Molecular and Cellular Biology, Division of Biomedical Sciences, St
Bartholomew's and the Royal London School of Medicine and
Dentistry, Queen Mary and Westfield College, University of London,
London E1 4NS, United Kingdom
The study investigated
whether a persistent impairment of insulin secretion resulting from
mild protein restriction predisposes to loss of glucoregulatory control
and impaired insulin action after the subsequent imposition of the
diabetogenic challenge of high-fat feeding. Offspring of dams provided
with either control (20% protein) diet (C) or an isocaloric restricted
(8%) protein diet (PR) were weaned onto the maintenance diet with
which their mothers had been provided. At 20 wk of age, protein
restriction enhanced glucose tolerance despite impaired insulin
secretion and an augmented and sensitized lipolytic response to
norepinephrine in adipocytes. C and PR rats were then transferred to a
high-fat diet (HF, 19% protein, 22% lipid, 34% carbohydrate) and
sampled after 8 wk. These groups are termed C-HF and PR-HF. Glucose
tolerance was impaired in PR-HF, but not C-HF, rats. Insulin-stimulated glucose disposal rates were significantly lower (by 30%;
P < 0.01) in the PR-HF group than in
the C-HF group, and a specific impairment of antilipolytic response of
insulin was unmasked in adipocytes from PR-HF, but not C-HF, rats. The
study demonstrates that antecedent protein restriction accelerates and
augments the development of impaired glucoregulation and insulin
resistance after high-fat feeding.
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INTRODUCTION |
PROTEIN-CALORIE MALNUTRITION impairs the insulin
secretory response of the pancreatic
-cell (1, 4, 16, 19, 20, 26,
27), and when imposed during pregnancy, mild protein restriction, even
in the absence of calorie restriction, elicits a profound impairment in
the structural and functional development of the fetal endocrine
pancreas (3). There has been considerable research interest in the
proposition that changes to nutrition during pregnancy can influence
the normal metabolic control mechanisms in the offspring. Deprivation
of specific macronutrients, in particular protein, has been suggested
to have effects on glucose metabolism in later life, particularly on
the secretion of insulin. The continued maintenance of offspring of
dams subjected to moderate protein restriction during pregnancy on a
protein-restricted diet after birth leads to impaired
glucose-stimulated insulin secretion in adulthood (3, 9).
Paradoxically, the blunted response of insulin secretion to glucose
challenge in rats maintained on low-protein diets is frequently
associated with normal or enhanced glucose tolerance and insulin action
(5, 10, 16, 19, 20, 28). For example, the imposition of protein-calorie
restriction (35% restriction, 5% protein) on 4-wk-old female rats for
4 wk leads to greatly impaired glucose-stimulated insulin secretion but
is associated with enhanced insulin-mediated glucose uptake (20). In
male rats, provision of a 5% protein diet from 4 wk to 15 wk of age
also results in slightly enhanced tolerance to intravenous glucose,
whereas the insulin response to glucose is severely blunted (19).
Furthermore, hypoglycemia induced by intravenous insulin is more
sustained in protein-malnourished rats compared with rats fed a normal
protein diet (19), suggesting increased whole body insulin sensitivity.
In a separate study, normal glucose tolerance (despite a failure to
release insulin after intravenous glucose) has been suggested to
indicate increased sensitivity to insulin and/or increased
peripheral glucose utilization in rats provided with a 6% protein diet
for 14 wk (16). Providing 4-wk-old rats with an isocaloric 5% protein
diet for 4 wk, while not affecting energy intake, enhances tolerance to
an intravenous glucose challenge and improves the actions of insulin to
stimulate peripheral glucose uptake and suppress hepatic glucose
output, whereas glucose-stimulated insulin secretion remains normal
(5). Thus, in general, the evidence to date does not support the
hypothesis that protein restriction necessarily elicits impaired
glucoregulatory control, despite an impaired insulin secretory
capacity, and an enhanced action of insulin action may help to limit
the deterioration of glucose tolerance in the face of impaired insulin
secretion (see Ref. 20).
