Instituto de Bioquímica, Centro Mixto Consejo Superior Investigaciones Científicas Universidad Complutense de Madrid, Facultad Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain
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ABSTRACT |
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Undernutrition in rats impairs secretion of insulin but maintains glucose normotolerance, because muscle tissue presents an increased insulin-induced glucose uptake. We studied glucose transporters in gastrocnemius muscles from food-restricted and control anesthetized rats under basal and euglycemic hyperinsulinemic conditions. Muscle membranes were prepared by subcellular fractionation in sucrose gradients. Insulin-induced glucose uptake, estimated by a 2-deoxyglucose technique, was increased 4- and 12-fold in control and food-restricted rats, respectively. Muscle insulin receptor was increased, but phosphotyrosine-associated phosphatidylinositol 3-kinase activity stimulated by insulin was lower in undernourished rats, whereas insulin receptor substrate-1 content remained unaltered. The main glucose transporter in the muscle, GLUT-4, was severely reduced albeit more efficiently translocated in response to insulin in food-deprived rats. GLUT-1, GLUT-3, and GLUT-5, minor isoforms in skeletal muscle, were found increased in food-deprived rats. The rise in these minor glucose carriers, as well as the improvement in GLUT-4 recruitment, is probably insufficient to account for the insulin-induced increase in the uptake of glucose in undernourished rats, thereby suggesting possible changes in other steps required for glucose metabolism.
undernutrition; muscle glucose transporters; insulin signaling
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INTRODUCTION |
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DIFFERENT STUDIES HAVE
SHOWN that deprivation of protein calories during early stages of
development has effects on glucose metabolism in adulthood, because
different aspects of this metabolism that can be affected by nutrition
are programmed during fetal and early postnatal periods, as indicated
by the "thrifty phenotype hypothesis" (19).
Early-malnourished humans and animals have reduced -cell secretory
responses as well as insulin resistance when normal food intake,
overnutrition, or high-fat feeding are established later in life.
Consequently, the development of type 2 diabetes may result (22,
23, 32). However, it is also well known that restriction in food
intake contributes to reduced insulin resistance in diabetics. In fact,
the restriction of protein calories imposed during limited periods of
time after suckling results in increases in insulin actions in humans
(14), rhesus monkeys (26), and rodents
(7-11, 16). The molecular mechanisms by which
food deprivation leads to changes in insulin sensitivity are not well
understood, and the development of animal models of caloric restriction
that resemble those of the human situation become important to study.
Diet protocols of food deprivation are commonly applied to animals for limited periods of their life span. We have previously established a rat model of undernutrition on the basis of a food restriction that begins in the fetal stage and continues until adulthood (12). This chronic deficiency better represents the condition of undernourished humans in developing countries. Food-restricted rats, according to this model, show normal glucose tolerance, despite the fact that the release of insulin is seriously impaired. Because skeletal muscle accounts for most of the whole body utilization of glucose, and white adipose tissue is severely reduced in these undernourished rats (12), an increased capacity to promote the uptake of glucose by muscle probably plays a major role in the enhanced insulin responses, as previously reported in other models of dietary restriction (7-9, 15).
The uptake of glucose depends on a facilitative glucose transporter family. GLUT-4, which is recruited to plasma membrane in response to insulin, is the main glucose carrier in skeletal muscle. Recent investigations have shown no effects of shorter dietary restriction on muscle GLUT-4 content in rats but a better recruitment after insulin (8, 9). Other GLUT isoforms, particularly GLUT-1, are also expressed in muscle fibers in a much lower proportion (reviewed in Ref. 41), and the effects of food restriction on them are not known at present. The goal of this work was to investigate the effect of chronic undernutrition on basal and insulin-induced glucose uptake in vivo in a representative skeletal muscle such as the gastrocnemius by means of a hyperinsulinemic euglycemic clamp and to correlate this uptake with the content of the different glucose transporters present in muscle. Key proteins in the insulin-signaling cascade can be affected by nutrition (32, 39). Consequently, another purpose of the present study was to assess the effects of chronic undernutrition on different transducers in insulin-mediated glucose transport, especially considering the activation of phosphatidylinositol (PI) 3-kinase by insulin in vivo, which is an essential step in the hormone stimulus on glucose transport (3).
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MATERIALS AND METHODS |
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Animals and diets. Wistar rats bred in our laboratory with controlled temperature and an artificial dark-light cycle (light from 0700 to 1900) were used throughout the study. Females were caged with males, and mating was confirmed by the presence of spermatozoa in vaginal smears. Each dam was housed individually from the 14th day of pregnancy. Food restriction was established from the 16th day of pregnancy. Control animals were fed a commercial standard laboratory diet ad libitum, containing by weight 19% protein, 56% carbohydrate (starch and sucrose), 3.5% lipid, 4.5% cellulose, 5% vitamin and mineral mix, and 12% water. Food-restricted animals were subjected to the following dietary pattern: pregnant rats received 10 g of the standard food daily until delivery. The number of pups in each litter was evened to eight. Lactating mothers received 15, 20, and 25 g of the standard diet daily during the 1st, 2nd, and 3rd wk of suckling, respectively. After weaning, only females were selected for this study. They received daily 35% of the diet consumed by controls until day 70 of their life. Water was given ad libitum. Food intake of control and undernourished rats has been previously reported (12).
