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Endocrinology Vol. 143, No. 11 4295-4303
Copyright © 2002 by The Endocrine Society

ARTICLE

Effects of Chronic Undernutrition on Glucose Uptake and Glucose Transporter Proteins in Rat Heart

M. Lucia Gavete, Maria Agote, M. Angeles Martin, Carmen Alvarez and Fernando Escriva

Instituto de Bioquímica, Centro Mixto: Consejo Superior de Investigaciones Científicas-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
 
The high energy demands of myocardium are met through the metabolism of lipids and glucose. Importantly, enhanced glucose utilization rates are crucial adaptations of the cardiac cell to some pathological conditions, such as hypertrophy and ischemia, but the effects of undernutrition on heart glucose metabolism are unknown. Our previous studies have shown that undernutrition increases insulin-induced glucose uptake by skeletal muscle. Consequently, we considered the possibility of a similar adaptation in the heart. With this aim, undernourished rats both in the basal state and after euglycemic hyperinsulinemic clamps were used to determine the following parameters in myocardium: glucose uptake, glucose transporter (GLUT) content, and some key components of the insulin signaling cascade. Heart membranes were prepared by subcellular fractionation in sucrose gradients. Although GLUT-4, GLUT-1, and GLUT-3 proteins and GLUT-4/1 mRNAs were reduced by undernutrition, basal and insulin-stimulated 2-deoxyglucose uptake were significantly enhanced. Phosphoinositol 3-kinase activity remained greater than control values in both conditions. The abundance of p85{alpha} and p85ß regulatory subunits of phosphoinositol 3-kinase was increased as was phospho-Akt during hyperinsulinemia. These changes seem to improve the insulin stimulus of GLUT-1 translocation, as its content was increased at the surface membrane. Such adaptations associated with undernutrition must be crucial to improvement of cardiac glucose uptake.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE HIGH PREVALENCE of undernutrition in developing countries as well as in patients hospitalized in Western societies constitutes a significant public health problem. Poor nutrition during the perinatal period has repercussions on health in adulthood. The cardiovascular system is among those affected by food restriction. Intrauterine exposition to maternal undernutrition may induce damages that could play a role in the subsequent development of cardiovascular alterations (1, 2). It has been suggested that coronary heart diseases associated with food restriction during growing periods could be a consequence of the subsequent transition to adequate nutrition (3). However, to our knowledge, few studies have investigated the impact of a permanent undernutrition state, which was established in the perinatal period and chronically continued until adulthood, on cardiac metabolism, a condition present in a significant number of undernourished humans.

Heart muscle meets its energy needs preferentially through the oxidation of fatty acids (4), but it is capable of using glucose as an energy-providing substrate. Moreover, there are physiological situations associated with high glucose utilization rates, such as late fetal life (5) and exercise (6). Importantly, many studies have suggested that glucose becomes a crucial fuel to support an optimal functional recovery after myocardial hypoxia and ischemia (7). Glucose utilization is initiated by glucose uptake, which depends on a series of carrier proteins, members of the family of facilitative glucose transporters (GLUTs). The main carrier isoforms present in heart, skeletal muscles, and adipose cells are GLUT-1 and GLUT-4, which are located in plasma as well as intracellular membranes. Insulin promotes the recruitment of both carriers to cell surface in heart and adipocytes, whereas in skeletal muscle only GLUT-4 is translocated in response to the hormone (reviewed in Ref. 8). Alterations in myocardial glucose uptake seen in different pathological states can be the result of concomitant changes in glucose transporters. Thus, myocardial glucose metabolism is impaired and heart GLUT-4 content reduced in untreated diabetes (9, 10), whereas overexpression of this carrier in mice results in an increased cardiac glucose utilization rate (11). Hypoxia and ischemia enhance heart glucose metabolism and glucose transporters (12, 13). Intrauterine growth retardation caused by utero-placental insufficiency reduces the expression of these carriers in heart muscle, as recently reported (14).

