of Feeding a High-Fat Diet During Pregnancy on Glucose Metabolism in the Rat Anne-Francoise
and Jean Girard
To alter glucose homeostasis in a period of great glucose demand, pregnant rats were submitted to a high-fat diet and compared to virgin rats. In virgin rats, blood glucose, ketone bodies, plasma insulin, and free fatty acids were not affected by the diet consumed. Glucose turnover measured in the postabsorptive period was slightly decreased in virgin rats fed a high-fat diet compared to rats fed a standard diet. Assuming that the glucose turnover rate is representative for the 24-hour average endogenous glucose production, in rats fed a standard diet the daily carbohydrate intake (9.2 ? 0.7 g/d) exceeded the glucose turnover rate (4 i 0.2 g/d) and could meet the glucose requirement. In rats fed a high-fat diet the carbohydrate intake (2.7 + 0.2 g/d) was lower than the glucose turnover rate (3.9 + 0.2 g/d), which demonstrated the need for an active endogenous glucose production. Blood glucose, ketone bodies, plasma insulin, and free fatty acid concentrations followed the same patterns during pregnancy in rats fed a standard diet compared to rats fed a high-fat diet. The glucose turnover rate in the postabsorptive period was no more decreased by the high-fat diet in pregnant rats compared to virgin rats despite the greater glucose demand. In late pregnancy the glucose turnover rate was increased up to 70%. Assuming that the glucose turnover rate is representative of the 24-hour average endogenous glucose production, the difference of daily carbohydrate intake 13 i 1 g/d for the standard diet and 3.7 * 0.2 g/d for the high-fat diet and of glucose turnover rates 6.1 k 0.9 g/d and 5.8 + 0.3 g/d suggest that (1) an increased endogenous glucose production and not a decreased glucose utilization occurred in rats fed a high-fat diet and (2) the endogenous glucose production must be increased twofold in pregnant rats fed a high-fat diet for maintaining glucose homeostasis. a 1987 by Grune & Stratton,
URING PREGNANCY, in the rat fed a standard diet with high carbohydrate and low fat composition, continuous glucose utilization by the fetus results in maternal hyperphagia and progressive alterations of glucose metabolism. Some of the metabolic adaptations described during this period are a postabsorptive hypoglycemia,‘*’ an increased glucose turnover rate3z4 and a state of insulin resistance in liver and peripheral tissues.s*6Late pregnancy is also associated with high levels of plasma free fatty acids’** providing alternative fuel for maternal tissues. Since the mammalian fetus is not able to oxidize free fatty acids: fetal growth is markedly dependent upon glucose availability from exogenous or endogenous origin. To alter glucose homeostasis in a period of great glucose demand, pregnant rats were submitted to a high-fat diet. Indeed high-fat feeding induces a decrease in glucose turnover rate” and the appearance of a state of insulin resistance characterized by a decreased insulin sensitivity of glucose transport and lipolysis in adipose tissue,““’ and in soleus muscle. I3 Concomitantly, liver gluconeogenesis’ I4 and ketogenesis” are enhanced. In the postabsorptive period, glucose homeostasis in pregnant rats fed a high-fat diet could be maintained by either an increased hepatic glucose production or by a diminished glucose utilization by maternal tissues or both. The present study was undertaken to answer the following
From the Centre de Recherches sur la Nutrition du CNRS Meudon-Bellevue, FRANCE. Supported by a grant from CNRS AIP 693175 and Inserm Cre 837010. Address reprint requests to Dr Armeile Leturque. Centre de Recherches sur la Nutrition. 9 rue Jules Hetzel. 92190 MeudonBellevue, France. D 1987 by Grune & Stratton, Inc. 0026SO495/87/3601~012%03.00/0
