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1 Department of Clinical
Biochemistry, We have hypothesized that permanent changes
caused by poor growth during early development due to maternal
malnutrition may be exacerbated by overnutrition of offspring in later
life. To test this hypothesis, rats were exposed to a maternal 20%
protein diet or an isocaloric 8% protein diet during fetal and
postnatal life. All offspring were weaned onto laboratory chow. At 6 wk, rats were fed laboratory chow or a highly palatable diet (high fat
and high calorie with adequate protein) and studied at 12 wk after a
48-h fast. The highly palatable diet resulted in excess weight gain and
higher plasma insulin levels in all animals. Plasma insulin
concentrations were significantly increased in male offspring of dams
fed a reduced-protein diet compared with male offspring of dams fed an
adequate-protein diet, but no differences were observed between the
female offspring. The key hepatic enzymes of glucose homeostasis
programmed in offspring of protein-restricted rat dams retained the
ability to respond to overnutrition during adult life. In these
offspring, however, the enzymes were regulated around a "set
point" that was different from that in the controls.
maternal diet; highly palatable diet; glucokinase; phosphoenolpyruvate carboxykinase; diabetes
RECENT EPIDEMIOLOGICAL studies have shown an
association between reduced growth in early life and an increased risk
of hypertension (2), hypertriglyceridemia, impaired glucose tolerance,
non-insulin-dependent diabetes, and insulin resistance syndrome in
adult life (reviewed in Ref. 17). These findings have led to the
thrifty phenotype hypothesis, which suggests that impaired glucose
tolerance and non-insulin-dependent diabetes may result from poor fetal
growth interacting adversely with abundant nutrition in adult life. It was proposed that the unfavorable nutritional environment during early
fetal development might induce insulin resistance (10). Subsequent
epidemiological evidence indicated that poor fetal growth was
associated with adult insulin resistance. Furthermore, subjects
who were thin at birth, as indicated by the ponderal index
(weight/length3), but obese as
adults, as judged by their body mass index, were the most
resistant to insulin (16).
Our previous studies demonstrated that protein deficiency during
pregnancy and lactation not only permanently altered the hepatic enzyme
activities involved in the regulation of glucose homeostasis (5, 7) and
hepatic insulin sensitivity (15) but also reduced glucose tolerance in
the adult offspring (11). In all these studies, however, the
consequences of maternal low-protein diet on the offspring have been
studied in the absence of adult obesity.
The aim of the present study was to investigate whether maternal
protein restriction during gestation and lactation combined with excess
weight gain of offspring in adult life induced by a highly palatable
diet (high fat and high calorie with adequate protein) produced
features of insulin resistance and affected the response of key enzymes
involved in glucose metabolism in later life.
Animals and diet.
All animal procedures were performed, under license, in accordance with
the Home Office Animal Act (1986). Virgin female Wistar rats with
initial body weight of 240 ± 20 g were maintained at 22°C on a
controlled 12:12-h light-dark cycle. They were mated, and
day 0 of gestation was taken as the
day on which vaginal plugs were observed. Thereafter, rats were fed a
diet containing 20% protein or an isocaloric diet containing 8%
protein throughout gestation and lactation. The details and full
dietary compositions have been previously reported (6). After birth at
3 days of age, the litter size was standardized to four males and four
females. At 21 days of age, all offspring were weaned onto normal
laboratory chow. For simplicity the two groups of offspring are termed
"control" and "low protein." Only the mothers underwent
dietary manipulation. At 6 wk of age, two males and two females from
each litter were placed on normal laboratory chow (PRD pellet, Labsure,
Poole, UK) and the remaining two males and two females on the highly palatable diet, the composition of which is given in Table
1. This high-fat and high-calorie,
cafeteria-style diet was designed to be highly palatable so that the
rats ate relatively large amounts of it (23). The respective diets
(Table 2) fed ad libitum were continued for
a period of 6 wk, and the animals were studied at the age of 12 wk.