Euglycemic insulin clamp.
These studies were performed in control rats ~15 h after removal of
food. In the undernourished group, they were performed 15 h after
the restricted amount of food had been consumed. Rats were anesthetized
with pentobarbital sodium (4 mg/100 g body wt), and after tracheotomy
(to prevent respiratory problems), one carotid artery was catheterized
for blood sampling. Once glycemia returned to the level observed before
anesthesia (~40 min), insulin (Actrapid; Novo, Copenhagen, Denmark)
was infused through a saphenous vein at a constant rate to reach an
insulin dose of 5.0 IU · h1 · kg
1. A solution
of glucose, 30 and 40% for control and food-restricted rats,
respectively, was also infused through the other saphenous vein 5 min
after the infusion of hormone was started. The difference in the
concentration of glucose was necessary to infuse similar final volumes
in both groups. The infusion rate was adjusted to clamp blood glucose
at the level present in the conscious animals. To achieve this rate,
blood samples were taken every 5 min from the carotid artery, blood
glucose was determined within 2 min using a Reflolux II glucose
analyzer (Boehringer Mannheim, Mannheim, Germany), and the pump dial
was adjusted according to the changes in the level of blood glucose.
Within 40 min of the start of the clamp, plasma insulin and glucose
levels remained constant without further adjustment of the pump dial.
At this steady state, insulin infusion was equal to insulin clearance,
and the overall glucose utilization reached a constant value. This
condition was maintained for 60 min, and then the rats were cervically
dislocated. The gastrocnemius muscle of both hindlimbs was quickly
excised, trimmed free of fat and connective tissue, freeze-clamped in
liquid N2, and stored at
80°C until assayed. The clamp
was also applied to a group of 35-day-old control rats that weighed
approximately the same as the 70-day-old undernourished rats to
quantify the glucose infusion rate to maintain euglycemia.
Estimation of glucose uptake. The uptake of glucose by gastrocnemius was estimated by measuring the accumulation of the phosphorylated form of the glucose analog 2-deoxy-D-glucose. A bolus of 80 µCi 2-deoxy-D-[1-3H]glucose (Amersham, Alesbury, UK) was injected intravenously 40 min after the clamp experiment was started, that is, under steady-state condition, as required by the theoretical model. The same bolus was administered 40 min after anesthesia to rats not infused with insulin to estimate basal uptake of glucose. Arterial blood was sampled for determination of the concentration of blood glucose and 2-deoxy-D-[1-3H]glucose radioactivity. At the end of the experiment, rats were killed, and gastrocnemius muscles were removed and stored as indicated. This tissue was digested at 60°C for 45 min in 1 M NaOH, and the 2-deoxy-D-[1-3H]glucose 6-phosphate content was determined as described previously (13). This method is based on the fact that both 2-deoxyglucose and 2-deoxyglucose 6-phosphate remain soluble in 6% HClO4 extracts, whereas 2-deoxyglucose 6-phosphate precipitates in the Somogyi reagent [BaSO4-Zn(OH)2]. The rate of glucose utilization was calculated by dividing the disintegrations per minute of 2-deoxy-D-[1-3H]glucose 6-phosphate in the tissue by the calculated integral of the ratio of arterial blood 2-deoxy-D-[1-3H]glucose to glucose concentration.
Muscle fractionation. The procedure used to isolate plasma and intracellular membranes was similar to that described by Gumá et al. (18), with some modifications. Approximately 6 g of muscle (for the undernourished rats, the two gastrocnemius muscles were pooled) were minced and homogenized at 4°C in a Polytron at low speed (setting 4.8) for 20 s in buffer A [20 mM HEPES, 0.15 M KCl, containing 1 µM leupeptin and 100 µM phenylmethylsulfonyl fluoride (PMSF) as protease inhibitors, pH 7.4]. A solution of KCl was then added to the homogenate to a final concentration of 0.65 M, and it was left on ice for 15 min. It was then centrifuged at 2,000 g for 10 min. The supernatant was collected and kept on ice. The pellet was resuspended in 7 ml of buffer A, rehomogenized as indicated above for 10 s, treated with the KCl solution, left on ice for 15 min, and centrifuged for 10 min at 2,000 g. The two supernatants were pooled and subjected to ultracentrifugation at 190,000 g for 1 h. The resulting pellet, which contained crude membranes, was resuspended using a tissue grinder in 3 ml of buffer (0.25 M sucrose, 10 mM NaHCO3, 5 mM NaN3, and 100 µM PMSF, pH 7.4). A 0.05-ml sample was removed for measurements of GLUT content, marker enzymes, and proteins. The rest was loaded onto the top of a discontinuous sucrose gradient, 25, 30, and 35% (wt/wt, in 20 mM HEPES, pH 7.4), and centrifuged for 16 h at 150,000 g.