questions: 1. Was fetal growth altered when availability of carbohydrate in the diet was reduced? 2. Did the high-fat diet induce changes in glucose turnover rate more prominent in pregnant than in virgin rats? MATERIALS
The rats were an albino Wistar strain bred in the laboratory. They were housed at 24 OCwith light from 7 AM to 7 PM and fed ad libitum on two semisynthetic diets (Table 1). Caloric distribution was 26% protein, 62% carbohydrate, and 12% fat for the standard diet and 26% protein, 19% carbohydrate, and 55% fat for the high-fat diet. The diets were administered from day 0 of pregnancy, and the rats were studied on days 7,19, and 2 1 of pregnancy. In each experiment, age-matched virgin rats were used as controls. Food intake and body weight were recorded daily. Maternal weight gain is total body weight gain minus the weight of uterus and its content. The rats were anesthetized with pentobarbital (30 mg/kg body weight IP). One carotid artery was catheterized and a tracheotomy was performed. Body temperature was maintained at 38 OC. Food was withdrawn at 7 AM and the measurements of glucose turnover rates were performed at 10 AM ie, in the postabsorptive period (when the blood glucose concentration in the carotid artery is slightly higher than in the portal vein as described previously).” In brief, [6-‘H] glucose was infused in the saphenous vein at a rate of 0.2 rCi/min after a priming dose of 4 PCi. Blood samples (200 /JCL) were obtained at 30, 40, 50, and 60 minutes after the beginning of the [6-‘H] infusion when a blood glucose steady-state specific radioactivity was established. They were deproteinized in Ba(OH)ZnSO,, an aliquot of the supernatant was used for determination of blood glucose by glucose oxidase method. Another aliquot of the supernatant was evaporated to dryness to remove ‘H,O and counted. Glucose turnover rates at steady state were calculated by dividing the [6-3H] glucose infusion rate (dpm/min) by the specific radioactivity of glucose in blood (dpm/mg). Glucose metabolic clearance rates were calculated as the ratio of glucose turnover rate and of blood glucose and were expressed in mL/min.
No 1 (Jaman/), 1987: pp 66-70
IN PREGNANT RATS
Table 1. Composition of the Semisynthetic Diets (g/kg) StandardDief
Peanut oil Lard
acids, and blood ketone bodies were each rat before turnover measurement. Plasma insulin was determined by radioimmunoassay using human insulin as tracer and rat insulin as standard.5 Plasma free fatty acids were Plasma
insulin, free fatty
measured by an enzymatic method using NEFA-C test WAK0.16 Acetoacetate and @-hydroxybutyrate were determined enzymatitally on blood deproteinized with 6% (w/v) perchloric acid.” Statistics Results are expressed as mean + SEM of six determinations. Significance of differences was determined by using Student’s t-test RESULTS
Effects of Pregnancy and High-Fat Diet on Food Intake and Maternal Weight Gain
Cumulative food intake expressed in grams and calories is shown in Table 2. In rats fed the standard diet an increased food intake (P < 0.01) was observed during pregnancy. In rats fed a high-fat diet, caloric consumption was similar to the rats fed a standard diet in both virgin and pregnant rats. Rats fed a standard diet gained weight during pregnancy of which 63 + 7 g represent maternal tissues (Table 2). After 21 days of diet consumption, virgin rats fed a high-fat diet tended to be heavier than control. Pregnant rats fed a high-fat diet gained 69 + 8 g of maternal tissues; the differences in maternal weight gain were not statistically significant (Table 2). On day 21 of pregnancy, the average number of fetuses [ 10.6 k 0.7 (n = 6)] for the rat fed a standard diet and [ 11.4 f 1.2 (n = 6)] for the rats fed a high-fat diet and the mean fetal weight (Table 2) were similar in the two groups.