They were deprived of food but not water for 48 h before the study. The
experimental study design is illustrated in Fig.
1.
ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
Table 1.
Composition of highly palatable diet
Table 2.
Dietary composition of experimental diets
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Fig. 1.
Experimental study design. Maternal and offspring diets were altered as
indicated.
Homogenization of liver. For glucokinase (EC 2.7.1.1), liver homogenate was prepared at 4°C in a medium containing (in mM) 50 sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid, 100 KCl, 1 EDTA (sodium salt), 5 MgCl2, and 2.5 dithiothreitol at final pH 7.4 (4). For phosphoenolpyruvate carboxykinase (EC 4.1.1.32), the liver was homogenized at 4°C in 0.25 M sucrose, 5.0 mM tris(hydroxymethyl) aminomethane · HCl, and 1.0 mM mercaptoethanol at final pH 7.4 (1). The homogenate was then centrifuged at 100,000 g for 60 min at 4°C, and the supernatant was assayed immediately.
Determination of enzyme activities. Enzyme activities were determined in the cytosolic fractions. Glucokinase activity was assayed as described previously (4). The contribution of hexokinases with low Michaelis-Menten constants was determined using 0.5 mM glucose. The final assay medium contained (in mM) 50 sodium N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid buffer, 100 KCl, 2.5 dithiothreitol, 7.5 MgCl2, 0.5 or 100 glucose, 5.0 ATP, and 0.5 NAD and 0.2 IU of glucose-6-phosphate dehydrogenase (Leuconostoc mesenteroides). Reagent blanks without liver supernatant were run for each medium, i.e., low and high glucose, without or with ATP. Sample blanks contained liver supernatant and complete assay medium except for ATP. Sample blanks (without ATP) were subtracted for high and low glucose concentrations. The glucokinase activity was determined from the difference in corrected rates between 100 and 0.5 mM glucose. Glucokinase activity was expressed as micromoles of NADH formed per minute under the assay conditions.
Phosphoenolpyruvate carboxykinase activity was assayed in the presence of NADH and malate dehydrogenase by measuring the rate of incorporation of [14C]bicarbonate into malate (1). The medium contained (in mM) 0.1 imidazole, 2 MnCl2, 1 dithiothreitol, 100 NaHCO3 (containing 2 µCi of NaH14CO3), 1.5 phosphoenolpyruvate, 1.25 IDP, and 0.5 malate dehydrogenase plus liver extract at final pH 7.0 in a volume of 1.0 ml. The reaction was started by the addition of extract after preincubation of medium for 2 min at 37°C. The reaction was stopped with 0.5 ml of 10% (wt/vol) trichloroacetic acid. The acid-stable 14C activity in a 0.5-ml aliquot was heated to dryness in a scintillation counting vial at 85°C for 60 min in a forced-draft oven (13). After addition of 0.5 ml of water and then 10 ml of liquid scintillator to the vial, acid-stable 14C activity was determined using a liquid scintillation counter. One unit of activity is the amount of enzyme that catalyzes the fixation of 1 µmol of [14C]bicarbonate per minute. Liver protein was determined by a dye-binding method using bovine serum albumin as standard (3).Glucose, insulin, triglycerides, and nonesterified fatty acids. Whole blood glucose was measured using a B-glucose photometer supplied by Hemocue (Angelholm, Sweden). Plasma insulin was assayed by radioimmunoassay with rat insulin standards (Linco, Biogenesis, Poole, UK). Plasma triglyceride was quantified using the RA 1000 (Bayer Diagnostics, Basingstoke, UK), with a standard enzymatic method. Plasma nonesterified fatty acid (NEFA) measurements were determined enzymatically on the basis of the activity of acyl-CoA synthase (Boehringer Mannheim, Lewes, Sussex, UK). The resultant acyl-CoA was oxidized to yield hydrogen peroxide, which was measured colorimetrically using a Monarch analyzer (20). The between-assay coefficient of variation was 10% at 0.40 mmol/l and 6% between 1.2 and 2.3 mmol/l.
