Regulation of hepatic enzymes and insulin levels in offspring
of rat dams fed a reduced-protein diet
Mina
Desai1,
Christopher D.
Byrne1,
Karim
Meeran2,
Nick D.
Martenz1,
Steven R.
Bloom2, and
C. Nicholas
Hales1
1 Department of Clinical
Biochemistry, University of Cambridge, Addenbrooke's Hospital,
Cambridge CB2 2QR; and 2 Division
of Endocrinology and Metabolism, Royal Postgraduate Medical School,
Hammersmith Hospital, London W12 0HS, United Kingdom
 |
ABSTRACT |
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
 |
INTRODUCTION |
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.
 |
MATERIALS AND METHODS |
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.
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).
 |
RESULTS |
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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Fig. 2.
Growth curves of male (A) and female (B) rats.
Values are means ± SE of weekly measured body weights of control
and low-protein rats fed laboratory chow or a highly palatable diet;
n = 10 males and 10 females from
5 litters per diet group. , Control rat fed laboratory chow; ,
control rat fed highly palatable diet; , low-protein rat fed
laboratory chow; low-protein rat fed highly palatable diet.
|
|
In male rats the organ weights that showed significant differences
between low-protein and control groups were muscle, kidney, and heart
(Table 3). When expressed relative to the
body weight, heart and brain showed differences between these groups
(Table 3). In female rats the organ weights that showed significant differences between low-protein and control groups were kidney and
heart (Table 4). When expressed relative to
the body weight, pancreas and brain showed differences between these
groups (Table 4).
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).
Conversely, the activity of the gluconeogenic enzyme
phosphoenolpyruvate carboxykinase was increased in the low-protein
group compared with the controls fed the laboratory chow diet. Rats from both groups fed the highly palatable diet showed a tendency toward
decreased enzyme activity, although this was not statistically significant. However, significantly increased activity was still evident in the low-protein group fed the highly palatable diet compared
with the controls fed the same diet (Table 5).
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).
The fasting plasma insulin concentrations were different between male
and female rats. Male rats in general tended to be more insulin
resistant than females, as determined by their higher fasting plasma
insulin concentrations. There was a significant difference between male
and female control rats fed a laboratory chow diet
(P < 0.01). Although the magnitude
of the differences in mean concentrations was similar in male and
female low-protein rats fed a laboratory chow diet, it did not reach
statistical significance because of greater variance in both groups
(P = 0.128). With the highly palatable
diet the difference in plasma insulin concentrations was more marked
between male and female rats, and this difference was observed in the
control (P < 0.001) and low-protein (P < 0.001) groups.
When the effects of maternal diet (i.e., control and low-protein
groups) were compared in animals of the same sex fed laboratory chow as
adults, no significant differences in plasma insulin concentrations were evident in rats of either sex. However, the highly palatable diet
caused a significant increase in fasting plasma insulin concentrations in male offspring compared with laboratory chow-fed males from the
control (P < 0.001) and low-protein
groups (P < 0.001). The effect of
the highly palatable diet was more marked in the low-protein male rats
than in the control male rats. The fasting plasma insulin concentrations were significantly increased in low-protein male rats
fed the highly palatable diet compared with control male rats fed the
same diet (P = 0.03).
In female rats the highly palatable diet also caused a significant
increase in fasting plasma insulin concentrations in the control rats
compared with laboratory chow-fed controls
(P < 0.001). In the low-protein
females, there was a similar trend toward increased fasting plasma
insulin concentration as a result of the highly palatable diet, but
this difference was not statistically significant compared with the
laboratory chow-fed low-protein females
(P = 0.102). There was also no
difference between control and low-protein female rats fed the highly
palatable diet (Table 6).
The maternal low-protein diet had no effect on fasting plasma
triglycerides and NEFA concentrations, inasmuch as there were no
significant differences between the control and low-protein rats fed
the laboratory chow or the highly palatable diet. The highly palatable
diet (independent of maternal low-protein diet) caused a significant
increase in triglyceride concentrations in the control male and female
rats compared with rats of the same sex fed the laboratory chow diet.
The increase in triglyceride concentrations was also observed in
low-protein male rats fed the highly palatable diet compared with
low-protein male rats fed the laboratory chow. However, in the
low-protein female rats the highly palatable diet did not cause a
significant increase in triglyceride concentration. Furthermore, the
highly palatable diet did not affect the fasting plasma NEFA
concentrations in any of the groups compared with rats fed the
laboratory chow (Table 6).
 |
DISCUSSION |
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.
 |
ACKNOWLEDGEMENTS |
We thank D. Hutt, A. Flack, and A. Wayman (Animal Unit, Dunn
Nutritional Laboratory) for invaluable assistance and S. Archer for
measuring the triglycerides.
 |
FOOTNOTES |
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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