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Endocrinology Vol. 146, No. 8 3368-3378
Copyright © 2005 by The Endocrine Society

Maternal Food Restriction Enhances Insulin-Induced GLUT-4 Translocation and Insulin Signaling Pathway in Skeletal Muscle from Suckling Rats

M. L. Gavete, M. A. Martín, C. Alvarez and F. Escrivá

Instituto de Bioquímica (Consejo Superior de Investigaciones Centíficas-Universidad Complutense de Madrid), Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain

Address all correspondence and requests for reprints to: Dr. Fernando Escrivá, Departamento de Bioquímica y Biología Molecular, Facultad de Farmacia, Universidad Complutense, Ciudad Universitaria, 28040 Madrid, Spain. E-mail: fescriva{at}farm.ucm.es.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Restriction of protein calories during stages of immaturity has a major influence on glucose metabolism and increases the risk of type 2 diabetes in adulthood. However, it is known that reduction of food intake alleviates insulin resistance. We previously demonstrated an improved insulin-induced glucose uptake in skeletal muscle of chronically undernourished adult rats. The purpose of this work was to investigate whether this condition is present during suckling, a period characterized by physiological insulin resistance as well as elucidate some of the underlying mechanisms. With this aim, 10-d-old pups from food-restricted dams were studied. We showed that undernourished suckling rats are glucose normotolerants, despite their depressed insulin secretion capacity. The content of the main glucose transporters in muscle, GLUT-4 and GLUT-1, was not affected by undernutrition, but fractionation studies showed an improved insulin-stimulated GLUT-4 translocation. p38MAPK protein, implicated in up-regulation of intrinsic activity of translocated GLUT-4, was increased. These changes suggest an improved insulin-induced glucose uptake associated with undernutrition. Insulin receptor content as well as that of both regulatory and catalytic phosphoinositol 3-kinase subunits was increased by food restriction. Insulin receptor substrate-1-associated phosphoinositol 3-kinase activity after insulin was enhanced in undernourished rats, as was phospho-glycogen synthase kinase-3, in line with insulin hypersensitivity. Surprisingly, protein tyrosine phosphatase-1B association with insulin receptor was also increased by undernutrition. These adaptations to a condition of severely limited nutritional resources might result in changes in the development of key tissues and be detrimental later in life, when a correct amount of nutrients is available, as the thrifty phenotype hypothesis predicts.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A SERIES OF epidemiological studies carried out worldwide indicate that undernutrition during critical stages of immaturity, especially in utero, is associated with increased risk for the development of impaired glucose tolerance and type 2 diabetes in adult life (1, 2). Although the biochemical basis of these deleterious consequences are not well defined, it has been indicated that a poor nutrition during gestation or lactation results in metabolic changes in the offspring, which can lead to permanent (so called programmed) alterations in the structure and/or function of certain organs and tissues. The thrifty phenotype hypothesis proposed by Hales and Barker (3) postulates that these changes occur to limit the use of nutrients by certain tissues to ensure sufficient supply of them to the brain and other vital organs. The endocrine pancreas is particularly affected by this adaptation; an inappropriate development of islets of Langerhans and ß-cells is a major factor in the etiology of type 2 diabetes (4). Early-malnourished animals have reduced ß-cell secretory responses as well as insulin resistance when normal food intake, overnutrition, or high-fat feeding, as shown in humans, are established later in life. Consequently, the development of type 2 diabetes may result (5). Permanent adaptations to a restricted diet imposed in immature periods may be unsuitable and therefore detrimental when the organism subsequently experiences a correct nutrition (2).

However, many studies have also shown that moderate calorie reduction imposed chronically or during limited periods of time results in an increased insulin action in humans (6), rhesus monkeys (7), and rodents (8, 9). In fact, it is well known that reduction in energy intake and weight loss are important therapeutic objectives for patients with type 2 diabetes because both performances are associated with decreased insulin resistance and better blood glucose tolerance (10). Evidence suggests that this beneficial effect of food restriction is largely due to improved muscle glucose use. In line with this, we previously demonstrated that insulin-induced glucose uptake is increased in both skeletal muscle and heart in a rat model of protein-calorie undernutrition based on a food restriction that begins in the fetal stage and continues until adulthood (11, 12). These conditions of malnutrition are comparable with those commonly seen in poorly nourished human populations, in particular of developing countries.

Most previous research in this area has investigated the effects of maternal undernutrition on the offspring in adulthood. Only a few studies have been devoted to the repercussions of undernutrition on glucose homeostasis during development, when crucial metabolic adaptations take place in mammals to sustain their characteristic high growth rate. Muscle glucose use is reduced, whereas liver glucose production is compulsory during suckling when compared with the adult period because milk does not meet all glucose requirements of other neonatal tissues, such as the brain (reviewed in Ref. 13).

