
, both male and female offspring of the stress group had greater body weight and showed signs of impaired glucose tolerance , suggesting that prenatal stress has early consequences for the offspring .
in contrast to the effect of high - fat diet on endocrine measures , the only hormone that was significantly different in the stress offspring was plasma corticosterone , which was lower in the stress group compared with control pups at birth .
this may be a compensatory response resulting from the stressed dams ' elevated plasma corticosterone at parturition .
it is not known whether negative feedback control of corticosterone secretion is operational at this early age .
prior studies examining the effects of prenatal stress on birth weights have produced mixed results .
some report decreased birth weights resulting from prenatal stress ( 26,35,36 ) , and others show increased birth weights ( 27 ) or no difference compared with controls ( 25 ) .
one possible explanation to account for some of the difference is that those studies reporting lower birth weights used the immobilization stress paradigm , which also results in decreased food intake and body weight gain in the dams . because stress - induced maternal undernutrition and decreased body weight gain during pregnancy has consequences similar to those of intrauterine growth restriction , parceling out the direct effects of stress is difficult in these models .
the variable stress schedule that we used did not significantly affect maternal food intake of dams during the stress period .
epidemiological reports in humans indicate that the birth weight  to  adult fat mass picture has developed into a u - shaped curve , such that being very light or very heavy at birth may predispose offspring to cardiovascular disease , diabetes , obesity , and some types of cancer ( 15,37 )
. therefore , although the findings regarding the effects of prenatal stress on birth weight are variable , the different models may operate through different mechanisms depending on the type , intensity , and timing of stress during gestation ( 38 ) .
maternal obesity and high - fat diet feeding have been associated with hyperphagia and obesity in offspring , particularly when challenged postnatally with a hypercaloric diet ( 39 ) .
we hypothesized that prenatal stress against a background of high - fat diet feeding would result in an additive or synergistic effect on the offspring .
the data at the time points examined suggest that there is no increased effect of the combination of stress and high - fat diet .
an alternative possibility was that maternal high - fat diet would have a  beneficial  effect in attenuating the stress response of the dam and , in turn , lessen its impact on the developing fetus .
( 40 ) have demonstrated that high - fat diet can attenuate the hpa axis response to stress .
we did not note either a detrimental or beneficial effect of maternal high - fat diet on top of the effects of stress . in adulthood ,
offspring exposed to stress or high - fat diet or both gained significantly more body weight and adiposity , but only if they were weaned onto a high - fat diet .
consistent with increased body weight and adiposity on high - fat diet in adulthood , animals from stressed or high - fat diet  fed dams also continued to have impaired glucose tolerance .
in contrast , when offspring were weaned on to standard chow , body weight , body composition , and glucose tolerance differences among the groups were no longer as pronounced .
it is important to note that the data reported here are from young adult offspring and do not preclude the possibility that metabolic disorders may occur more slowly , emerging later in life .
overall , our data suggest that prenatal conditions can alter the susceptibility of offspring to future metabolic challenge , in this case high - fat diet resulting in obesity .
this concept has been proposed as the  two - hit  model and suggests that genetic or environmental factors disrupt early development and produce increased susceptibility to disease , including parkinson 's disease ( 41,42 ) and schizophrenia ( 43,44 ) .
first hit  disruptions that occur during early development set the stage for long - term vulnerability to a 
thus , it appears that prenatal stress or maternal high - fat diet is not the direct cause of increased body weight or disturbances in energy homeostasis , but instead increases future susceptibility .
identification of the pathways and mechanisms that produce long - term vulnerability in response to early environmental disruption will facilitate development of clinical intervention and prevention strategies to reduce the incidence of disease . using these animal models
, we are now able to extend our studies to determine the potential mechanisms through which prenatal stress or high - fat diet may program systems that regulate body weight and control food intake .
there is evidence that hormones such as corticosterone , insulin , and leptin can cross the placental - fetal barrier to potentially affect fetal development ( 4547 ) , and thus it is likely that fetuses have hormone levels that mirror those of their dams .
the significant changes in endocrine parameters in offspring throughout the early postnatal period reported here represent reasonable candidates for  metabolic programming  that occurs in response to high - fat diet consumption or maternal stress ( 48 ) .
both leptin and insulin are trophic factors that act during the pre- and postnatal periods and can significantly affect development of neural systems important in the maintenance of energy homeostasis ( 49 ) . similarly ,
stress and excess glucocorticoids may have an effect prenatally by programming increased susceptibility to stress in adulthood . although placental 11-hydroxysteroid dehydrogenase ( 11-hsd ) serves to buffer the developing fetus from excess glucocorticoids in response to acute stress , its upregulation of 11-hsd is often insufficient during chronic stress ( 50 ) .
future studies are required to explore the role of 11-hsd in the phenotypes we observed .
the intrauterine period is a critical time of development , and evidence shows that disturbances to the fine balance in utero can have significant and persistent consequences for the offspring .
the development of relevant animal models is required to examine the etiology of metabolic disorders and to determine the mechanisms for greater predisposition to those disorders .
 objectiveperturbations to the prenatal environment have been associated with the development of adult chronic disease , findings that gave rise to the  barker hypothesis  or the  developmental origins of adult disease  concept . in this study 
 , we used an animal model to determine the metabolic consequences of maternal prenatal stress and high - fat feeding on the developing offspring.research design and methodspregnant female sprague - dawley rats were maintained on standard chow or 60% high - fat diet throughout gestation and lactation . 
 half of each group were exposed to a novel variable stress paradigm during the 3rd week of gestation , whereas control dams were left undisturbed . 
 body weight , body composition , glucose tolerance , and endocrine parameters were measured in offspring through early adulthood.resultsmale and female pups from dams that experienced prenatal stress and/or were on a high - fat diet weighed more beginning on postnatal day 7 compared with standard chow  
 control pups . 
 access to high - fat diet at weaning increased the body weight effect through early adulthood and was attributable to greater adiposity . 
 pups weaned onto standard chow diet showed no significant difference in glucose clearance or insulin secretion . 
 however , pups weaned onto high - fat diet had impaired glucose tolerance if their dams were on a high - fat diet , experienced prenatal stress , or both.conclusionsour data demonstrate that prenatal stress and/or high - fat diet during the intrauterine or postnatal environment affects offspring in a manner that increases their susceptibility to diet - induced obesity and leads to secondary adverse metabolic consequences . 
RESEARCH DESIGN AND METHODS
Variable stress.
Body weight.
Glucose tolerance test.
Death.
Endocrine assays.
Statistical analysis.
RESULTS
Pregnant dams.
Neonatal offspring.
Adult offspring.
DISCUSSION