Fractions were collected from the top of the 25% gradient (25% fraction) and from interphases 25-30% (30% fraction) and 30-35% (35% fraction). The pellet was also collected (35P fraction). All of the fractions were diluted 10-fold with buffer A and centrifuged at 190,000 g for 90 min. The resulting pellets were resuspended in 20 mM HEPES, pH 7.4. Proteins were assayed, and fractions were kept frozen atWestern blot analyses.
The fractions of muscle membrane were subjected to SDS-PAGE on
7-10% polyacrylamide gels according to Laemmli (28).
Proteins were then electrophoretically transferred to polyvinylidene
difluoride filters (PVDF protein sequencing membrane, Bio-Rad
Laboratories, Alcobendas, Spain) for 2 h. After transfer,
the filters were blocked with 5% (wt/vol) nonfat dry milk in
phosphate-buffered saline with 3% bovine serum albumin and 0.02%
sodium azide. Antibodies against the GLUT-1 and GLUT-4 glucose
transporters were purchased from Biogenesis (Sandown, NH) and were used
at dilutions 1:5,000 and 1:1,000, respectively. Antibodies against
GLUT-3 and GLUT-5 (1:2,500 dilutions) were obtained from Chemicon
(Temecula, CA). Anti-insulin receptor, -subunit, and anti-rat
1-subunit of N+-K+-ATPase
(Upstate Biotechnology, Lake Placid, NY) were diluted at 1:250. The
PVDF filters were next washed four times for 10 min at 37°C with
phosphate-buffered saline with 0.1% Tween 20, followed by a 1-h
incubation with goat anti-rabbit immunoglobulin G conjugated to
horseradish peroxidase (Sigma BioSciences, St. Louis, MO). The PVDF
membranes were then washed as already indicated. Detection of
antibody-antigen complexes was accomplished by the enhanced
chemiluminescence method (BM Chemiluminescence, Boehringer Mannheim,
Mannheim, Germany). Optical density of bands was determined by laser
scanning densitometry (Molecular Dynamics, Sunnyvale, CA).
RNA isolation and Northern blot analysis.
RNA was extracted from gastrocnemius (500 mg) obtained from rats in the
basal condition, by use of the guanidinum
isothiocyanate-phenol-chloroform method (6). After
quantification, total RNA (30 µg) was subjected to Northern blot
analysis, following the method previously described (38).
A 2.47-kb rat GLUT-4 cDNA cloned into the EcoRI site of pBluescript KS+ (Stratagene, Merck Farma y Química, Barcelona, Spain) and a 2.6-kb rat GLUT-1 cDNA insert subcloned from prGT3 into pBluescript KS at the EcoRI site (Promega
Innogenetics Diagnostica y Tecnología, Spain) were
kindly provided by Dr. A. Zorzano (Dept. of Biochemistry and Molecular
Biology, University of Barcelona, Barcelona, Spain) and were used as
probes. Membranes were autoradiographed, and relative densities of
signals were determined by densitometric scanning of the autoradiograms
in a laser densitometer.
PI 3-kinase assay.
To determine insulin-stimulated PI 3-kinase, rats were
anesthetized with pentobarbital sodium as indicated, the abdominal cavity was opened, the portal vein was exposed, and 5 IU of insulin were injected. After 90 s, gastrocnemius muscles were quickly removed and freeze-clamped with liquid N2 and stored at
80°C until assayed. Muscles (100 mg) from basal and
insulin-injected rats were homogenized with a Polytron operated at
maximum speed in 1 ml of lysis buffer, composed of 50 mM HEPES (pH
7.4), 1% Triton X-100, 50 mM sodium pyrophosphate, 100 mM sodium
fluoride, 10 mM EDTA, 10 mM sodium vanadate, 2 mM PMSF, 2 mM
benzamidine, and 20 µM leupeptin. The homogenates were left on ice
for 30 min and then subjected to centrifugation at 180,000 g
for 60 min at 4°C. The supernatants were used as samples for protein
and PI 3-kinase determinations. Aliquots containing 2 mg of protein
were immunoprecipitated with monoclonal anti-phosphotyrosine antibody (Santa Cruz Quimigranel, Madrid, Spain), and immunocomplexes were collected with anti-mouse IgG agarose (Sigma BioSciences).