The restriction of protein calories results in increased insulin action in obese humans (15), rhesus monkeys (16), and rodents (17). We have previously developed a rat model of undernutrition based on a food restriction that begins in the fetal stage and continues until adulthood. These rats show normal glucose tolerance despite the fact that the release of insulin is impaired. It is mainly due to an enhanced insulin capacity to promote glucose uptake by skeletal muscle, as well as to an increase in GLUT-1 content in such a tissue (18). However, a detailed knowledge of the effects elicited by chronic food restriction on cardiac glucose uptake and GLUTs is presently lacking.

In the present work, we used a rat model of permanent undernutrition to study the effects of this perturbation on the heart, regarding basal and insulin-induced glucose uptake, glucose transporters content and recruitment, insulin receptor, and several postreceptor signals implicated in this recruitment. Our key findings show that chronic undernutrition leads to higher rates of glucose uptake by heart, a similar adaptation to that characteristic of hypoxia and ischemia. The mechanism for such an improvement seems to be provided by an increase in GLUT-1 located at the cardiac cell surface membrane. This better glucose utilization may contribute to the preservation of myocardial function when a long-lasting food restriction status is established.


   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 (lights on from 0700–1900 h) were used throughout the study. Females were caged with males, and mating was confirmed by the presence of spermatozoa in vaginal smear. 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 with 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 week of suckling, respectively (50% of the control diet). After weaning, only females were selected for this study. They received daily 35% of the diet consumed by controls until d 70 of life. Water was given ad libitum. The food intake of control and undernourished rats has been previously reported (18, 19).

Euglucemic insulin clamp
These studies were performed in control rats about 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 (4 mg/100 g body weight), 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 0.4 or 5.0 IU/h•kg, respectively. A solution of glucose (30% and 40% for control and food-restricted rats, respectively) was infused through the other saphenous vein 5 min after starting the infusion of hormone. The difference in glucose concentration was necessary to infuse a final volume roughly proportional to the body weight in both groups of rats (19). The infusion rate was adjusted to clamp blood glucose at the level present in 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 (Roche Molecular Biochemicals, Mannheim, Germany), and the pump dial was adjusted according to changes in the level of blood glucose. Within 40 min of starting the clamp, plasma insulin and glucose levels remained constant without further adjustment with 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 rats were then cervically dislocated. The heart was rapidly removed, freeze-clamped in liquid N2, and stored at –80 C until use.

Estimation of glucose uptake
The uptake of glucose by heart was estimated by measuring 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 International, Aylesbury, UK) was injected iv 40 min after starting the clamp experiments, 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 the basal uptake of glucose. Arterial blood was sampled for determination of the concentration of blood glucose and 2-deoxy-D-[3H]glucose radioactivity. At the end of experiment, rats were killed, and hearts were removed and stored as indicated. The heart 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 (20). 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 into the Somogy reagent [BaSO4/Zn(OH)2]. The rate of utilization of glucose 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.

Heart fractionation
The procedure used to isolate plasma and intracellular membranes was similar to that described by Gumá et al. (21) for skeletal muscle, with some modifications. Approximately 2 g heart muscle were minced and homogenized at 4 C in a Polytron (Brinkmann Instruments, Inc., Westbury, NY) at low speed for 20 sec in buffer A [20 mM HEPES and 0.15 M KCl, containing 1 µM leupeptin and 100 µM phenylmethylsulfonylfluoride (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, then centrifuged at 2,000 x g for 10 min. The supernatant was collected and kept on ice. The pellet was resuspended in 7 ml buffer A, rehomogenized as indicated above for 10 sec, treated with the KCl solution, left on ice for 15 min, and centrifuged for 10 min at 2,000 x g. The two supernatants were pooled and subjected to ultracentrifugation at 190,000 x g for 1 h. The resulting pellet, which contains crude membranes, was resuspended using a tissue grinder in 3 ml 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 on 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 x g. Fractions were collected from the top of the 25% gradient (25% fraction) and from the interphases 25–30% (30% fraction) and 30–35% (35% fraction). The pellet was also collected (35P fraction). All fractions were diluted 10-fold with buffer A and centrifuged at 190,000 x g for 90 min. The resulting pellets were resuspended in 20 mM HEPES, pH 7.4. Proteins were assayed, and fractions were kept frozen at -80 C.