The present study examined whether the persistent impairment in the
insulin secretory response of the pancreas introduced as a consequence
of protein restriction predisposes to altered insulin action, either in
vivo or in vitro, and/or influences the development of
peripheral insulin resistance through compromising the response to the
potentially diabetogenic challenge of a high-fat diet that in rats
leads to the development of whole body insulin resistance
(23).
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METHODS |
Kits for determination of plasma insulin concentrations were from
Phadeseph Pharmacia, Uppsala, Sweden. Collagenase was from Lorne
Laboratories, Twyford, Berkshire, UK. Human Actrapid insulin was from
Novo Nordisk, Bagsvaerd, Denmark. Kits for determination of plasma
nonesterified fatty acid (NEFA) concentrations were supplied by Alpha
Laboratories, Eastleigh, Hants, UK. Glycerol kinase and
glycerol-3-phosphate dehydrogenase were from Boehringer Mannheim,
Lewes, East Sussex, UK. Other biochemicals and chemicals, including
glucose assay kits, were from Boehringer Mannheim or from Sigma, Poole,
Dorset, UK. Female Wistar rats were from Charles River, Margate, Kent, UK.
The composition of the diets is presented in Table
1. Isocaloric control and
protein-restricted diets were prepared (pellet form) by Hope Farms BV,
Woerden, Netherlands. The control diet contained 20% protein
[casein (22 g/100 g diet; 0.88 g protein/g) supplemented with
DL-methionine (0.2 g/ 100 g
diet)], 63.15% carbohydrate [cerelose (55.15 g/100 g diet)
and corn starch (8 g/100 g diet)], 4.3% lipid (soybean oil) by
weight, and other dietary components as specified in Table 1 (see also
Ref. 12). The isocaloric protein-restricted diet contained 8% protein
[casein (9 g/100 g diet; 0.88 g protein/g) supplemented with
DL-methionine (0.08 g/100 g
diet)], 76.17% carbohydrate [cerelose (68.17 g/100 g diet) and corn starch (8 g/100 g diet)], and 4.3% lipid (soybean oil) by weight. Isocaloricity was maintained by increasing the carbohydrate content of the protein-restricted diet to 76% (Table 1; see also Refs.
12 and 24). The high-energy, high saturated fat diet, henceforth
referred to as high-fat diet, contained 19% protein [casein
(20.6 g/100 g diet; 0.88 g protein/g) supplemented with DL-methionine (0.40 g/100 g diet)] but 34%
carbohydrate (maize starch) and 22% lipid [lard as the major
source of lipid (20.1 g/100 g diet), together with corn oil (1.9 g/100
g diet) to prevent essential fatty acid deficiency] by weight
(Table 1; see also Ref. 7). The lipid component of the high-fat diet
comprised 16% saturated fatty acids (mainly stearic), 16%
monounsaturated fatty acids (mainly oleic), and 7% polyunsaturated
fatty acids (mainly linoleic) by energy. The high-fat diet was prepared
at 3-day intervals with components supplied by Special Diet Services (Witham, Essex, UK), with the exception of the saturated fat component (lard), which was purchased
locally.
Female Wistar rats were housed in a temperature-controlled room (21 ± 2°C) on a standard 12:12-h light-dark cycle (light from 8:00
AM). Rats were time mated by the appearance of sperm plugs (day
0 of pregnancy) (15), immediately
randomly assigned to either the control or protein-restricted diets,
and maintained on these diets throughout pregnancy and lactation. The
provision of the protein-restricted diet did not influence maternal
food intake or body weight gain during pregnancy (24), and litters of
normal numerical size were produced. Mean litter sizes were 12 ± 1 (n = 22) and 12 ± 1 (n = 23) in control and
protein-restricted groups, respectively. Any litters containing <10
pups or >15 pups were excluded from the study. Preliminary studies
indicated that small litters (<10 pups) were associated with
accelerated neonatal growth. It is unclear whether pups in the larger
litters grew slowly during suckling because of greater competition for
nursing or whether pups in litters of <10 pups gain weight during
suckling more rapidly because of less competition for nursing.