The present work investigated whether the improved muscle insulin response that we previously demonstrated in chronically undernourished adult rats (12) had already been established during suckling period. The offspring from restricted dams, as well as their controls, were studied on d 10 of our present investigation. We performed glucose tolerance tests and analyzed two main glucose transporter present in muscle, GLUT-4 and GLUT-1, because the uptake of glucose depends on these facilitative carriers. After insulin stimulation, GLUT-4 was translocated to plasma membrane (reviewed in Ref. 14) and as our previous work showed, food restriction increased this response (11); we therefore also studied GLUT-4 subcellular redistribution. The effects of a poor nutritional status during suckling on key steps of insulin signaling were incompletely defined, and, accordingly, we investigated whether they were influenced by undernutrition. Furthermore, it is well known that insulin receptor and its substrates, which are activated by tyrosine phosphorylation, can be inhibited by specific protein tyrosine phosphatases (PTPases) (15). Although a number of PTPases are potentially involved in the physiological regulation of the insulin action pathway, PTP1B has been suggested to be of most importance, and even a putative role in the insulin resistance associated with type 2 diabetes has been postulated for this phosphatase (16). Therefore, in the present work, the possible effects of undernutrition on PTP1B were also studied. Our results showed that food restriction established on dams had significant effects in the suckling rats, namely a deep reduction in insulin secretion capacity and a sensitization of skeletal muscle to insulin responses, the latter being associated with marked increases in a number of steps in insulin signaling pathway. These results explain why the malnourished developing rats are able to maintain normal glucose tolerance, despite reduced insulin secretion.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals and diets
Wistar rats bred in our laboratory with controlled temperature and artificial dark-light cycle (light from 0700 to 1900 h) were used throughout this 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, 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. Lactating mothers received 15 g of this food during the first week of suckling and 20 g during the next 3 d. Water was given ad libitum. The number of pups in each litter was evened to eight. Offspring from dams that received normal diet and those from food-restricted dams were studied at age 10 d. Only females were selected. Food intake of control and undernourished pregnant and lactating mothers were previously reported (17).

All studies were conducted according to the principles and procedures outlined in the Committee for Animal Experimentation of the Universidad Complutense, Madrid.

Glucose tolerance tests
Tolerance tests were performed in nonfasted conscious rats. They were separated from the mother and normal body temperature was maintained with heating lamps. A 35% glucose solution was injected ip at a dose of 1 g/kg body weight. Rats were decapitated at different times, as indicated, and blood withdrawn from the neck was collected in heparinized tubes. An aliquot was deproteinized for glucose determination and the rest was used to obtain the plasma and to analyze insulin. We calculated the integrated glucose and insulin responses, which were the incremental values above basal levels of their respective concentrations over a period of 60 min after the glucose injection.

Insulin stimulation
Nonfasted conscious rats were injected ip with 4 U per 100 g body weight of insulin (Actrapid, Novo Nordisk Pharma SA, Copenhagen, Denmark). The control group was injected with vehicle only. Twenty minutes after this treatment, the animals were decapitated. The mixed muscle from hind legs was quickly exposed and rapidly removed, cleaned of visible fat and connective tissue and frozen in liquid N2. Muscles were kept at –70 C until used.

Analytic procedures
Glucose was measured in supernatants of Ba(OH)2-ZnSO4 deproteinized blood by a glucose oxidase method (Byosistems, Barcelona, Spain). Immunoreactive insulin in serum samples was determined by RIA using rat insulin as standard (INCSTAR, Stillwater, MN). This method allows the determination of 2.0 ng/ml, with coefficients of variation within and between assays of 10%. The concentration of protein was determined by the Bradford method (18) using a protein assay (Bio-Rad Laboratories, Inc., Hercules, CA), using {gamma}-globulin as standard. 5'-nucleotidase-specific activity was measured as a marker for plasma membranes and was determined as described by Avruch et al. (19).

Antibodies used
Antibodies included the following: anti-GLUT-4 (Biogenesis, Sandown, NH); anti-GLUT-1, anti-GLUT-3 and anti-GLUT-5 (Chemicon, Temecula, CA); antiinsulin receptor ß-subunit (Upstate Biotechnology, Lake Placid, NY, and Oncogene Research Products, Boston, MA); anti-insulin receptor substrate (IRS)-1 and anti-IRS-2 (Upstate Biotechnology and Santa Cruz Biotechnology, Inc., Santa Cruz, CA); anti-p85{alpha}- and anti-p85ß-specific antibodies (Abcam Limited, Cambridge, UK); anti-p85, which recognizes all variants of p85{alpha} and p85ß (Upstate Biotechnology); anti-p110{alpha} and anti-p110ß (Santa Cruz Biotechnology); anti-phosphotyrosine (Upstate Biotechnology and Santa Cruz Biotechnology); anti-Akt (or protein kinase B) and anti-phospho-Akt recognizing phosphorylated Ser473 of Akt (Cell Signaling Technology, Beverly, MA); anti-protein kinase C (PKC){zeta}/{lambda} (Santa Cruz Biotechnology); anti-phospho-glycogen synthase kinase (GSK)-3{alpha}/ß recognizing phosphorylated Ser21 of GSK-3{alpha} or Ser9 of GSK-3ß (Cell Signaling Technology); anti-p70S6 kinase (Cell Signaling Technology); anti-phospho-p70S6 kinase recognizing phosphorylated Thr421/Ser424 of p70S6 kinase (Cell Signaling Technology); anti-p38 MAPK (Santa Cruz Biotechnology); anti-phospho-p38 MAPK recognizing phosphorylated Thr180/Tyr182 of p38 MAPK (Cell Signaling Technology); and anti-PTP1B (Santa Cruz Biotechnology).