Effects of High-Fat Diet on Glucose Kinetics and Free Fatty Acids and Ketone Body Concentrations in Virgin Rats
The effects of feeding a high-fat diet were measured in virgin rats after 7 days, 19 days, and 21 days of diet consumption. In rats fed a standard diet, glucose turnover rates were 2.6 i 0.3 mg/min (n = 6) after 7 days, 3.2 2 0.3 mg/min (n = 6) after 19 days, and 2.8 k 0.2 mg/min (n = 6) after 21 days of diet consumption. In rats fed a high-fat diet, glucose turnover rates were 2.6 + 0.2 mg/min (n = 6) after 7 days, 2.7 +_0.2 mg/min (n = 6) after 19 days, and 2.6 I 0.2 mg/min (n = 6) after 21 days of diet consumption. No statistical difference arose between the rats submitted to various lengths of diet consumption on the parameters tested. Therefore, the three groups have been pooled and the results are shown in Table 3. The blood glucose concentration was similar in rats fed a high-fat diet and rats fed a standard diet (Table 3). Glucose turnover rate, expressed as mg/min/kg was lower (P < 0.05) in rats high-fat fed than in rats standard fed (Table 3). Glucose clearance rate expressed as mL/min or mL/min/kg was decreased by 10% to 20% in rats fed a high-fat diet (P < 0.01) (Table 3). Plasma insulin, plasma free fatty acid, and blood ketone body concentrations were unaffected by the composition of the diet consumed (Table 3). Effects of High-Fat Diet on Glucose Kinetics and Plasma Free Fatty Acid and Blood Ketone Body Concentrations in Pregnant Rats
In pregnant rats fed a standard diet, blood glucose concentration was identical at 0 and 7 days of pregnancy and was decreased by 30% (P < 0.001) after seven days of pregnancy onwards (Fig 1). The glucose turnover rate increases slightly (P < 0.01) after seven days of pregnancy, to reach a value 70% higher than in virgin rats on day 19 of pregnancy. On day 21 a decrease in the glucose turnover rate was observed. The glucose metabolic clearance rate followed the same pattern; it was increased 2.4-fold on day 19 of pregnancy but remained elevated on day 21 compared to virgin values. The plasma insulin concentration in the postabsorptive state was not altered by pregnancy; a significant drop (P < 0.01) of plasma insulin concentration was observed on day 21 of pregnancy compared to day 19 of pregnancy. Plasma free
Table 2. Effects of Pregnancy snd High-Fat Diet on Food Intake and Weight Gain Total Food Intake During 2 1 Days Cal
Mean Fetal Wmght
335 f 14
237 k 3
36 t 5
302 f 3.
247 k 6%
53 t 4$
2 1-Day pregnant rats Standard diet
f 21 14’
Results are expressed as mean k SEM for six animals. lp < 0.05,
standard fed Yhigh-fat fed rats.
tf’ c 0.01,
standard fed v high-fat fed rats.
346 5 12
63 r 7
350 2 9$
69 r 8$
LETURQUE ET AL
Table 3. Effect of a High-Fat Diet on Blood Glucose, Glucose Turnover end Clearance Rates, Plasma Insulin, Free Fatty Acids, and Blood Ketone Bodies in Postabsorptive Virgin Rats
Blood Glucose (mo/mL)
Glucose Metabdic cbarana Rats
GlucoseTwnmw Rate ma/min
1.10 * 0.02
2.9 * 0.1
12.2 * 0.5
2.6 t 0.1
1.15 * 0.02.
2.6 * 0.1.
10.6 * 0.47
2.3 * O.lt
mL/min/kg 11.1 zt 0.5 9.2 * 0.3$
Plasma Insulin WJlmil
Plasma Free Fatty Acids bmol/mLl
139 * 13
0.36 * 0.03
0.620 * 0.080
121 * 8.
0.35 * 0.04.
0.880 tt 0.120.