At 26 days after birth, sexes were separated. The female offspring were
then weaned onto the maintenance diet (control or protein restricted)
with which their mothers had been provided and maintained on this diet
until ~20 wk of age. These are termed the C and PR groups,
respectively. Rats from each of the groups were then transferred to a
high-fat diet (HF) and studied after a further 8 wk, except for the
adipocyte studies that were undertaken after 4 wk. These rats are
termed the C-HF and PR-HF groups.
For intravenous glucose tolerance tests, each rat was fitted with a
chronic indwelling jugular cannula under Hypnorm [fentanyl citrate (0.315 mg/ml)-fluanisone (10 mg/ml); 1 ml/kg ip] and
diazepam (5 mg/ml; 1 ml/kg ip) anesthesia at 5-7 days before study
(see Refs. 10, 12). Food was removed at the end of the dark (feeding) phase at 8:00 AM, and intravenous glucose tolerance tests (0.5 g
glucose/kg body wt; 150 µl/100 g body wt) were performed in awake,
unstressed rats at 6 h after food withdrawal (see Refs. 10, 12).
Glucose was injected and blood samples (100 µl) were withdrawn at
intervals from the indwelling cannula, which was flushed with saline (2 × 250 µl) after the injection of glucose to remove residual
glucose. Samples of whole blood (150 µl) were deproteinized with
ZnSO4-Ba(OH)2
and centrifuged (10,000 g) at 4°C, and the supernatant was retained for subsequent assay of blood
glucose. The remaining sample was immediately centrifuged (10,000 g) at 4°C, and plasma was stored
at
20°C until assayed for insulin. The insulin and glucose
responses during the intravenous glucose tolerance tests were
calculated as the incremental areas under the plasma insulin and blood
glucose curves, respectively, from data obtained during the 30-min
period after the glucose injection. The rate of glucose disappearance
(K) was calculated from the slope of the regression line obtained with
the log-transformed blood glucose values between 2 and 15 min after
glucose administration and expressed as percent per minute.
For euglycemic-hyperinsulinemic clamp studies, each rat was fitted with
two chronic indwelling cannulas. One cannula was placed in the right
jugular vein, and the other cannula was placed in the left jugular vein
(for infusion and sampling, respectively) under Hypnorm [fentanyl
citrate (0.315 mg/ml)-fluanisone (10 mg/ml); 1 ml/kg ip] and
diazepam (5 mg/ml; 1 ml/kg ip) anesthesia. The infusion
studies were conducted at 5-7 days after cannulation. Food was
withdrawn at the end of the dark (feeding) phase at 8:00 AM, and rats
were studied at 6 h after food withdrawal. Whole body glucose kinetics
were estimated in awake, unstressed, freely moving rats in the basal
(postabsorptive) state and during euglycemic hyperinsulinemia by use of
primed (0.5 µCi) continuous (0.2 µCi · min
1 · rat
1)
intravenous infusion of
[3-3H]glucose as
described in Refs. 12 and 24. A steady state of glucose specific
activity in the basal state was achieved by 60 min. Blood samples were
obtained at 60, 75, and 90 min after the commencement of the tracer
infusion for determination of basal glucose specific activity. From 90 min, animals were infused with insulin (human Actrapid) at a fixed dose
of 4 mU · kg
1 · min
1
while blood glucose was maintained at euglycemia for a further 120 min
with a variable rate of 30% glucose infusion, which was initiated at 1 min after the start of insulin infusion. Blood was sampled from the
right jugular vein at 5- to 10-min intervals, and blood glucose
concentrations were determined. Adjustments in the exogenous glucose
infusion rate (GIR) were made to maintain glucose concentrations
constant. The
[3-3H]glucose infusion
was continued for a further 120 min. Steady-state conditions were
achieved after 90 min, after which three blood samples (100 µl) were
taken at 15-min intervals (90, 105, and 120 min) for measurement of
glucose specific activity. Whole body glucose disposal rates
(Rd) were calculated as
described previously (12). Glucose metabolic clearance rate (GMR) was
calculated as Rd divided by the
blood glucose concentration.