Muscle membrane preparation
The isolation of surface and intracellular membranes was always carried out in parallel with muscles from control and undernourished rats. Approximately 3 g muscle were minced and homogenized at 4 C in a Polytron (Brinkmann Instruments, Inc., Westbury, NY) at low speed for 8 sec in 10 ml of buffer A [10 mM NaHCO3 (pH 7.0), 0.25 M sucrose, 5 mM NaN3, and 100 µM phenylmethylsulfonylfluoride]. The resulting crude muscle homogenate was centrifuged at 1300 x g for 10 min. The supernatant was collected and kept on ice. The pellet was resuspended with 7 ml buffer A, rehomogenized, and centrifuged as previously indicated, keeping on ice the low-speed membrane pellet. The two supernatants were pooled and centrifuged at 9000 x g for 10 min. The pellet was discarded and the supernatant was subjected to ultracentrifugation at 190,000 x g for 1 h. The resulting pellet was kept on ice (F1). The low-speed membrane pellet was rehomogenized with 20 ml buffer A in a Potter-Elvehjem homogenizer. Solid ClK and sodium pyrophosphate were added to final concentrations of 300 and 25 mM, respectively, to solubilize the contractile proteins and liberate GLUT-4-enriched inner membranes. The homogenate was vigorously mixed and then incubated at 4 C for 2 h with gentle rotation. Next, it was centrifuged at 1200 x g for 5 min, and the supernatant was centrifuged at 10,000 x g for 30 min. The supernatant was spun at 53,000 x g for 1 h, and the resulting supernatant was again centrifuged at 190,000 x g for 1 h, discarding the supernatant and saving the pellet (F2). The pellets F1 and F2 were resuspended using a Potter-Elvehjem homogenizer in 1.5 ml buffer A, and they were loaded on top of discontinuous sucrose gradients: 10 and 40% for F1; 25, 32, and 35% for F2. They were centrifuged at 150,000 x g for 16 h. Fractions were collected at the 10–40% interphase, and on the 25% sucrose layer, they were diluted 10-fold with buffer A and spun at 200,000 x g for 90 min. The resulting pellets were resuspended in appropriate volume of 20 mM HEPES (pH 7.4) and used fresh for proteins and enzyme activity measurements. Finally, they were kept at –80 C until used for Western blot analysis.

Preparation of muscle lysates
Muscles (150 mg) were homogenized with a Polytron operated at maximum speed in 1.5 ml ice-cold lysis buffer, composed of 50 mM HEPES (pH 7.4), 150 mM NaCl, 1 mM MgCl2, 10 mM sodium pyrophosphate, 10 mM sodium fluoride, 2 mM EDTA, 10% glycerol, 1% Nonidet P-40, 2 mM phenylmethylsulfonylfluoride, 2 mM benzamidine, 10 µM leupeptin, 10 µg/ml aprotinin, and 2 mM sodium orthovanadate. The tissue homogenate was incubated for 60 min at 4 C with gentle stirring and then centrifuged at 100,000 x g for 60 min. The supernatants were collected, assayed for protein concentration, aliquoted and stored at –80 C until used for Western blot analyses, immunoprecipitation, and phosphatidylinositol 3-kinase (PI 3-kinase) determination.

Immunoprecipitation
Muscle lysates containing 500-2000 µg proteins were immunoprecipitated overnight at 4 C with gentle rotation in presence of 2–5 µg of the corresponding primary antibody, followed by the addition of protein A-agarose (Roche Diagnostics, Indianapolis, IN); GammaBind Plus Sepharose (Amersham Biosciences, Uppsala, Sweden); or antimouse IgG-agarose (Sigma, St. Louis, MO) for the rabbit polyclonal, goat polyclonal, and mouse monoclonal antibodies, respectively. After mixing for 2 h, the pellets were collected by centrifugation and the supernatants were discarded. Then the pellets were washed and saved for Western blot analyses or PI 3-kinase activity determination.

Western blot analyses
The samples were subjected to SDS-PAGE on 7–10% polyacrylamide gels according to Laemmli (20). Proteins were then electrophoretically transferred to polyvinylidene difluoride (PVDF) filters (PVDF protein sequencing membrane, Bio-Rad Laboratories Inc., Alcobendas, Spain) for 2 h. After transferring, the filters were blocked with 5% nonfat dry milk (for general antibodies) or 3% BSA (for antiphosphotyrosine antibodies) in PBS followed by incubation with primary antibodies overnight. The PVDF filters were next washed four times for 10 min each time with PBS and 0.1% Tween 20, followed by 1 h incubation with appropriate secondary antibody conjugated to horseradish peroxidase (Sigma). The PVDF filters were then washed as indicated above and subsequently exposed to an enhanced chemiluminescence reagent (Amersham Life Science, Little Chalfont, Buckinghamshire, UK). The bands were quantified by laser scanning densitometry (Molecular Dynamics, Sunnyvale, CA). The presence of linearity between the time of x-ray film exposure and the OD of the bands was initially ensured. Finally, the PVDF membranes were stained with Coomassie blue to confirm that equal amounts of protein were analyzed in the same Western assay.

IRS-1-associated PI 3-kinase activity
Aliquots of lysates containing 2 mg protein were immunoprecipitated with anti-IRS-1 antibody, as indicated above. Immunocomplexes were collected with protein A-agarose.