Western blot analyses
The membrane fractions were subjected to SDS-PAGE on 7–10% polyacrylamide gels according to Laemmli (22). 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% (wt/vol) nonfat dry milk in PBS with 3% BSA and 0.02% sodium azide. Antibodies against the GLUT-1 and the GLUT-4 glucose transporters were purchased from Biogenesis (Sandown, NH) and were used at dilutions of 1:5000 and 1:1000, respectively. Antibodies against GLUT-3 (1:2500 dilution) were obtained from Chemicon (Temecula, CA). Antiinsulin receptor, ß-subunit, and antirat {alpha}1-subunit of the Na+,K+-adenosine triphosphatase (N+,K+-ATPase), from Upstate Biotechnology, Inc. (Lake Placid, NY), were diluted at 1:250. The PVDF filters were next washed four times for 10 min each time at 37 C with PBS and 0.1% Tween 20, followed by 1-h incubation with goat antirabbit IgG conjugated to horseradish peroxidase (Sigma, St. Louis, MO). The PVDF membranes were then washed as indicated above. Detection of antibody-antigen complexes was accomplished by the enhanced chemiluminescence method (BM chemiluminescence, Roche).The ODs of bands were determined by laser scanning densitometry (Molecular Dynamics, Inc., Sunnyvale, CA). Immunoblots were performed under linear conditions according to the amount of protein loaded on the gel. The PVDF filters were finally stained with Coomassie Blue to confirm that in the same Western assay equal amounts of protein were analyzed as well to confirm as the heterogeneity of the protein composition pattern of the different sucrose fractions.

RNA isolation and Northern blot analysis
RNA was extracted from heart (200–300 mg) obtained from rats in the basal condition by use of the guanidium isothiocyanate-phenol-chloroform method (23). After quantification, total RNA (20 µg) was subjected to Northern blot analysis following the method previously described (24). A 2.47-kb rat GLUT-4 cDNA cloned into the EcoRI site of pBluescript KS+ (Stratagene, Barcelona, Spain), a 2.6-kb rat GLUT-1 cDNA insert subcloned from pGT3 into pBluescript KS- at the EcoRI site (Promega Corp., Barcelona, Spain), and a 0.6-kb mouse GLUT-3 cDNA cloned into the HincII site of pGEM 4Z (also from Promega Corp.) were provided by Dr. A. Zorzano (Department of Biochemistry and Molecular Biology, University of Barcelona, Barcelona, Spain) and were used as probes. Membranes were autoradiographed, and the relative densities of the signals were determined by densitometric scanning of the autoradiograms with a laser densitometer. An 18S rRNA probe was used as a control for RNA loading.

Phosphatidylinositol 3-kinase (PI 3-kinase) assay
To determine insulin-stimulated PI 3-kinase, rats were anesthetized with pentobarbital as indicated, the abdominal cavity was opened, the portal vein was exposed, and 5 IU insulin were injected. After 90 sec the heart was quickly removed, freeze-clamped with liquid N2, and stored at -80 C until assayed. Cardiac muscles (100 mg) from basal and insulin-injected rats were homogenized with a Polytron operated at maximum speed in 1 ml 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 30 min and then subjected to centrifugation at 180,000 x g for 60 min at 4 C. The supernatants were used as samples for protein and PI 3-kinase determinations. Aliquots containing 2 mg protein were immunoprecipitated with monoclonal antiphosphotyrosine antibody (Quimigranel, Santa Cruz Biotechnology, Inc., Madrid, Spain). Immunocomplexes were collected with antimouse IgG-agarose (Sigma). PI 3-kinase activity was assayed by phosphorylation of phosphatidylinositol with [32P]ATP (Amersham International). The phosphorylated phosphatidylinositol was analyzed by thin layer chromatography using previously described procedures (24). The products of radioactive reaction were visualized by autoradiography and were quantified by densitometry.