For measurements of lipolytic activity, samples of parametrial adipose
tissue were removed from rats anesthetized with pentobarbitone (60 mg/kg body wt), and adipocytes were prepared by collagenase digestion
as described in Ref. 6. There was no evidence for adipocyte cell
breakage during preparation. Adipocyte cell diameter was measured with
an Olympus microscope with an OSM-1 eyepiece micrometer. Cell diameters
before high-fat feeding for C and PR groups were 29.5 ± 2.1 µm
(n = 8) and 26.8 ± 1.9 µm
(n = 10), respectively. Cell diameters
after high-fat feeding for C and PR groups were 24.9 ± 0.3 µm
(n = 9) and 23.3 ± 0.3 µm
(n = 8), respectively. Adipocytes were
resuspended to 20% lipocrit, and aliquots (100 µl) of adipocyte
suspension were added to round-bottomed polypropylene tubes containing
0.7 ml of HEPES-buffered Krebs-Henseleit saline (in mM: 10 HEPES, 1.25 MgSO4, 2.5 CaCl2, 140 NaCl, 2 K2HPO4, 0.5 KH2PO4,
pH 7.4) containing 2% (wt/vol) BSA and 2 mg/ml glucose. All subsequent
procedures were performed at 37°C. Tubes were preincubated for 10 min (200 oscillations/min) before the addition of norepinephrine and
insulin, at the concentrations indicated, to a final volume of 1 ml.
Samples were incubated for 30 min (200 oscillations/min), at the end of
which period the cell suspension was centrifuged (1,200 g for 1 min). The infranatant was
heated at 100°C for 20 min and then centrifuged at 10,000 g for 10 min. The supernatant was
assayed spectrophotometrically for glycerol.
Plasma insulin concentrations were measured by radioimmunoassay with a
kit from Phadeseph Pharmacia. Blood glucose concentrations during the
clamp were determined with a glucose analyzer (YSI, Yellow Springs,
OH). In other studies, blood glucose concentrations were measured in
deproteinized samples by the glucose oxidase method with kits supplied
by Boehringer Mannheim. Plasma NEFA concentrations were measured with a
spectrophotometric method with a WAKO
c-test kit (supplied by Alpha
Laboratories) according to the instructions of the manufacturer.
Results are expressed as means ± SE. Statistical comparisons were
made with StatView (Abacus Concepts, Berkeley, CA). Multiple comparisons were made by analysis of variance (ANOVA) and individual comparisons by Fisher's post hoc tests.
 |
RESULTS |
Exposure to the protein-restricted diet during fetal life and suckling
resulted in a 33% reduction (P < 0.001) in body weights of the female offspring at weaning (Fig.
1; see also Ref. 9). Body weights of the PR rats were
consistently lower than C rats from weaning to adulthood and, at 20 wk
of age, were ~14% lower than control. Weight differences between the
C and PR groups were maintained during high-fat feeding (Fig. 1), and
body weight gains after transfer to the high-fat diet, expressed as a
percentage of initial body weight, were unaffected by antecedent
protein restriction [C, 7.2 ± 1.1 % (n = 11); PR, 10.1 ± 1.0 % (n = 16)].

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Fig. 1.
Growth curves for control (C) and protein-restricted (PR) rats from
birth to adulthood and after subsequent transfer to high-fat diet. Rats
were maintained on either standard (20% protein) diet (C; ) or PR
(8% protein) diet ( ) from weaning until 20 wk of age and were then
transferred to a high-fat diet for 8 wk. Values are means ± SE for
8-10 rats. Effects of protein restriction were significant at all
time points studied (P < 0.01).