PI 3-kinase activity was assayed by phosphorylation of PI with [32P]ATP (Amersham Life Science). The phosphorylated PI was analyzed by thin-layer chromatography by use of previously described procedures (21). The products of the radioactive reaction were visualized by autoradiography and quantified by densitometry.

Expression of the results
All the data are reported as the mean ± SE. The difference between two mean values is assessed with t test. For multiple comparisons, significance was evaluated by ANOVA, followed by the protected least significant difference test.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Undernourished rats weighed roughly 50% less than controls at 10 d of age: 11.7 ± 2.8 and 24.7 ± 3.1 g, respectively. Blood glucose levels were also reduced: 111 ± 13 mg per 100 ml for the food-restricted and 160 ± 5 mg per 100 ml for the controls (P < 0.001). Plasma insulin in the group subjected to undernutrition was 50% of that in the control group: 5.7 ± 2.4 vs. 13.2 ± 2.4 µU/ml (P < 0.05). Figure 1Go shows the results obtained in the glucose tolerance tests. The control group underwent a high increase in plasma insulin levels, whereas the increase in restricted rats was slight. Consequently, the integrated insulin response was 4-fold lower in restricted rats than in controls. However, blood glucose levels after glucose load changed in a similar fashion in the two groups so that the integrated glucose responses were not statistically different. The insulinogenic index, which is the ratio between integrated glucose and integrated insulin responses, was 90 in food-restricted and 19 in control rats.



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  		FIG. 1. Blood glucose and plasma insulin concentration-time profiles after a glucose load in control and undernourished suckling rats. Blood glucose, integrated glucose responses (A) and plasma insulin and integrated insulin responses (B) were determined in control ({blacksquare}) and undernourished ({square}) 10-d-old rats that received an ip glucose load as described in Materials and Methods. Values are mean ± SE for six to eight animals. Significant differences between control and undernourished rats: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
As shown in Fig. 2AGo, undernutrition did not alter the content of glucose transporter GLUT-4 or that of GLUT-1 in the whole skeletal muscle. Unexpectedly, GLUT-5 content underwent a sharp decrease in the undernourished rats: 2-fold less than the control value. GLUT-3 protein in the muscle from suckling rats was not detected. To study the distribution of GLUT-4, subcellular membrane fractions from this tissue were prepared by sucrose-gradient centrifugation. The amounts of protein recovered in the two fractions collected from sucrose gradients showed no differences between undernourished and control rats and was not affected by insulin treatment (Table 1Go). The fractions were characterized by the presence of a known plasma membrane marker, i.e. 5'-nucleotidase, whose specific activity was higher in the 25% sucrose fractions, compared with that in 40% sucrose preparations in both groups of rats. These results indicate that 25% fraction was enriched in plasma membrane, whereas 40% was relatively poor and contained mainly low-density membranes. No changes were produced in this enzyme distribution by insulin administration, indicating that the purity of membrane fractions was not altered by this treatment. It should be noted, however, that food restriction led to a significant increase in the 5'-nucleotidase-specific activity determined in plasma as well as in intracellular membrane fractions (Table 1Go).



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  		FIG. 2. Effect of undernutrition on glucose transporters content and GLUT-4 translocation in muscle from suckling rats. GLUT-4, GLUT-1, and GLUT-5 proteins content in the muscle from control (C) and undernourished (U) suckling rats. A total of 5, 10, and 80 µg of protein from muscle lysates (for GLUT-4, GLUT-1, and GLUT-5, respectively) were subjected to SDS-PAGE and subsequent Western blotting (A). GLUT-4 content in plasma (PM) and intracellular (IM) membranes (25 and 40% sucrose fractions, respectively) from muscle of control (C) and undernourished (U) rats in basal (–) or insulin-stimulated (+) conditions, as indicated in Materials and Methods. Ten micrograms of protein were immunoblotted (B). Representative blots are shown at top. Each bar represents mean ± SE of five to seven determinations. Differences between basal and undernourished rats: *, P < 0.05 and **, P < 0.01. Differences between basal and insulin-stimulated within the same group of rats: a, P < 0.05; b, P < 0.01; c, P < 0.001.

 

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  		TABLE 1. Protein recovery and 5'-nucleotidase activities in skeletal muscle from control and undernourished suckling rats submitted to fractionation in sucrose gradients

 
Figure 2BGo illustrates the distribution of GLUT-4 in surface and intracellular membranes. The content of this transporter was not affected by undernutrition under basal conditions in each type of membrane fraction, in accordance with the previous result obtained by analyzing the whole muscle homogenate. As can be seen, insulin stimulation induced significant increases of GLUT-4 in the surface membrane in both groups of rats, whereas the intracellular membranes experienced a concomitant decrease in this carrier. However, the relative quantity of GLUT-4 translocated upon insulin stimulation was higher in the food-restricted animals so that the level of this protein analyzed on plasma membrane was significantly higher in undernourished than control rats. The result obtained in intracellular membranes was consistent with that: after insulin treatment, the decline in GLUT-4 content was more marked in the food-restricted than control rats.