Determination of insulin-receptor substrate-1 (IRS-1), total p85, p85{alpha}, p85ß, p110{alpha}, Akt, and phospho-Akt
Hearts from rats in the basal state were extracted as indicated above and samples containing 0.5 mg protein were immunoprecipitated with one of the following antibodies: polyclonal antirat IRS-1, broad specificity polyclonal antirat p85 (both from Upstate Biotechnology, Inc., Lake Placid, NY), or polyclonal antimouse Akt (New England Biolabs, Inc., Beverly, MA). The complexes were bound to antimouse IgG agarose, as described above. The agarose beads were treated with Laemmli sample buffer with 100 mM dithiothreitol at 95 C for 5 min and subjected to SDS-PAGE. The contents of p85{alpha}, p85ß, p110{alpha}, and phospho-Ser473-Akt were analyzed, in the basal condition, directly in 75 µg protein from extracts obtained as indicated above and also subjected to SDS-PAGE; phosphorylated Akt was also determined in the rats treated with insulin in the way described above. We used the following antibodies: polyclonal antihuman p85{alpha}, polyclonal antihuman p110{alpha} (both from Santa Cruz Biotechnology, Inc.), monoclonal antirat p85ß (a gift from P. Parker Imperial Cancer Research Foundation, London, UK), and polyclonal antimouse phospho-Ser473-Akt (Cell Signaling Technology, Beverly, MA). The rest of the Western blot procedure was as described for GLUT determinations, with the following antibody dilutions: anti-IRS-1, 1 µg/ml; anti-p85, 1:2000; anti-Akt, 1:1000; anti-p85{alpha}, 1:1000; anti-p85ß, 1:1000; anti-p110{alpha}, 1:500; and anti-phospho-Ser473-Akt, 1:1000.

Other analytical procedures
The concentration of protein was determined by the Bradford method (25) using a protein assay (Bio-Rad Laboratories, Inc.), using {gamma}-globulin as standard. The specific activity of phosphodiesterase I was assayed as a plasma membrane marker (26). Plasma insulin was determined by RIA, using rat insulin as standard (INCSTAR Corp., Stillwater, MN). This method allows the determination of 2.0 ng/ml, with coefficients of variation within and between assays of 10%.

Expression of the results
All the data are reported as the mean ± SE. The difference between two mean values was 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 50% less than controls at 70 d of age (Table 1Go). The weights of hearts were 0.61 ± 0.05 and 0.34 ± 0.05 g for the control and undernourished rats, respectively. Nevertheless, the heart weight to body weight ratio was slightly, but significantly, greater for the undernourished rats than for their well nourished controls (0.0035 ± 0.0005 vs. 0.0032 ± 0.0006; P < 0.01). The blood glucose concentration was not different between the groups of animals, although insulinemia of the food-restricted animals was 70% lower than that of the controls. The uptake of 2-deoxyglucose by the heart was 2.5-fold higher in undernourished rats than in controls in the basal state. During the clamps, plasma insulin was raised to similar levels in both groups. In these conditions the rates of glucose infusion required to maintain euglycemia were higher in the undernourished rats than in controls. With insulin treatments, cardiac 2-deoxyglucose uptakes were activated in both groups of rats; the increases were higher in the control than in the undernourished animals at the two doses administered (0.4 and 5.0 IU/h•kg). However, glucose uptakes under these stimulated conditions were still above the control values in the food-restricted rats (Table 1Go).


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  		Table 1. Weight, blood glucose, plasma insulin, glucose infusion rate, and heart glucose uptake in control (C) and undernourished (U) 70-d-old rats

 
The amount of protein recovered in each of the fractions derived from sucrose gradients was not different between undernourished and control rats, nor was it affected by insulin treatment (Table 2Go). The fractions were characterized by the presence of known plasma membrane markers, as phosphodiesterase I and the {alpha}1-subunit of Na+,K+-ATPase. The specific activities of phosphodiesterase I were roughly 15-fold higher in the fraction enriched in plasma membrane (25% sucrose), compared with those in crude membranes, in both groups of rats. No changes were produced by insulin on this enzyme. However, undernutrition significantly increased the activity, as shown in the 25% and 30% sucrose fractions (Table 2Go). The {alpha}1-subunit of Na+,K+-ATPase was found predominantly in fraction 25%; it was much lower proportion in the other fractions in both groups of rats, and this distribution remained unaltered after insulin treatment (Fig. 1Go). As a whole, these results indicate that 25% sucrose preparation was enriched in plasma membrane, while the other fractions were relatively poorer in this surface membrane.