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The general metabolic characteristics of C and PR rats in the
postabsorptive state before and after transfer to the high-fat diet for
8 wk are shown in Table 2. Plasma insulin and blood glucose concentrations did not differ significantly between the groups
(C, PR, C-HF, and PR-HF). The insulin-to-glucose ratio, an index of the
relative response of insulin to fasting glycemia, was also not
significantly affected by protein restriction, either before or after
high-fat feeding. Kinetic studies, in which rats were infused with
[3-3H]glucose, were
undertaken to evaluate the effects of mild protein restriction on whole
body glucose turnover before and after challenge by the provision of
the high-fat diet. Rd values
(expressed per rat) in the postabsorptive state did not differ
significantly between PR rats and C rats before transfer to the
high-fat diet. Basal Rd values
also did not differ significantly between PR-HF and C-HF groups. GMR
values (which take into account differences in basal blood glucose
concentrations) also did not differ significantly between C and PR rats
before or after transfer to the high-fat diet.
To evaluate glucoregulatory control, blood glucose (Fig
2, A and
B) and plasma insulin concentrations
(Fig. 2, C and
D) were measured at intervals after
the intravenous administration of a glucose bolus (500 mg/kg body wt).
Protein restriction enhanced glucose tolerance (Fig.
2A) with a significantly higher
(1.5-fold, P < 0.01) K value for
glucose disappearance (Table 3), despite impaired
insulin secretion as demonstrated by significantly lower insulin
concentrations at 2 min (41%; P < 0.05) after intravenous glucose administration (Fig.
2C; see also Ref. 9). Blood glucose concentrations in PR-HF rats were significantly higher than in C-HF
rats at 15 min (by 36%, P < 0.05)
and 30 min (69%; P < 0.05) after
intravenous glucose administration (Fig.
2B). Plasma insulin concentrations
were significantly lower at 5 min (by 41%;
P < 0.01) and 10 min (29%;
P < 0.05) after intravenous glucose
administration in PR-HF, compared with C-HF, rats (Fig.
2D). Switching to the high-fat diet
for 8 wk resulted in a significant decrease in the K value (45%;
P < 0.001) and a significant
increase in the incremental area under the curve (IAUC) for glucose
(61%; P < 0.01) in rats previously
subjected to protein restriction, but it did not alter glucose
tolerance, the K value, or the IAUC for glucose in control rats (Table
3). As a consequence, whereas K values before high-fat feeding were
higher in PR compared with C rats, K values of PR-HF rats were
significantly lower (by 29%; P < 0.05) than those of C-HF rats (Table 3).

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Fig. 2.
Blood glucose (A,
B) and plasma insulin
(C,
D) during an intravenous glucose
tolerance test in C rats ( ) and PR rats ( ) before
(A,
C) and after
(B,
D) high-fat feeding. Further details
are given in METHODS. Values are means ± SE for 5-9 rats in each group. * Statistically
significant effects of protein restriction,
P < 0.05.
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Table 3.
Characteristics of intravenous glucose tolerance tests undertaken in
C and PR rats before and after transfer to HF diet for 8 wk
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Because it was not clear whether impaired glucose tolerance in PR-HF
rats was a consequence of impaired insulin secretion or insulin
resistance, whole body glucose kinetics were measured during
hyperinsulinemia. Insulin was infused into conscious, unrestrained rats
at a fixed rate to raise steady-state insulin concentrations approximately sixfold (Table 4). Clamped blood glucose
concentrations in the four groups were similar (Table 4). The
coefficients of variance for blood glucose concentrations were <15%
for all groups (results not shown). The steady-state GIRs required to
maintain euglycemia between 90 and 120 min of hyperinsulinemia were
similar in the C and PR rats. There were also no statistical
differences between GIR values for C-HF and PR-HF rats (Table 4), but
high-fat feeding resulted in a significant decline in GIR in both
groups [43% (P < 0.01) in
C-HF rats and 36% (P < 0.05) in
PR-HF rats].