To study the effects of undernutrition on the mechanisms regulating glucose transport, we first examined the more proximal steps in the insulin signaling pathway, i.e. in the hormone receptor and IRS-1 and -2 in skeletal muscle of food-restricted and control rats. Insulin receptor (ß-subunit) abundance in muscle was remarkably increased by undernutrition. In contrast, there was no difference between food-restricted and control groups for IRS-1 (Fig. 3AGo); the same was true for the IRS-2 (data not shown). Under basal condition, the amount of tyrosine-phosphorylated insulin receptor was scarcely detectable, and it was not affected by food restriction. In this basal condition, we also determined the amount of phosphotyrosine content of IRS-1, finding that it was not altered in undernourished animals. Twenty minutes after insulin injection, tyrosine phosphorylation of insulin receptor as well as that of IRS-1 increased in the two groups of rats, but this stimulatory effect of insulin was significantly improved in the muscle from undernourished animals for both proteins (Fig. 3BGo).



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  		FIG. 3. Effects of undernutrition on muscle content and phosphorylation of insulin receptor (IR) and IRS-1 in suckling rats. Muscle lysates from control (C) or undernourished (U) suckling rats in basal (–) or insulin-stimulated (+) condition, as described in Materials and Methods, were immunoprecipitated (IP) with antiinsulin receptor antibody (ß-subunit) and Western blotted with antiinsulin receptor or antiphosphotyrosine (pY) antibodies (A). Other aliquots of these muscle lysates were immunoprecipitated with anti-IRS-1 antibody followed by Western blotting (IB) with anti-IRS-1 or antiphosphotyrosine antibodies. Representative blots and the bars corresponding to densitometric quantification of six to eight independent determinations are shown. Results are expressed as mean ± SE. Differences between control and undernourished: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
Analysis of the different isoforms of PI 3-kinase regulatory p85{alpha}, p85ß, and catalytic p110{alpha}- and p110ß-subunits revealed that undernutrition caused marked increases in the muscle content of them all, ranging from 50 to 150% above control levels (Fig. 4AGo). Because insulin action involves docking of tyrosine-phosphorylated IRS proteins to the p85 regulatory subunit, we examined the amount of this subunit associated with IRS-1. We found no differences in the IRS-1/p85 complex between the two groups of rats in the basal condition. However, 20 min after hormone injection, insulin-stimulated association of both proteins was enhanced by food restriction (Fig. 4BGo). It was also observed that undernutrition did not affect the p85 association with IRS-2 (data not shown). PI 3-kinase activity was then determined using immunoprecipitates with anti-IRS-1, and the results are also shown in Fig. 4CGo. Under basal condition, no differences were found in this activity between the two groups of rats. Nevertheless, insulin-induced PI 3-kinase stimulation was significantly higher in the muscle from food-restricted than control rats.



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  		FIG. 4. A, Content of p85 and p110 subunits of PI 3-kinase in muscle from control and undernourished suckling rats. To determine p85 forms, muscle lysates from control (C) or undernourished (U) rats were submitted to immunoprecipitation (IP) performed with an antibody directed against both p85{alpha} and p85ß, analyzed by SDS-PAGE, and then immunoblotted (IB) with specific antibodies. p110 subunits were analyzed in muscle lysates previously immunoprecipitated with either specific antibodies against p110{alpha} or p110ß, followed by Western blotting with p110{alpha} or p110ß antibodies, respectively. B, IRS-1-associated p85 subunit of PI 3-kinase in muscle from control and undernourished suckling rats. The analysis of association of p85 subunits with IRS-1 was made in muscle lysates proceeding from control (C) or undernourished (U) rats under basal (–) or insulin-stimulated (+) conditions, as indicated in Materials and Methods, previously immunoprecipitated with anti-IRS-1 antibody, and then immunoblotted using an antibody that reacts with both p85{alpha} and p85ß. C, IRS-1-associated PI 3-kinase activity in muscle from control and undernourished suckling rats. The activity of IRS-1-associated PI 3-kinase was assayed in immunocomplexes from muscle lysates treated with anti-IRS-1 antibody and collected with protein A-Sepharose beads. PIP, Phosphatidylinositol 3-phosphate. Suckling control (C) and undernourished (U) rats were in basal (–) or insulin-stimulated (+) conditions, as indicated in Materials and Methods. Representative immunoblots and autoradiogram are shown in the upper panels. Bars, in the lower panels, represent mean ± SE for five to six independent experiments. Differences between control and undernourished: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
The impact of undernutrition was measured on a series of downstream effectors in the insulin signaling system. Two of the potential targets of PI 3-kinase are the protein kinases B/Akt and PKC{zeta}/{lambda}. Figure 5AGo shows that both muscle Akt content and insulin-induced Akt phosphorylation were increased by food restriction, whereas the content of PKC{zeta}/{lambda} remained unchanged. Downstream of Akt, both {alpha}- and ßGSK-3 were phosphorylated on insulin treatment. Our data in Fig. 5BGo indicate that ßGSK-3 was the most abundant isoform contained in muscle from suckling rats and that an appreciable amount of phosphorylated isoforms was already present in basal condition, the proportion being higher in undernourished rats than controls. In response to insulin, the increases in {alpha}- and ßGSK-3 phosphorylation were significantly improved by food restriction.