Determination of insulin receptor substrate-1. Gastrocnemius muscles from rats in the basal state were extracted as aforementioned, and samples containing 0.5 mg of protein were immunoprecipitated with polyclonal anti-rat insulin receptor substrate-1 (IRS-1; Upstate Biotechnology). The complexes were bound to anti-mouse IgG agarose as described. The agarose beads were treated with Laemmli sample buffer with 100 mM dithiothreitol at 95°C for 5 min and subjected to SDS-PAGE (6%). The rest of the Western blot procedure was performed as described for GLUT determinations, with polyclonal anti-rat IRS-1 as primary antibody, diluted at 1 µg/ml.
Other analytical procedures.
The concentration of protein was determined by the Bradford method
(2) with the Bio-Rad protein assay and with -globulin as standard. The specific activity of phosphodiesterase-I was assayed
as a plasma membrane marker (27). Plasma insulin was determined by RIA with rat insulin as standard (Incstar, Stillwater, MN). This method allows the determination of 2.0 ng/ml, with a coefficient of variation within and between assays of 10%.
Expression of the results. All of the data are reported as means ± SE. A difference between two mean values was assessed with the Student's t-test. For multiple comparisons, significance was evaluated by ANOVA, followed by the protected least significant difference test.
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RESULTS |
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Body weight of food-restricted rats was reduced to 50%
compared with the control value at 70 days of life (Table
1). No differences in blood glucose
levels were found between control and food-restricted rats at the
moment in which the clamps were performed. However, plasma insulin in
the group subjected to a restricted diet was 70% of that in the
control group. During the clamp, plasma insulin was raised to the same
level in both groups. In this condition, the rate of glucose infusion
required to maintain euglycemia was significantly higher in the
undernourished rats than in the controls (Table 1). A group of
35-day-old control rats, whose body weights (88.6 ± 3.3 g)
were not significantly different from those of the undernourished, was
subjected to a clamp in the same conditions. The rate of glucose
infusion to maintain euglycemia in this group was 26.6 ± 2.1 mg · min1 · kg
1, which is
similar to that obtained for the 70-day-old control rats (Table 1). The
uptake of 2-deoxyglucose in the food-deprived rats in the basal state
was 50% that of controls. After insulin, glucose utilization
was activated in the two groups of rats. However, the food-restricted
group underwent a remarkably higher increase (12- vs. 4-fold), and
their values exceeded those of controls (Table 1).
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Undernutrition did not affect the recoveries of protein in the
fractions of membrane obtained from gastrocnemius (Table
2). Specific activities of the enzyme
marker phosphodiesterase-I were 7- to 15-fold higher in the fraction
enriched in the plasma membranes (25% sucrose) compared with those in
the crude membranes. No changes in this enzyme activity were produced
by insulin, whereas undernutrition significantly increased
phosphodiesterase-I activity (Table 2).
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The 1-subunit of Na+-K+-ATPase
was barely detected in the fraction enriched in intracellular
membranes, being predominant in the plasma membranes. Insulin did not
affect the content of this protein marker (Fig.
1A).
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The distribution of GLUT-4 and GLUT-1 is shown in Fig. 1B.
Every fraction of membrane contained significant amounts of GLUT-4. However, when the proteins recovered were considered (Table 2), this
carrier isoform was found predominantly in intracellular membranes. In
contrast, GLUT-1 was mainly collected in the plasma membrane fraction
(Fig. 1B). As shown in Fig.
2A, the content of GLUT-1 in
plasma membranes was increased 2- to 3.5-fold in food-restricted rats
above control values, without changes after insulin treatment. The
whole content of GLUT-4 underwent a remarkable decrease (~70%) after
undernutrition, as shown in Fig. 2B. When gastrocnemius
muscle was fractionated, this decrease was evident in plasma as well as
in intracellular membranes (Fig. 3). In
response to insulin, an increase in GLUT-4 present in plasma membranes was concomitant to a decrease in the intracellular content in both
groups of rats. However, the relative quantity of GLUT-4 translocated
was different: whereas a 1.6-fold increase in plasma membrane and a
1.3-fold decrease in intracellular membrane with respect to the
contents found in the basal state were observed in control rats, a
3.0-fold increase and a 3.3-fold decrease, respectively, were observed
in those food deprived (Fig. 3).
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Figure 4 depicts representative
experiments showing the Northern blot analysis of muscle GLUT-4 and
GLUT-1 mRNA. The GLUT-4 mRNA content was decreased in gastrocnemius
muscle of undernourished rats compared with controls. In contrast,
GLUT-1 mRNA remained unchanged. The mean content of mRNA corresponding
to -actin, used for normalization, was similar in both groups.
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The presence of GLUT-3 and GLUT-5 was barely evident in muscle crude
membranes of control rats (Fig. 5).