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Table 4.
Whole body glucose kinetics during euglycemic hyperinsulinemia in C
and PR rats before and after transfer to HF diet for 8 wk
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Rd values, estimated at
steady-state hyperinsulinemia with
[3-3H]glucose,
expressed either relative to body weight (Fig. 3) or per
rat [C, 7.4 ± 0.7 mg · min
1
· rat
1
(n = 5); PR 6.3 ± 0.3 mg · min
1
· rat
1
(n = 6)], did not differ
significantly between C and PR rats before high-fat feeding. Plasma
insulin-glucose disposal relationships before
(A) and after
(B) high-fat feeding are shown in
Fig. 3. Before transfer to a high-fat diet, the gradients of the lines were similar in the C and PR groups, indicating that the response of
whole body glucose disposal to this degree of hyperinsulinemia is
unaffected by mild protein restriction.
Rd values after high-fat feeding
were significantly (30%; P < 0.01) lower in PR-HF rats than in C-HF rats. This arises as a
consequence of an almost twofold greater decline in insulin-stimulated
Rd (47%;
P < 0.001) in response to high-fat
feeding in previously PR rats than in the C group (27%;
P < 0.01). In contrast, there was
greater suppression of endogenous glucose production in the PR-HF group
compared with the C-HF group (results not shown).

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Fig. 3.
Responses of whole body glucose disposal rate
(Rd) to plasma insulin in C rats
( ) and PR rats ( ) before (A)
and after (B) high-fat feeding.
Further details are given in METHODS.
Values are means ± SE for 5 rats in each group.
Statistically significant effects of protein restriction,
P < 0.01.
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The sensitivity and responsiveness of adipose tissue lipolysis
(assessed from glycerol release) to stimulation by norepinephrine in C
rats and PR rats before and after transfer to the high-fat diet are
shown in Table 5. Basal rates of adipose
tissue lipolysis before and after high-fat feeding were unaffected by
protein restriction. Lipolysis was maximally stimulated by
norepinephrine at 1 µM for adipocytes from all four groups (results
not shown). With adipocytes from C rats, 0.1 µM norepinephrine led to
a 3.6-fold (P < 0.01) increase in
lipolysis. In contrast, the response of lipolysis to stimulation by 0.1 µM norepinephrine was increased by 17.2-fold (P < 0.001) in adipocytes prepared
from PR rats. Lipolysis rates at 1 µM norepinephrine were
approximately twofold higher (P < 0.001) with adipocytes from PR rats compared with adipocytes from C
rats. The increased sensitivity of lipolysis to stimulation by
norepinephrine at low concentrations and the increased responsiveness to norepinephrine at the maximal concentration were retained in adipocytes prepared from PR-HF rats compared with C-HF controls.
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Table 5.
Basal and norepinephrine-stimulated rates of adipose tissue lipolysis
(assessed from glycerol release) for C and PR rats before and after
HF feeding
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To assess the antilipolytic action of insulin, adipocytes prepared from
rats in each of the groups were incubated with norepinephrine at a
fixed concentration of 1 µM in the presence or absence of insulin at
75 µU/ml (a concentration comparable to that attained during the
insulin infusion studies in vivo) (Table 5). This concentration of
insulin produced 80 and 90% inhibition of norepinephrine-stimulated lipolysis in adipocytes prepared from C and PR rats, respectively. The
response to 75 µU/ml insulin was unimpaired in the C-HF group (80%
inhibition of lipolysis). In contrast, the response of
norepinephrine-stimulated lipolysis to suppression by 75 µU/ml
insulin was markedly and selectively blunted in adipocytes prepared
from the PR-HF rats, with only ~50% inhibition of
norepinephrine-stimulated lipolysis.