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  		FIG. 5. A, Effect of undernutrition on Akt, Akt phosphorylation, and PKC {zeta}/{lambda} in skeletal muscle from 10-d-old rats. Aliquots of muscle lysates from control (C) and undernourished (U) rats were subjected to SDS-PAGE and immunoblotted (IB) with anti-Akt or antiphospho-Akt antibodies. For phospho-Akt analysis the rats were stimulated with insulin, as described in Materials and Methods. The analysis of PKC{zeta}/{lambda} was performed after immunoprecipitation (IP) with anti-PKC{zeta}/{lambda} antibody and collection of immunocomplexes; subsequently, immunoblotting was carried out with anti-PKC{zeta}/{lambda}. B, Effect of undernutrition on GSK-3 insulin-induced phosphorylation: phospho-GSK-3{alpha} and -ß were determined in muscle lysates from control (C) and undernourished (U) suckling rats and treated with saline (–) or insulin (+) as indicated in Materials and Methods. The analysis was performed by Western blotting with a phospho-GSK-3{alpha}/ß(Ser 21/9) antibody. Immunoblots representative of six to seven independent determinations are shown. Bars in the lower panels, Mean ± SE. Differences between control and undernourished: ***, P < 0.001.

 
Figure 6Go shows that the protein expression of p70 S6 kinase, which is involved in the insulin-mediated activation of protein synthesis, was not affected by undernutrition. Moreover, there were no differences in the insulin stimulus on Thr421/Ser424 phosphorylation of this kinase between food-restricted and control groups (Fig. 6AGo). p38MAPK has recently been proposed to play a role in the insulin-stimulated glucose transport; consequently, we determined the effect of undernutrition on muscle content of this protein, finding that it resulted in a 1.7-fold increase over the values seen in control rats (Fig. 6BGo). In our experimental conditions, no increase was detected in the phospho-p38MAPK on insulin injection, compared with basal values; however, the level of this phosphorylated form was found to be markedly enhanced in the undernourished rats under basal as well as stimulated conditions.



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  		FIG. 6. Effects of undernutrition on muscle contents and phosphorylation of p70S6K and p38MAPK. A, p70S6K and phospho-p70S6K content in skeletal muscle from 10-d-old control (C) and undernourished (U) suckling rats, treated with saline (–) or insulin (+) as indicated in Materials and Methods. The analysis was performed by Western blotting in muscle lysates. Phosphorylation status of p70S6K was measured with an antibody that detects p70S6K when phosphorylated at Thr 421/Ser 424. B, p38MAPK and phospho-p38MAPK content in skeletal muscle from 10-d-old control (C) and undernourished (U) suckling rats, treated with saline (–) or insulin (+) as indicated in Materials and Methods. Muscle lysates were prepared and analyzed by Western blotting. Phospho-p38MAPK was measured with an antibody that recognizes p38MAPK only when activated by dual phosphorylation at Thr180/Tyr1282. Representative immunoblots are shown, and the bars represent mean ± SE of six to eight determinations. Differences between control and undernourished: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
As shown in Fig. 7AGo, PTP1B abundance in muscle was similar in the two groups of rats. However, the association of PTP1B with insulin receptor was significantly enhanced by undernutrition in the basal condition as well as after insulin administration. (Fig. 7BGo). In contrast, there were no differences in the association of PTP1B with IRS-1 (Fig. 7CGo) or IRS-2 (data not shown).



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  		FIG. 7. Effect of undernutrition on PTP1B content and association with insulin receptor or IRS-1 on muscle from suckling rats. A, Muscle lysates from control (C) or undernourished (U) rats were immunoprecipitated (IP) with anti-PTP1B antibody followed by Western blotting (IB) with anti-PTP1B. B, Muscle lysates from control (C) or undernourished (U) rats in basal (–) or insulin-stimulated (+) condition, as indicated in Materials and Methods, were immunoprecipitated with anti-PTP1B antibody and Western blotted with antiinsulin receptor (IR) antibody (ß-subunit). C, Muscle lysates from control (C) or undernourished (U) rats in basal (–) or insulin-stimulated (+) condition, as indicated in Materials and Methods, were immunoprecipitated with anti-IRS-1 antibody and analyzed by Western blotting using anti-PTP1B antibody. Representative immunoblots are shown, and the bars represent mean ± SE of six to eight determinations. Differences between control and undernourished: *, P < 0.05; **, P < 0.01.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Maternal nutrition is important for the course and outcome of pregnancy. We have previously shown that the body weights of term fetuses, from rats food restricted according to the present experimental model, are significantly reduced (22). Important also is that maternal diet meets all nutritional needs during lactation; food restriction during this period causes a large reduction in milk volume (23), probably resulting in the negative impact of undernutrition on the litter growth seen in suckling rats from food-restricted mothers studied in the present work.

Reduced growth associated with malnutrition in fetal and neonatal periods could induce glucose intolerance later in life (1, 5). The thrifty phenotype hypothesis postulates that this is a consequence of programming, whereby an insult at a critical period of growth has long-term effects on glucose homeostasis that may be detrimental in times of normal nutrition in adulthood (3). However, we previously demonstrated that glucose tolerance is not altered when the food-restricted status is extended throughout the entire life (17). In the present study, our results indicate that undernutrition lowers basal blood glucose as well as plasma insulin in suckling rats; moreover, these animals exhibited a very poor insulin secretion in response to glucose. It is well known that food restriction damages pancreatic ß-cell function, as reported in most of the published studies in animal models of malnutrition (24, 25). However, despite their depressed insulin response, undernourished suckling rats are capable of maintaining a normal glucose tolerance. Taken together, these results suggest that insulin sensitivity is increased by undernutrition. Therefore, we confirm that the effect of chronic undernutrition on glucose homeostasis, i.e. impaired insulin secretion and glucose normotolerance (17), displayed by adult rats is already established during development.