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  		Table 2. Protein recovery and phosphodiesterase I activities in hearts from control and undernourished rats submitted to fractionation in sucrose gradients

 


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  		Figure 1. Representative immunoblots showing the distribution of {alpha}1-ATPase in different heart fractions from control (C) and undernourished (U) rats. Protein per lane, 20 µg.

 
Undernutrition led to a decrease in cardiac GLUT-4 content (55% of control value), as shown in the unfractionated membrane preparation (Fig. 2Go) and in both plasma and intracellular membranes (Fig. 3Go). As expected, insulin yielded an increase in the GLUT-4 protein present in plasma membrane and induced a concomitant decrease in the transporter content in the intracellular compartment. Because of the fact that the amounts of carrier translocated, relative to basal contents, were similar in both groups of animals, GLUT-4 from restricted rats was still under control values in the plasma and intracellular membranes following insulin treatment (Fig. 3Go). In the heart from food-restricted animals, GLUT-1 protein was reduced to 55% of the level present in controls, as shown in membranes obtained before separation (Fig. 2Go) as well as in the intracellular membranes (Fig. 3Go). Nevertheless, it is important to point out that the level of GLUT-1 located on the plasma membrane from undernourished rats was markedly increased in the basal state compared with that in controls (Fig. 3Go). Translocation of GLUT-1 in response to insulin led to the expected opposite changes of this protein in both surface and intracellular membranes. This transporter remained increased in plasma membrane from undernourished rats in this stimulated condition compared with controls (Fig. 3Go). The heart was found to contain GLUT-3. However, taking into account the amount of protein loaded for the Western blot analysis, this carrier isoform was present in much less proportion than the other investigated in this study. Reduced amounts of cardiac GLUT-3 were detected in the food-restricted rats (70% of control value; Fig. 2Go).



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  		Figure 2. GLUT-4, GLUT-1, and GLUT-3 contents in crude membrane isolated from heart muscle of control (C) and undernourished (U) rats. A, Blots show a representative experiment. B, Bars correspond to ODs normalized to the value in control rats. Fifteen, 20, and 90 µg protein for GLUT-4, GLUT-1, and GLUT-3, respectively, were laid on gels. Data are the mean ± SE from six independent analysis. Differences between C and U rats: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 


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  		Figure 3. GLUT-4 (A) and GLUT-1 (B) contents in plasma (PM) and intracellular (IM) membranes from heart muscle of control (C) and undernourished (U) rats, under basal (-) or insulin-stimulated (5 IU/h•kg; +) conditions. Five and 15 µg protein were analyzed for GLUT-4 and GLUT-1, respectively. Blots correspond to a representative determination. Bars show the averaged results for six independent experiments. Data are expressed as the mean ± SE. *, P < 0.05; **, P < 0.01 (vs. control). a, P < 0.001; b, P < 0.01 (vs. insulin-stimulated).

 
Representative autoradiograms of Northern blot analysis of RNA from heart are depicted in Fig. 4Go. Total RNA content was not altered by undernutrition (873 ± 103 vs. 992 ± 113 µg/g for control and food-restricted rats, respectively). Both GLUT-4 and GLUT-1 mRNA levels from undernourished animals were 40–50% of control values. We found that GLUT-3 mRNA was present in rat heart, although its content was much lower than that in the other carriers. GLUT-3 mRNA remained unchanged by undernutrition.



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  		Figure 4. A, Representative Northern blot showing heart GLUT-4, GLUT-1, and GLUT-3 mRNA as well as 18S rRNA from control (C) and undernourished (U) rats. Twenty micrograms of total RNA were loaded in each lane. B, Densitometric analysis of Northern blot data after correcting for loading with 18S rRNA. Results represent the mean ± SE for five or six independent experiments. Differences between C and U rats: *, P < 0.05; ***, P < 0.001.