Dose-response curves for the antilipolytic action of insulin are shown
in Fig. 4. Adipocytes prepared from C and PR rats
responded to insulin with suppression of norepinephrine-stimulated
lipolysis at all insulin concentrations tested (Fig. 4,
A and
C). Insulin at concentrations from 5 to 75 µU/ml led to significant (P < 0.05) suppression of norepinephrine-stimulated rates of lipolysis
with adipocytes prepared from C-HF rats (Fig. 4,
B and
D). In contrast, insulin at 5 µU/ml was without significant effect on norepinephrine-stimulated lipolysis in adipocytes from PR-HF rats, and there was little or no
enhancement of the antilipolytic action of insulin when the insulin
concentration was increased from 10 to 75 µU/ml (Fig. 4B and
D). A significant
(P < 0.05) effect of insulin to
suppress lipolysis was observed only at 75 µU/ml insulin. As a
consequence, rates of lipolysis were significantly higher in the PR-HF
group compared with the C-HF group at all insulin concentrations
studied (Fig. 4B). The percentage
suppression of lipolysis was significantly less in the PR-HF group
(C-HF, 80%; PR-HF, 47%; P < 0.01)
(Fig. 4D); however, by virtue of the
higher response of lipolysis to norepinephrine stimulation in the PR-HF
group, the absolute decrease in glycerol release at the highest insulin
concentration studied (75 µU/ml) was similar in the C-HF and PR-HF
groups (Fig. 4B).

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Fig. 4.
Dose-response curves for effects of insulin on
norepinephrine-stimulated lipolysis in parametrial adipocytes from C
and PR rats, before (A,
C) and after 4 wk of high-fat
feeding (B,
D). Adipocytes prepared from C ( )
or PR ( ) rats were incubated with norepinephrine (1 µM) and
various concentrations of insulin as indicated. Rates of lipolysis were
assessed as glycerol release in parametrial adipocytes. Results are
expressed as absolute rates of lipolysis
(pg · h 1 · cell 1;
A and
B) and as percentages of maximal
norepinephrine-stimulated lipolysis (C
and D). Further details are given in
METHODS. Results are means ± SE
for 5 preparations of adipocytes, with individual incubations in
triplicate. Statistically significant effects of protein restriction,
P < 0.05;
P < 0.01;
* P < 0.001.
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Plasma NEFA concentrations in vivo did not differ significantly between
the C-HF and PR-HF groups in either the basal (postabsorptive) state
[C-HF, 0.69 ± 0.20 mM (n = 6); PR-HF, 0.42 ± 0.07 mM (n = 6)] or after insulin infusion [C-HF, 0.11 ± 0.02 mM
(n = 12); PR-HF, 0.10 ± 0.01 mM (n = 6)].
 |
DISCUSSION |
The present study was undertaken to evaluate whether antecedent protein
restriction predisposes to the later development of glucose intolerance
and insulin resistance. The effects of antecedent protein restriction
on whole body glucose homeostasis and insulin action were examined
after the dietary challenge of the provision of a high-fat diet for 8 wk. A previous study also demonstrated a modest, but not statistically
significant, loss of glucose tolerance after intraperitoneal glucose
administration after 4 wk of high-fat feeding in the adult offspring of
mothers fed a low (5%) protein diet during pregnancy and lactation
(28). The present results demonstrate that mild protein restriction
from conception to adulthood (20 wk of age) leads to significantly
enhanced tolerance to intravenous glucose, despite an impaired insulin
response and a greatly augmented and sensitized lipolytic response of
the adipocyte to norepinephrine, but does not predispose to either
glucose intolerance or impaired insulin action with respect to glucose
disposal. However, an effect of high-fat feeding to elicit a
deterioration in the action of insulin to stimulate peripheral glucose
disposal is exaggerated by antecedent protein restriction; Fig.