The amount of white adipose tissue is very low in rats during suckling (13). Moreover, a significant reduction in fat pads has been reported in the offspring of rats submitted to protein restriction (26). These facts led us to suppose that glucose normotolerance in restricted suckling rats could be associated with an increased glucose use by muscle; we have therefore undertaken a study to determine whether the insulin response is improved in skeletal muscle after undernutrition, as previously shown in adult rats (11, 17). In any case, this idea does not rule out the possibility that undernutrition could also have repercussions on liver, modifying the insulin capacity to suppress hepatic glucose production.

We determined the effect of maternal food restriction on the amount of muscle glucose transporters in the offspring. This condition does not influence GLUT-4 or GLUT-1 contents, a result in contrast to the effect of chronic undernutrition in the adult rats, of which muscles undergo increases and decreases in GLUT-1 and GLUT-4, respectively. Muscle GLUT-3 and GLUT-5 contents are increased by food restriction in adult rats (11). However, we have not detected GLUT-3 protein in the muscle from 10-d-old rats, whereas GLUT-5 was found decreased after undernutrition. This fructose carrier, which has a low capacity to transport glucose, is mainly expressed in the intestine after weaning, but fructose can enhance its mRNA abundance during suckling (27). Because little is known about muscle GLUT-5 regulation, the significance of the decrease found in this work deserves further attention. Insulin ability to recruit GLUT-4 is improved in chronically undernourished adult rats as well as rats calorie restricted during a limited period of life span (9, 11). We therefore studied the effect of insulin on muscle subcellular distribution of this transporter in developing rats. In newborn rats, sarcolemma GLUT-4 concentration is greater than in adult, a fact associated with the minor insulin responsiveness of this transporter (28). At d 10, GLUT-4 is more abundant within muscle than in sarcolemma, as shown by our data. The amount of this carrier translocated to plasma membrane on insulin stimulation is significantly higher in undernourished rats than controls. Consequently, GLUT-4 protein abundance in muscle surface membranes after insulin treatment is increased in restricted rats. This result differs from that obtained by Sampaio et al. (29), who detected a decrease in the insulin-induced glucose uptake in muscle from suckling rats submitted to maternal undernutrition. In that case, insulin failed to induce GLUT-4 translocation. These discrepancies can be attributed to the fact that the undernutrition pattern used by these authors, a protein-free diet supplied only during early lactation, differed from that employed in the present work. Moreover, they assayed glucose transport in isolated muscle strips, a condition in which possible influences of factors present in vivo are discarded.

The enhanced GLUT-4 translocation must contribute to glucose normotolerance of undernourished rats. However, it is known that translocation alone cannot quantitatively account for the stimulation of glucose uptake (30). An insulin-induced increase in GLUT-4 intrinsic activity has been suggested as an additional factor regarding the stimulus on glucose transport, possibly via p38 MAPK-dependent pathway (31, 32). We found that p38 MAPK protein as well as its activated form, phosphorylated, is markedly increased in muscle from undernourished developing rats. This result presents the possibility that activation of translocated GLUT-4 was increased in these rats, contributing to the improved glucose uptake. The important role of p38 MAPK in regulation of glucose transport is stressed by a recent report (33) showing that insulin exposure does not increase p38 MAPK phosphorylation in type 2 diabetic patients, typically insulin resistant. The fact that p38 MAPK phosphorylation was rapid and transient (31, 33) might explain the lack of increase in phospho-p38 MAPK content after insulin found in this work.

Alterations in insulin sensitivity have been associated with modifications of receptor and postreceptor signaling. This prompted us to assess the effect of food restriction on different steps of the insulin pathway. We found dramatic increases in the insulin receptor content and phosphorylation in muscle from undernourished developing rats. In contrast, IRS-1 protein content did not change, but its phosphorylation as well as its association with the p85 regulatory subunit of PI 3-kinase after insulin was enhanced. Consistently, stimulation of PI 3-kinase activity associated with IRS-1 was also increased. Such association is depressed in muscle from diabetic insulin-resistant patients (34). The increases found in these early steps of insulin action may be instrumental in the improvement of muscle insulin sensitivity evident in undernourished suckling rats.

Our results indicate that undernutrition markedly increases the two isoforms of both regulatory and catalytic PI 3-kinase subunits analyzed. A reduced expression of p85{alpha} or p85ß improves insulin sensitivity. The proposed mechanism is based on a competition between these isoforms and the p85-p110 heterodimer to bind phosphorylated IRS-1 proteins (35, 36). An increase in p85{alpha} has recently been suggested as the cause of muscle insulin resistance associated with pregnancy (37). However, despite the increased p85 content, we found an improved activation of PI 3-kinase in undernourished rats, which is probably due to the concomitant increase in p110 catalytic subunits.