 
Insulin receptor (ß-subunit) was decreased in the heart from undernourished rats (70% of control value), and the cardiac level of IRS-1 was not significantly altered (Fig. 5Go). The heart content of total p85 regulatory subunit protein of PI 3-kinase was markedly raised in undernourished rats, which resulted from the increased abundance of both p85{alpha} and p85ß isoforms (2.2- and 1.6-fold, respectively, over the control values; Fig. 5Go). In contrast, the p110{alpha} isoform of catalytic subunit remained unchanged. Cardiac Akt content was increased by food restriction (1.5-fold over the control value). Phospho-Ser473-Akt was practically undetectable in the basal condition (result not shown), and 90 sec after a single insulin dose it was much higher in food-restricted than in well-nourished rats (Fig. 5Go).



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  		Figure 5. Effect of undernutrition on the cardiac content of insulin receptor (ß-subunit); IRS-1; total p85, p85{alpha}, p85ß, and p110{alpha} subunits of PI 3-kinase; Akt; and phospho-Ser473-Akt. The results show a representative autoradiograph. The bars correspond to densitometric quantification of five or six independent determinations. Insulin receptor was analyzed in crude membranes (70 µg protein). The other parameters were determined in samples prepared as described in Materials and Methods. Results are expressed as the mean ± SE for six independent experiments. Differences between control (C) and undernourished (U) rats: *, P < 0.05; **, P < 0.01; ***, P < 0.001.

 
The basal activity of antiphosphotyrosine immunoprecipitable PI 3-kinase was approximately 3-fold higher in hearts from undernourished rats than in controls. The enzyme was stimulated in the two groups of rats after insulin administration; the peak was still 2-fold higher in undernourished in such a condition (Fig. 6Go).



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  		Figure 6. PI 3-kinase activity in the heart muscle from control (C) and undernourished (U) rats. Rats were in the basal condition (-) or treated with 5 IU insulin (+). Heart extracts were immunoprecipitated with antiphosphotyrosine antibodies, immunocomplexes were collected with IgG-agarose, and enzyme activity was assayed by 32P incorporated into PI. The phosphorylated PI was resolved by thin layer chromatography. Blots corresponding to migrated PI 3-phosphate (PIP) in a representative determination are shown. Bars correspond to the mean ± SE for six independent experiments. **, P < 0.01, significantly different from the corresponding control value.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Heart preferentially uses lipid-derived fuels as energy substrates (4), but it also uses glucose and at higher rates than the skeletal muscle (27). As shown in the present work, undernutrition, a condition that cuts by half the basal glucose uptake in rat skeletal muscle (18), markedly increases it in the heart. An obvious difference between both tissues, which can explain these divergent adaptations, is that cardiac cells continuously work and depend on a constant supply of energy. In prolonged fasting, glucose uptake decreases in both tissues (4, 28), because plasma nonesterified fatty acids rise and inhibit glucose utilization (28). However, hepatic and adipose lipid contents as well as the amount of white adipose tissue are largely reduced in undernourished rats. In fact, circulating nonesterified fatty acids and ketone bodies are not increased in these animals (19), in concert with the marked up-regulation of cardiac glucose uptake found in them. This response can be compared with that observed in other pathological states, as hypoxia or ischemia, both increasing the cardiac glucose metabolism (7, 29), and suggests that this adaptation is important to maintain heart function.

Glucose transport constitutes the major rate-limiting step for cardiac glucose utilization. Because GLUT-4 and GLUT-1 are the main glucose carriers present in heart, we have determined their myocardial expression and abundance to determine whether they are affected by food restriction, which could explain the changes produced in glucose uptake. We have also analyzed the heart subcellular distribution of both carriers, as available information about this subject is contradictory with regard to the GLUT-1 isoform. It is well known, as shown by our data, that protein abundance is much higher in intracellular than in surface membranes. To quantify heart GLUT-4 and GLUT-1 levels by Western blotting, equivalent amounts of proteins from both preparations have been loaded. Considering this fact and the differences obtained when we compare the content of each GLUT isoform between surface and intracellular fractions, it can be concluded that these carriers are more abundant inside heart muscle fibers than in sarcolemma, a well established fact for GLUT-4. The location of GLUT-1 in heart was less clear, as it has mostly been found as much in plasma (30) as in intracellular membranes (31) from isolated, but not contracting, cardiomyocytes. Our results were obtained in working hearts and thus support the statement that a substantial part of cardiac GLUT-1 remains inside the cell in vivo. This is in contrast to the skeletal muscle fibers, where GLUT-1 is mainly found in the sarcolemma even in an unstimulated condition (8).