However, both protein transporters underwent large increases after food
restriction: three- and sixfold above the control values for GLUT-3 and
GLUT-5, respectively. Insulin receptor (-subunit) was significantly
increased in gastrocnemius muscles of undernourished rats, as shown in
Fig. 6A. However, food
restriction did not alter the muscle content of IRS-1 (Fig.
6B).
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Anti-phosphotyrosine immunoprecipitable PI 3-kinase activities were the
same in both groups of rats in the basal state. The enzyme was
remarkably stimulated 90 s after a single insulin dose (see
MATERIALS AND METHODS), although the peak was significantly smaller in undernourished rats than in controls. In the
hyperinsulinemic condition established for 60 min (clamp technique), PI
3-kinase was decreased compared with the levels found immediately after insulin injection, and again enzyme activity was lower in the food-restricted rats (Fig. 7).
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DISCUSSION |
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In a previous study (12), we showed that rats
subjected to undernutrition from the fetal stage until adulthood are
glucose tolerant, despite the fact that food restriction alters
-cell function (22, 30) and induces hypoinsulinemia.
This result is linked to the fact that both muscle and adipose tissues
from chronically undernourished rats present a compensatory increase in
insulin-induced glucose uptake (12). Because the livers of food-restricted rats seem to undergo insulin resistance and the amount
of adipose tissue is largely reduced, the increased glucose disposal in
vivo after insulin treatment observed in the present as well as in the
previous study (12) must be contributed mainly by skeletal
muscle. In fact, gastrocnemius muscle from these rats exhibits a much
greater increment in uptake of 2-deoxyglucose under the euglycemic
insulin clamp than in their controls. A similar insulin-sensitizing
effect of calorie restriction on the transport of muscle glucose has
been found in rats and mice subjected to reduced food consumption for
shorter periods (7, 9, 15, 25). However, in such cases,
the uptake of basal glucose by incubated muscles remains unaltered,
whereas it is significantly diminished in vivo in the present model of
chronic undernutrition. Because a primary regulated step in glucose
utilization is the membrane transport, the possibility arises that this
discrepancy could result from a different effect of the type of food
deprivation on the amount and/or functional activity of muscle glucose
carriers. The abundance of GLUT-4 is not altered in skeletal muscle
from rats or rhesus monkeys subjected to caloric restrictions without malnutrition (8, 9, 16). Consequently, we studied this and
other glucose carrier isoforms in gastrocnemius muscles from chronically undernourished rats, analyzing specifically the subcellular distribution of GLUT-4 and GLUT-1.
Undernutrition leads to a large decrease in GLUT-4 protein and mRNA in skeletal muscle. On the other hand, GLUT-1 is significantly increased without changes in the corresponding mRNA. The decline in GLUT-4 probably leads to the impaired basal uptake of glucose shown in undernourished rats, which would not be sufficiently compensated for by the rise in GLUT-1. Although it is currently accepted that GLUT-1 is the main carrier for unstimulated glucose transport, our results support the idea that GLUT-4 might also be important in mediating this transport, as recently proposed by other authors (16). In favor of this idea, it could be argued that GLUT-4 intrinsic activity is higher than that of GLUT-1 (21), which accounts for only a minor part of total glucose transporters (41) and seems to be primarily present in the perineural sheaths rather than in muscle fiber (20). Moreover, although GLUT-4 is located mainly in intracellular membranes in the basal state, our data as well as those of the others (31) show a significant presence of this carrier in the plasma membrane in such a condition, probably due to the exposure of muscle fibers to basal insulinemia and tonal contraction, both inducers of GLUT-4 translocation (29).
Because the muscle uptake of glucose is increased in hyperinsulinemic, undernourished rats over the control values, we studied insulin's effect on the translocation of GLUT-4 from the intracellular to the plasma membrane. When the decrease in the abundance of GLUT-4 in the chronically food-restricted rats is taken into account, the ability of insulin to recruit this carrier seems to be improved by undernutrition. An increase in GLUT-4 translocation in response to insulin has also been reported in skeletal muscle from calorie-restricted rats, and in this case, it leads to a higher amount of this carrier in the plasma membrane (9). However, in the present undernutrition model, the final amount of GLUT-4 located at the cell surface in hyperinsulinemic state is still lower than in control rats. Consequently, we thought that merely a better GLUT-4 translocation cannot be sufficient to explain the insulin-sensitizing effect of chronic undernutrition on the muscle glucose uptake. Thus we studied other GLUT isoforms to explore whether they could explain the enhanced muscle uptake of glucose. Undernutrition led to an increase in both GLUT-3, which is barely detectable in control rats, and GLUT-5, which is mainly a fructose carrier but has a low capacity to transport glucose (17). To the best of our knowledge, we have established the first evidence that GLUT-5 is present in rat gastrocnemius muscle and that it is enhanced by undernutrition. Because fructose contributes to carbohydrate metabolism in muscle (40), the increase in GLUT-5 could be an adaptation to the chronic reduction in food consumption, perhaps to favor a better utilization of fructose supplied in the diet. In any case, we think that the improvement in muscle insulin-induced glucose uptake, as seen in this work, cannot be accounted for by increases in these minor carrier isoforms. The possibility arises from the fact that chronic undernutrition will affect the GLUTs' intrinsic activity, although this parameter is not altered in rats food restricted for limited periods of life (9).