The PKC and Akt, which lie downstream of PI 3-kinase, seem to be implicated in the stimulus on glucose transport (38). Our results show that Akt and its insulin-induced phosphorylation are markedly increased in muscle from undernourished rats, whereas there are no changes in the level of atypical forms of PKC{zeta}/{lambda}. These findings suggest that Akt is involved in the improvement in insulin sensitivity observed in undernourished rats. However, we cannot exclude that undernutrition may alter PKC{zeta}/{lambda} activation, which is impaired in muscle cells from patients with glucose intolerance (39).

Glycogen synthesis in skeletal muscle plays an important role in blood glucose homeostasis (40). The rate-limiting enzyme, glycogen synthase, is activated by insulin-induced dephosphorylation. Insulin triggers the inhibition of GSK-3 by phosphorylation, a process catalyzed by Akt (reviewed in Ref. 41). Our data show that the levels of both {alpha}- and ß-Ser-phosphorylated GSK-3 isoforms present in muscle are increased in restricted rats in basal as well as stimulated condition. It has been shown that GSK3 also phosphorylates IRS-1 on Ser residues, a type of phosphorylation that impairs insulin receptor kinase activity (42). Consequently, the more efficient insulin-induced GSK-3 phosphorylation in undernourished rats, leading to its inhibition, fits the increased IRS-1 tyrosine-phosphorylation found in these animals. GSK-3 activity is elevated in fat and muscle from type 2 diabetic mice and human patients, which are insulin resistant (43, 44). Therefore, the restricted rats studied in the present work may constitute a useful model of insulin hypersensitivity concomitant to a higher degree of insulin-induced GSK-3 inhibition. In fact, the use of GSK-3 inhibitors might be a promising therapy in the states of resistance associated with increased GSK-3 activity (45).

The PI 3-kinase/mammalian target of rapamycin/p70S6K is one of the signaling pathways initiated after insulin binding to its receptor, important in regulating the rate of protein synthesis. To be active, p70S6K must be phosphorylated in multiple Ser/Thr residues (46). We investigated the effect of undernutrition on this step downstream of PI 3-kinase. Despite the increased insulin-induced PI 3-kinase activation associated with undernutrition, the rate of p70S6K phosphorylation, as well as p70S6K protein content, remains unaffected by this condition. Nevertheless, we analyzed only the Thr421/Ser424 phosphorylated p70S6K, but its activity is also regulated by phosphorylation on a number of other residues that have not been studied here.

PTP1B plays a major role in the negative regulation of insulin signaling (15). Because undernourished rats represent a condition of improved muscle insulin responses, we examined muscle PTP1B protein content and its association with both insulin receptor and IRS-1. Undernutrition altered neither PTP1B protein abundance nor PTP1B/IRS association. However, we have found a higher degree of IRS-1-associated PI 3-kinase activity after insulin in food-restricted rats, despite the fact that this association inhibits the interaction between IRS-1 and PI 3-kinase (47). As discussed below, phosphatase activity must be considered to interpret these results. PTP1B association with insulin receptor was markedly increased in undernourished rats in both conditions explored, basal and insulin stimulated. This result is surprising because PTP1B-insulin receptor interaction is increased in muscle of insulin-resistant rats (48) and insulin-induced glucose uptake is elevated in skeletal muscle of PTP1B-deficient mice (49). However, the rate of association between PTP1B and insulin receptor or IRS-1 does not necessarily parallel the intensity of their dephosphorylation because PTP1B activity can be regulated by a number of mechanisms not yet well understood, including tyrosine and serine residues phosphorylation (50) as well as oxidative inhibition (51). Consequently, the improved PTP1B/insulin receptor association in coincidence with enhanced receptor phosphorylation could reveal an underregulated phosphatase activity in undernourished rats, considering that food restriction might influence the PTP1B phosphorylation or oxidative status. In our view, this increased association might be part of a mechanism present in muscle of restricted rats, eventually to prevent an aggravation of the increased insulin actions.

In conclusion, the data presented herein indicate that undernutrition promotes an outstanding modification of the glucose homeostasis characteristic of the suckling period: a pronounced insulin resistance, mediated by low insulin responses of muscle (reviewed in Ref. 13), which allows the maintenance of euglycemia despite the low carbohydrate content of milk. In normal conditions of nutrition, insulin sensitivity is enhanced after weaning to a high-carbohydrate diet (52). The increased muscle insulin sensitivity established in the rats from food-restricted dams perhaps allows the maximal possible growth of insulin-sensitive tissues under conditions of severely limited resources but might be detrimental to the correct development of other tissues that have more important requirements for glucose. Other than modifications in insulin signaling, it must be pointed out that tissue and serum factors, such as amino acids, lipids, or counterregulatory hormones, can also modulate insulin sensitivity and have an influence on the changes associated with undernutrition shown in this work.


   Acknowledgments
 
We are especially grateful to Susana Fajardo for excellent technical help. We thank Dr. Luis Goya for critical review of this manuscript.


   Footnotes
 
This work was supported by Grant BFI2002-00253 from Ministerio de Ciencia y Tecnología, Spain.

First Published Online May 19, 2005

Abbreviations: GSK, Glycogen synthase kinase; IRS, insulin receptor substrate; PI 3-kinase, phosphatidylinositol 3-kinase; PKC, protein kinase C; PTP, protein tyrosine phosphatase; PVDF, polyvinylidene difluoride.

Received December 23, 2004.

Accepted for publication May 5, 2005.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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