Reaulo are mean * SEM of 18 experiments
lNS. tP c 0.05, aMdad tP < 0.01.
fed Y high-fat fad rats.
standard fsd Y hi&-fat fed rats.
fatty acid and blood ketone body concentrations were decreased on day 7 of pregnancy and were increased by 150% (P < 0.001) and 65% (P < 0.05), respectively, in late pregnancy (Fig 1). During pregnancy a high-fat diet (Fig 1) did not alter the normal metabolic response of the rat. The glucose turnover rate was not increased by day 7 of pregnancy, and plasma free fatty acid concentrations were decreased on day 21 of pregnancy compared with pregnant rats fed a standard diet. In contrast, blood ketone body concentrations were increased in rats fed a high-fat diet compared to control in 21-day pregnant rats. DISCUSSION
The composition of the diets was chosen to alter the dietary carbohydrate to fat ratio while preserving the daily protein intake” and food palatability. Consequently, the animals ingested the same amount of total calories in the pregnant group, whereas it was only slightly higher for the high-fat diet in the virgin rats. Thus, the changes observed could be mainly attributed to the ratio of dietary carbohydrate to fat and not to food restriction or overfeeding. No difference in weight gain was observed in the Wistar
Fig 1. Blood glucose, glucose turnover rate, metabolic clearance rate, plasma insulin, plasma free fatty acids, and total ketone bodies in virgin rats (day 0 of pregnancy) and in 7-day, IS-day. and Il-day pregnant rats fed a standard diet (0) or a high-fat diet (0). Values are means t SEM of six to 18 experiments. lP < 0.01 when rats fed a standard diet were compared with rats fed a high-fat diet.
strain after 21 days of consumption of a high-fat diet, contrary to Osborne-Mendel rats in which fat deposition was shown to be promoted in virgin rats fed with high-fat diet.ig Virgin rats fed a high-fat diet had blood glucose and plasma insulin concentrations close to those of virgin rats fed a standard diet. This is in agreement with previous reports.2h 22Nevertheless, the glucose turnover rate in the postabsorptive period is decreased by 10% to 20% (P < 0.05) as previously reported.” The decrease in glucose utilization is modest compared to the 50% to 80% decrease in glucose utilization by adipose tissue in vitro’O~” and to the 20% to 30% decrease in glucose utilization by soleus muscle.13 Assuming that the glucose turnover rate is representative of the 24-hour average endogenous glucose production, the difference in daily carbohydrate intake (9.2 2 0.7 g/d in rats fed a standard diet) v (2.7 + 0.2 g/d in rats fed a high-fat diet) (P < 0.001) in front of a daily glucose turnover rate of 4.0 + 0.2 g/d v 3.8 + 0.2 g/d, respectively, shows that in rats fed a standard diet, the glucose requirements are exogenously met whereas they are not in rats fed a high-fat diet, and emphasizes the need of an active endogenous glucose production of about 1.1 g/d in high-fat fed rats. In rats high-fat fed, liver glycogen content is reduced twofold23*24 suggesting that the source of endogenous glucose production could have been exhausted more rapidly in these rats. Thus, in rats fed high-fat, glucose homeostasis must be maintained by an active hepatic gluconeogenesis. The activity of gluconeogenic enzymes is high in the liver of rats fed a high-fat diet.24*2’The gluconeogenic substrates used to sustain a high rate of gluconeogenesis have not been identified and could depend on the composition of the diet. An elevated gluconeogenesis in liver of rats fed a high-fat diet is observed despite normal plasma insulin and glucagon.14 The plasma free fatty acid concentration is not modified in the postabsorptive state in virgin rats fed a high-fat diet. The diurnal measurements of free fatty acids26 have shown that major differences appear mainly during the feeding period. The concentrations of free fatty acids were lower in the feeding period in rats fed a high-fat diet, whereas in the postabsorptive state similar concentrations are observed in rats fed a standard or a high-fat diet, in agreement with this study. In contrast to De Gasquet et a1,26we have not been able to see any difference in blood ketone body concentrations, although in rats fed a high-fat diet, high ketogenic rates have been shown in perfused liver.15 The discrepancy is probably due to the higher (73% and 90%) fat content of the diet used in these two studies’5*26than in the present study (55%).