Improvements in the uptake of glucose might be secondary to several systemic changes. One of them could be the basal rate of blood flow, which is also increased by insulin (33). This hormonal effect is improved in muscle after physical training (37). A similar change elicited by undernutrition, which in turn would affect the uptake of glucose, may also be considered to explain our results. Plasma fatty acids can also influence insulin sensitivity (5, 36), but in a previous study (12), we did not find differences between control and undernourished rats in plasma fatty acids and ketone body. Changes in counterregulatory hormones might improve insulin actions in food-restricted rats. However, increased insulin-induced glucose uptake has been reported in isolated muscle preparations in vitro from rodents submitted to dietary restriction (7-9, 15), a result which seems to be consistent with our in vivo results.
Nonetheless, few studies have determined the effects of undernutrition on the insulin-signaling pathways, an important question to identify the cellular mechanism that could explain the improved muscle insulin sensitivity. Therefore, in the present work, we have shown that this condition upregulates the insulin receptor, as occurs in prolonged fasting (34). An increase in tyrosine-phosphorylated insulin receptor has been reported in gastrocnemius from calorie-restricted rats injected with insulin (10). In the present model, undernutrition did not alter muscle IRS-1 content, whereas it decreased in rodents subjected to calorie restriction for 20 days (9, 15). Despite the increase in GLUT-4 recruitment, we found that insulin activation on PI 3-kinase, which plays an important role in this recruitment (3), was partially impaired in undernourished rats, in contrast with the lack of changes after shorter periods of food restriction (9, 10). These results could indicate, as previously suggested by others (10), that the improved uptake of glucose secondary to undernutrition does not necessarily involve a concomitant change in the PI 3-kinase sensitivity to insulin. An alternative explanation could be based on the fact that PI 3-kinase is compartmentalized in adipocytes (35), which opens up the possibility of a similar situation in the muscle and suggests different effects of undernutrition on distinct PI 3-kinase pools. The increase in insulin action secondary to undernutrition could also be derived from alterations in other pathways related to the effects of insulin on the uptake of glucose. Food restriction may affect protein kinase C isoforms involved in insulin signaling, leading to GLUT-4 translocation in muscle primary cultures (4) or influence c-Cbl-associated proteins, recently proposed as another pathway (PI 3-kinase independent) to recruit GLUT-4 (1). Finally, a new glucose carrier, designated GLUT-X1 (24) may be enhanced by food restriction. This carrier seems not to be involved in basal uptake of glucose, which is decreased in our undernourished rats.
An understanding of the adaptive mechanisms elicited to compensate for the partial loss of GLUT-4 when glucose normotolerance is maintained could be beneficial in the treatment of insulin resistance states in the long run. To this effect, undernutrition represents a useful experimental model to be added to others currently studied.
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ACKNOWLEDGEMENTS |
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We are grateful to Dr. M. A. Martín for help in some of the studies reported in this article. We especially thank Drs. M. Lorenzo and R. Conejo for the Northern blot analyses included in the present work.
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FOOTNOTES |
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This work was supported by a grant from Dirección General de Investigación Científica y Técnica, Ministerio de Educación y Cultura, Spain, reference no. PM97-0017.
Address for reprint requests and other correspondence: F. Escrivá, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain (E-mail: fescriva{at}eucmos.sim.ucm.es).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 1 December 2000; accepted in final form 9 July 2001.
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REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Baumann, CA,
Ribon V,
Kanzaki M,
Thurmond DC,
Mora S,
Shigematsu S,
Bickel PE,
Pessin JE,
and
Saltiel AR.
CAP defines a second signalling pathway required for insulin stimulated glucose transport.
Nature
407:
202-207,
2000[ISI][Medline].
2.
Bradford, MM.
A rapid and sensitive method for the quantification of microgram quantities of protein, utilizing the principle of protein-dye binding.
Anal Biochem
72:
248-254,
1976[ISI][Medline].
3.
Brady, JB,
Pessin JE,
and
Saltiel AR.
Spatial compartmentalization in the regulation of glucose metabolism by insulin.
Trends Endocrinol Metab
10:
408-413,
1999[ISI][Medline].
4.
Braiman, L,
Shefi-Friedman L,
Bak A,
Tennebaum T,
and
Sampson SR.
Tyrosine phosphorylation of specific protein kinase C isoenzymes participates in insulin stimulation of glucose transport in primary cultures of rat skeletal muscle.