5 illustrates the more rapid and exaggerated decline in
the incremental insulin-stimulated increase in peripheral glucose
disposal elicited by the provision of a high-fat diet in rats
previously exposed to protein restriction. This effect is observed in
conjunction with a specific impairment of the antilipolytic response of
insulin in adipocytes and manifests as impaired glucose tolerance after
intravenous glucose administration. Although plasma NEFA concentrations
did not differ significantly between the C-HF and PR-HF groups in
either the basal (postabsorptive) state or after insulin infusion,
steady-state concentrations give no indication of relative flux. We
have demonstrated previously that an increased supply of fatty acid
leads to suppression of glucose utilization in vivo in a range of
skeletal muscles, implying accelerated fatty acid utilization (11), and
the present results are compatible with the concept that increased
supply and utilization of NEFA underlie the more rapid decline in
insulin-stimulated glucose disposal in the PR-HF group. The present
results therefore provide clear evidence that protein restriction from
conception to adulthood can accelerate and augment the development of
impaired glucoregulation and insulin resistance, but only in response
to the diabetogenic stimulus of high-fat feeding. The results are important within the context of previous studies that have failed to
demonstrate a deterioration of insulin action in rats subjected to
protein restriction alone and the role of subsequent dietary-lifestyle factors in determining the onset of impaired glucose tolerance.

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Fig. 5.
Progressive impact of high-fat feeding on the incremental increase in
whole body glucose disposal in response to hyperinsulinemia in C ( )
and PR ( ) rats. Further details are given in
METHODS. Values are means ± SE for
5 rats in each group. Statistically significant effects of
protein restriction, P < 0.01.
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Impaired insulin secretion and reduced insulin action are both thought
to contribute to the pathogenesis of type 2 (non-insulin-dependent) diabetes mellitus (2, 14, 22). The degree to which glucoregulatory control deteriorates varies as a function of the magnitude of loss of
in vivo insulin action in relation to the capacity of the
-cell to
compensate for this defect by insulin hypersecretion (21). Growth
retardation evoked by suboptimal protein nutrition during early life
leads to decreased basal glucose disposal and glucose utilization by a
range of tissues and the fetus during a subsequent pregnancy, but this
effect is not associated with any major permanent impairment of
glucose-stimulated insulin secretion or whole body insulin action at
day
19 of pregnancy (12), even though it
is recognized that late pregnancy is an insulin-resistant state (13,
17) (reviewed in Ref. 25). The physiological stimulus of pregnancy is
associated with an enhanced insulin secretory response to glucose (18)
(reviewed in Ref. 25), and this adaptation appears to be intact in
pregnant rats that have experienced early growth retardation (12). The
present study involves separate analyses of two dietary interventions
known to target either insulin secretion (protein restriction) or
insulin action (high-fat feeding). The results indicate that protein
restriction alone, while impairing insulin secretion, enhances whole
body and peripheral insulin action. As a consequence, there is no
adverse effect on glucose tolerance. Conversely, a low-carbohydrate,
high-fat diet leads to a modest impairment in peripheral insulin action
without a deterioration of glucose tolerance, because insulin secretion is enhanced (see also Ref. 9). However, the sequential imposition of
the two dietary interventions results in synergistic interactions, leading to a marked impairment in peripheral insulin action in combination with a blunted insulin secretory response, leading to
glucose intolerance. The study has relevance to the interpretation of
studies in humans demonstrating an increased incidence of diabetes on
change from a nutritionally poor diet to a high-energy diet containing
a relatively high percentage of lipid, characteristic of Western
affluent societies, and may also be germane to the hypothesis that
diseases that manifest in adult life, such as non-insulin-dependent
diabetes mellitus, may originate in infant or early life as a
consequence of poor early nutrition (for a recent review see Ref. 8).
This work was supported in part by funds from the British Diabetic
Association. We are grateful to Lee G. D. Fryer for adipocyte preparation and incubation.
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Address for reprint requests: M. J. Holness, Dept. of Biochemistry,
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Mile End Road, London E1 4NS, UK.