Statistical analysis. Statistical analysis included analysis of variance, Mann-Whitney U-test, and unpaired t-tests. Values are means ± SE or geometric means with 95% confidence intervals. Mann-Whitney U-tests were undertaken for nonnormal distributions (i.e., insulin, triglycerides, and NEFA).
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RESULTS |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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Body and organ weights. The pups from the low-protein group had lower body weights than pups from the control group at 3 days after birth (6.4 ± 0.4 and 8.8 ± 0.40 g, respectively, P < 0.001). This pattern was evident throughout the preweaning period [low-protein vs. control group: 10.5 ± 0.5 vs. 16.2 ± 0.7 g at 7 days of age (P < 0.001); 15.9 ± 0.8 vs. 33.3 ± 1.0 g at 14 days of age (P < 0.001); 29.2 ± 0.7 vs. 46.6 ± 1.2 g at 21 days of age (P < 0.001)]. After weaning at 21 days of age, the low-protein group fed a normal laboratory chow or a highly palatable diet had consistently lower body weights than the controls fed the same diet. In both groups, male and female rats fed a highly palatable diet attained a greater increase in body weight than those fed the laboratory chow diet (Fig. 2).
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Hepatic enzymes. The activity of the glycolytic enzyme glucokinase was significantly reduced in the low-protein group fed laboratory chow diet compared with the controls fed the same diet. Feeding the highly palatable diet caused a significant increase in glucokinase activity in both groups. However, the rats fed the low-protein diet maintained a reduction in glucokinase activity compared with the control group (Table 5).
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Blood glucose, plasma insulin, triglycerides, and NEFA. In both sexes, neither the previous exposure to maternal low-protein diet nor feeding the subsequent highly palatable diet in later life had a significant impact on fasting blood glucose concentrations (Table 6).
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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The present study shows that excess weight gain induced by the highly palatable diet resulted in higher plasma insulin concentrations. Fasting insulin concentration is a good proxy measure of insulin resistance, as determined by hyperinsulinemic-euglycemic clamp study, where insulin induces glucose uptake in the skeletal muscle (12). Generally, male rats had higher plasma insulin concentrations than female rats. In male rats, exposure to maternal protein restriction during early life followed by the highly palatable diet led to much higher plasma insulin levels. Interestingly, this phenomenon was not evident in the female offspring of protein-restricted rat dams, suggesting that some factor(s) associated with gender affected these rats. Despite the fact that the low-protein male rats fed the highly palatable diet had lower body weight (16% reduction) than the control males fed the same diet, they had higher fasting plasma insulin concentrations. Consistent with our observation that there is a different response to maternal protein-restricted diet between the sexes, we previously noted a greater deterioration in glucose tolerance in 15-mo-old male offspring of dams fed a protein-restricted diet during pregnancy and lactation (11). Thus the combination of fetal growth retardation and excess weight gain in adult life is particularly detrimental to male rats. This finding is also consistent with observations in humans that showed men to be more insulin resistant than women (16). It is known that the distribution of fat is important in the etiology of insulin resistance. Men have greater fat deposition around the waist, and intra-abdominal obesity is associated with increased insulin resistance (9, 14), although whether male rats have a different pattern of fat distribution is uncertain. In the present study, body fat was not measured. However, it is possible that the low-protein and highly palatable diets may affect the amount, location, and type of body fat that may contribute to the findings.
It is known that, during starvation, glycolysis is decreased and gluconeogenesis and lipolysis are increased (18). Studies have also shown that in rats fasted for 48 h the glucokinase activity is decreased (8), whereas phosphoenolpyruvate carboxykinase activity is increased (19). Our previous findings of permanently altered activities of key hepatic enzymes of glycolysis (glucokinase) and gluconeogenesis (phosphoenolpyruvate carboxykinase) (5, 7) in the offspring of protein-restricted rat dams in the nonfasted state were also observed in the present study in which animals were subjected to 48 h of starvation. Therefore the changes in hepatic enzyme activities in the low-protein offspring are evident in fasted and starved states. For this reason, it is unlikely that the observed changes in low-protein rats can be attributed solely to starvation.