Diabetes
48:
1922-1929,
1999[Abstract].
5.
Boden, G.
Role of fatty acids in the pathogenesis of insulin resistance and NIDDM.
Diabetes
45:
3-10,
1996.
6.
Chomczynski, P,
and
Sacchi N.
Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction.
Anal Biochem
162:
156-159,
1987[ISI][Medline].
7.
Cartee, GD,
and
Dean DJ.
Glucose transport with brief dietary restriction: heterogenous responses in muscles.
Am J Physiol Endocrinol Metab
266:
E946-E952,
1994
8.
Cartee, GD,
Kietzke EW,
and
Briggs-Tung C.
Adaptation of muscle glucose transport with caloric restriction in adult, middle-aged, and old rats.
Am J Physiol Regulatory Integrative Comp Physiol
266:
R1443-R1447,
1994
9.
Dean, DJ,
Brozinick JTJR,
Cushman SW,
and
Cartee GD.
Calorie restriction increases cell surface GLUT-4 in insulin-stimulated skeletal muscle.
Am J Physiol Endocrinol Metab
275:
E957-E964,
1998
10.
Dean, DJ,
and
Cartee GD.
Calorie restriction increases insulin-stimulated tyrosine phosphorylation of insulin receptor and insulin receptor substrate-1 in rat skeletal muscle.
Acta Physiol Scand
169:
133-139,
2000[ISI][Medline].
11.
Escrivá, F,
Kergoat M,
Bailbé D,
Pascual-Leone AM,
and
Portha B.
Increased insulin action in the rat after protein malnutrition early in life.
Diabetologia
34:
559-564,
1991[ISI][Medline].
12.
Escrivá, F,
Rodríguez C,
Cacho J,
Alvarez C,
Portha B,
and
Pascual-Leone AM.
Glucose utilization and insulin action in adult rats submitted to prolonged food restriction.
Am J Physiol Endocrinol Metab
263:
E1-E7,
1992
13.
Ferré, P,
Leturque A,
Burnol AF,
and
Girard J.
A method to quantify glucose utilization in vivo in skeletal muscle and white adipose tissue of the anesthetized rat.
Biochem J
228:
103-110,
1985[ISI][Medline].
14.
Friedman, JE,
Dohm GL,
Legget-Frazier N,
Elton CW,
Tapscott EB,
Pories WP,
and
Caro JF.
Restoration of insulin responsiveness in skeletal muscle of morbidly obese patients after weight loss.
J Clin Invest
89:
701-705,
1992[ISI][Medline].
15.
Gazdag, AC,
Dumke CL,
Kahn CR,
and
Cartee GD.
Calorie restriction increases insulin-stimulated glucose transport in skeletal muscle from IRS-1 knockout mice.
Diabetes
48:
1930-1936,
1999[Abstract].
16.
Gazdag, AC,
Sullivan S,
Kenmitz JW,
and
Cartee GD.
Effect of long-term caloric restriction on GLUT4, phosphatidylinositol-3 kinase, p85 subunit, and insulin receptor substrate 1 protein levels in rhesus monkey skeletal muscle.
J Gerontol A Biol Med Sci
55:
B44-B46,
2000[ISI].
17.
Gould, GW,
and
Holman GD.
The glucose transport family: structure, function and tissue-specific expression.
Biochem J
295:
329-341,
1993[ISI][Medline].
18.
Gumá, A,
Zierath JR,
Wallberg-Henriksson H,
and
Klip A.
Insulin induces translocation of GLUT-4 glucose transporters in human skeletal muscle.
Am J Physiol Endocrinol Metab
268:
E613-E622,
1995
19.
Hales, CN,
and
Barker DJP
Type 2 (non-insulin-dependent) diabetes mellitus: the thrifty hypothesis.
Diabetologia
35:
595-601,
1992[ISI][Medline].
20.
Handberg, A,
Kayser L,
Hoyer PE,
and
Vinten J.
A substantial part of GLUT-1 in crude membranes from muscle originates from perineural sheaths.
Am J Physiol Endocrinol Metab
262:
E721-E727,
1992
21.
Holman, GD,
Kozka IJ,
Clark AE,
Flower C,
Saltis J,
Habberfield AD,
Simpson IA,
and
Cushman SW.
Cell surface labeling of glucose transporter isoform GLUT-4 by bismannose photolabel. Correlations with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester.
J Biol Chem
265:
18172-18179,
1990
22.
Holness, MJ,
Langdown ML,
and
Sugden MC.
Early-life programming of susceptibility to dysregulation of glucose metabolism and the development of type 2 diabetes mellitus.
Biochem J
349:
657-665,
2000[ISI][Medline].
23.
Holness, MJ,
and
Sugden MC.