In pregnant rats, high-fat feeding does not alter the fetal weight at term nor the normal maternal metabolic response. The decrement of the blood glucose concentration which occurs after the first week of pregnancy is similar in rats fed standard or high-fat diets. When the metabolic requirement of the conceptus are very low at seven days of pregnancy, glucose turnover rates compared to virgin values are increased in rats fed a standard diet whereas no increase is observed in rats fed a high-fat diet. This suggests that in pregnant rats fed a high-fat diet free fatty acids can be used as an alternative fuel of glucose for maternal tissues. Thus, the extraglucose produced by pregnant rats fed a standard diet could support increased maternal tissue lipogenesis during this anabolic stage. The postabsorptive glucose turnover rate is increased by 70% in 19-day pregnant rats-’ to provide glucose for the pregnant uterus. The modest 10% decrease in glucose turnover rate of rats fed high-fat is not amplified during pregnancy, suggesting that in the postabsorptive state, the strategy of the pregnant organism for maintaining glucose homeostasis during high-fat feeding is an increased endogenous glucose production rather than a decreased peripheral glucose utilization. Thus, the state of insulin resistance induced by pregnancy is not further enhanced by high-fat feeding. In 19-day pregnant rats, the daily carbohydrate intake was 13 c 1 g/d in rats fed a standard diet v 3.7 + 0.2 g/d (P < 0.001) in rats fed a high-fat diet. Assuming that the glucose turnover rate is representative of the 24-hour average endogenous glucose production, the daily glucose turnover rate was 6.1 + 0.8 g/d and 5.8 -+ 0.3 g/d, respectively, in pregnant rats fed a standard and a high-fat diet. This suggests that the glucose requirements are exogenously met in rats fed a standard diet whereas they are not in rats fed a high-fat diet and emphasizes the need of an active endogenous glucose production of 2.4 g/d. An active endogenous glucose production was also observed in pregnant rats fasted for 24 hours4 since glucose turnover was enhanced compared to nonpregnant rats despite the absolute absence of carbohydrate intake during 24 hours. During pregnancy the enhanced liver glucose production is not due to glycogenolysis since the liver
glycogen content is similar in pregnant and virgin rats,*‘,*’ but rather to a high gluconeogenic capacity of the liver during this physiologic state.27 In the postabsorptive state, plasma insulin concentrations and glucose clearance rates are not affected by the diet during pregnancy; this confirms that a high-fat diet does not worsen insulin resistance during pregnancy. Circulating concentrations of free fatty acids and ketone bodies are a reflection of the two phases in lipid metabolism described previously.’ The initial phase (0 to 7 days) is characterized by a decrease in plasma fatty acid concentrations in agreement with previous reports4s6but also in blood ketone bodies which suggests a fat storage. The second phase observed on days 19 and 21 of pregnancy is characterized by an increase in plasma free fatty acid concentrations and in blood ketone bodies which suggests a fat mobilization. High-fat feeding does not prevent fat storage or fat mobilization since the concentrations of free fatty acids and ketone bodies are not altered by the diet consumed during pregnancy. On day 21 of pregnancy in rats fed a high-fat diet, plasma levels of free fatty acids are decreased and total ketone bodies are increased compared to standard fed pregnant rats; this could probably be explained by the fact that food intake is maintained in this group (16 + 1 g/d at 19 days and 16 t 1 g/d at 20 days), whereas it decreases significantly in the standard fed group (20 2 1 g/d at 19 days and 17 + 1 g/d at 20 days of pregnancy). A decrease in food intake is classically reported two days prior to delivery in the rat.29 In conclusion, this study demonstrates that the characteristic metabolic adaptations of pregnancy are not altered by a decreased availability of carbohydrate in the diet of the rat. To maintain glucose homeostasis an active gluconeogenesis must be present in pregnant rats without amplifying the decreased peripheral glucose utilization in maternal tissues.
ACKNOWLEDGMENT We are indebted to D. Chamereau for taking care of the animals and to I. Coquelet for the preparation of the manuscript.
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