The present data add to the above data showing that these permanent changes in hepatic enzymes can themselves be subject to regulation. In the low-protein offspring as well as in the controls, increased nutrition and enhanced weight gain in adult life led to a significant increased activity of glucokinase and decreased activity of phosphoenolpyruvate carboxykinase of approximately the same order of magnitude. Nevertheless, the relative changes seen as a consequence of being an offspring of a protein-restricted rat dam were maintained, despite the regulatory effect due to enhanced nutrient intake. It is therefore apparent that although the offspring of protein-restricted rat dams have permanently changed activities of key hepatic enzymes of glucose metabolism, these enzymes remain susceptible to regulation in adult life. The main effect of programming (a permanent change in the offspring) therefore is not to prevent further metabolic and endocrine control but to alter the "set point" about which this control takes place. Thus we suggest that rat pups exposed to early maternal malnutrition have their metabolic control point shifted in the direction of poor nutrition, since the activity of a glucose-utilizing enzyme is decreased and the activity of a glucose-producing enzyme is increased. It is possible that these changes may be an integral part of processes that may enhance survival of the animal under conditions of poor nutrition in postnatal life.
An interesting mechanism for the short-term control of glucokinase activity has been described (22). These studies have identified a regulatory protein capable of binding to and inhibiting glucokinase in the presence of fructose 6-phosphate. Inhibition is relieved in the presence of fructose 1-phosphate. The regulatory protein behaves as a fully competitive inhibitor that by itself does not affect the kinetic properties of glucokinase. It has further been shown that the inhibition exerted by the regulatory protein was influenced by changes in pH and salt concentrations (21). In the present study the assay conditions used for glucokinase were clearly unfavorable for the regulatory protein to exert its effect. In view of this, it seems unlikely that the changes observed in glucokinase activity can be explained by the effect of inhibitor protein.
Despite the changes in hepatic enzymes and insulin concentrations in the low-protein offspring, there were no alterations in plasma glucose, triglyceride, and NEFA concentrations. However, in this experiment the animals were fasted for 48 h before the study. Possibly because of this and/or stress, plasma NEFA concentrations were relatively high, and we cannot rule out the possibility that changes in these parameters might be observed in the fed, unstressed state.
In conclusion, the data suggest that the combination of fetal growth retardation and excess weight gain in adult life has particularly adverse consequences for insulin sensitivity in male but not female rats. Our results imply that maternal protein-restricted male offspring fed the highly palatable diet were more insulin resistant than controls fed the same diet, although this was not sufficient to affect their blood glucose concentration or lipid profile at 12 wk of age. We have demonstrated that key hepatic enzymes of glucose homeostasis programmed in offspring of protein-restricted rat dams are regulated around a different set point by overnutrition during adult life.
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ACKNOWLEDGEMENTS |
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We thank D. Hutt, A. Flack, and A. Wayman (Animal Unit, Dunn Nutritional Laboratory) for invaluable assistance and S. Archer for measuring the triglycerides.
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FOOTNOTES |
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This work was supported by the British Diabetic Association and the Medical Research Council (MRC). C. D. Byrne is an MRC Clinician Scientist Fellow.
Address for reprint requests: M. Desai, Dept. of Clinical Biochemistry, University of Cambridge, Addenbrooke's Hospital, Hills Rd., Cambridge CB2 2QR, UK.
Received 18 February 1997; accepted in final form 27 June 1997.
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Materials & Methods
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Discussion
References
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, Control rat fed laboratory chow; 
,
control rat fed highly palatable diet; 
, low-protein rat fed
laboratory chow; 
low-protein rat fed highly palatable diet.