Antecedent protein restriction exacerbates development of impaired insulin action after high-fat feeding.
Am J Physiol Endocrinol Metab
276:
E85-E93,
1999
24.
Ibberson, M,
Uldry M,
and
Thorens B.
GLUTX1, a novel mammalian glucose transporter expressed in the central nervous system and insulin-sensitive tissues.
J Biol Chem
275:
4607-4612,
2000
25.
Ivy, JL,
Young JC,
Craig BW,
Kohrt WM,
and
Holloszy JO.
Ageing, exercise and food-restriction: effects on skeletal muscle glucose uptake.
Mech Ageing Dev
61:
123-133,
1991[ISI][Medline].
26.
Kemmitz, JW,
Roecker EB,
Weindruch R,
Elson DF,
Baum ST,
and
Bergman RN.
Dietary restriction increases insulin sensitivity and lowers blood glucose in rhesus monkeys.
Am J Physiol Endocrinol Metab
266:
E540-E547,
1994
27.
Klip, A,
and
Walker D.
The glucose transport system of muscle plasma membranes: characterization by means of [3H]cytochalasin B binding.
Arch Biochem Biophys
221:
175-187,
1983[ISI][Medline].
28.
Laemmli, UK.
Cleavage of structural proteins during assembly of the head of bacteriophage T4.
Nature
227:
680-685,
1970[ISI][Medline].
29.
Lund, S,
Holman GD,
Schmitz O,
and
Pedersen O.
Contraction stimulates translocation of glucose transporter GLUT4 in skeletal muscle through a mechanism distinct from that of insulin.
Proc Natl Acad Sci USA
92:
5817-5821,
1995
30.
Martín, MA,
Alvarez C,
Goya L,
Portha B,
and
Pascual-Leone AM.
Insulin secretion in adult rats that had experienced different underfeeding patterns during their development.
Am J Physiol Endocrinol Metab
272:
E634-E640,
1997
31.
Muñoz, P,
Rosemblatt M,
Testar X,
Palacín M,
and
Zorzano A.
Isolation and characterization of distinct domains of sarcolemma and T-tubules from rat skeletal muscle.
Biochem J
307:
273-280,
1995[ISI][Medline].
32.
Ozanne, SE.
Programming of hepatic and peripheral tissue insulin sensitivity by maternal protein restriction.
Biochem Soc Trans
27:
94-97,
1998[ISI].
33.
Pendergrass, M,
Koval J,
Vogt C,
Yki-Järvinen H,
Iozzo P,
Pipek R,
Ardehali H,
Printz R,
Granner D,
DeFronzo RA,
and
Mandarino LJ.
Insulin-induced hexokinase II expression is reduced in obesity and NIDDM.
Diabetes
47:
387-394,
1998[Abstract].
34.
Saad, MJA,
Araki E,
Miralpeix M,
Rothenberg PL,
White MF,
and
Kahn CR.
Regulation of insulin receptor substrate 1 in liver and muscle of animal models of insulin resistance.
J Clin Invest
90:
1839-1849,
1992[ISI][Medline].
35.
Sharon, FC,
Molero JC,
and
James DE.
Release of insulin receptor substrate proteins from an intracellular complex coincides with the development of insulin resistance.
J Biol Chem
275:
3819-3826,
2000
36.
Shulman, GI.
Cellular mechanisms of insulin resistance.
J Clin Invest
106:
171-176,
2000
37.
Stallknecht, B,
Larsen JJ,
Mikines KJ,
Simonsen L,
Bülow J,
and
Galbo H.
Effect of training on insulin sensitivity of glucose uptake and lipolysis in human adipose tissue.
Am J Physiol Endocrinol Metab
279:
E376-E385,
2000
38.
Valverde, AM,
Navarro P,
Teruel T,
Conejo R,
Benito M,
and
Lorenzo M.
Insulin and insulin-like growth factor I up-regulate GLUT4 gene expression in fetal brown adipocytes in a phosphoinositide 3-kinase-dependent manner.
Biochem J
337:
397-405,
1999[ISI][Medline].
39.
Zierath, JR,
Houselknecht KL,
Gnudi L,
and
Kahn BB.
High-fat feeding impairs insulin-stimulated GLUT4 recruitment via an early insulin-signaling defect.
Diabetes
46:
215-223,
1997[Abstract].
40.
Zierath, JR,
Nolte LA,
Wahlström E,
Galuska D,
Shepherd PR,
Kahn BB,
and
Walberg-Henriksson H.
Carrier-mediated fructose uptake significantly contributes to carbohydrate metabolism in human skeletal muscle.
Biochem J
311:
517-521,
1995[ISI][Medline].
41.
Zorzano, A,
Fandos C,
and
Palacín M.
Role of plasma membrane transporters in muscle metabolism.
Biochem J
349:
667-688,
2000[ISI][Medline].