The present study shows that chronic food restriction reduces the cardiac content and mRNA expression of GLUT-4 and GLUT-1, suggesting that these down-regulations occur primarily at the transcriptional step. Cardiac abundance of glucose carriers changes in response to several physiopathological conditions. Fasting and diabetes repress GLUT-4 and GLUT-1 levels, thus limiting glucose availability (reviewed in Ref. 29), whereas ischemia and hypoxia increase myocardial GLUT-1 expression (12). Recently, a low cardiac gene expression of both carriers has been found in rats born with utero-placental insufficiency, suggesting that these changes could be programmed in utero (14). The regulation of cardiac expression of both GLUTs is not well understood; plasma insulin, which is low in undernourished rats (18, 19), seems to play a major role in vivo on GLUT-1 and has less influence on GLUT-4 (27, 28).

The findings described herein confirm that both GLUT-4 and GLUT-1 are redistributed to heart plasma membrane following insulin treatment. Concerning GLUT-1, this response has been previously reported in cardiomyocytes or perfused working rat heart preparations (reviewed in Ref. 29), but in vivo only in canine heart (32), and contrasts with the lack of such an effect in skeletal muscle (18). In the case of GLUT-4, however, it is a well known event for both muscles. According to our data, the relative amount of GLUT-1 translocated to plasma membrane is higher in food-restricted rats than in controls, considering the large decrease in the intracellular reserve of this isoform. Such an improvement is not produced in GLUT-4 translocation, which is proportionally similar, relative to the basal values, in both groups of animals. GLUT-3, preferentially expressed in cells with high energy demand, is present in rat heart, but in lower proportion than GLUT-4 and GLUT-1, a fact previously reported in human myocardium (33). Little is known about heart GLUT-3 regulation. The present work is the first to show that food restriction reduces this carrier without altering the corresponding mRNA level, in contrast to the effect of other perturbations, such as glucose deprivation (34) or hypoxia (35), which increase GLUT-3 in rat brain. This carrier is the most efficient among the different GLUTs, as it has the lowest Km (36). However, its reduction does not prevent the improved heart glucose uptake associated with undernutrition, probably because it is a minor isoform.

Our results suggest that the most likely cause of improved myocardial glucose uptake found in food-restricted rats is the increased GLUT-1 content in plasma membrane, its functional site. This proposal is consistent with the important role played by this transporter isoform in the heart. In skeletal muscle GLUT-1 accounts for only 5–10% of total glucose carriers, but in rat cardiomyocytes it accounts for 30% (31). In addition, cardiac GLUT-1 resides largely within these cells, near structures in contact with the extracellular space, in sharp contrast to the skeletal muscle in which most of it is present in the nerve sheets (30, 37). Laybutt et al. (28) have shown a close association between cardiac GLUT-1 content and glucose utilization rate in basal as well as insulin-stimulated conditions. Finally, it has been shown that in mice in which cardiac GLUT-4 expression is abolished, both GLUT-1 and basal glucose uptake are increased in a similar magnitude (38).

PI 3-kinase plays an essential role in insulin-stimulated glucose transport and GLUT-4/GLUT-1 translocation (39). Our results show a good correlation between both basal and stimulated heart glucose uptake in undernourished rats and the activity of this enzyme, all of which are greater than control values. p85{alpha} and p85ß proteins are the main regulatory subunits of PI 3-kinase present in rat cardiomyocytes, in which the splice variants of p85{alpha} are undetectable (40). It has been recently shown that reduced expression of either p85{alpha} or p85ß increases insulin sensitivity in mice, suggesting that these adapter subunits play negative roles in both liver and skeletal muscle insulin signaling (41, 42). The mechanisms proposed are based on competition between these monomeric isoforms and the p85-p110 heterodimer to bind to phosphorylated IRS proteins and a stimulation of PI 3-phosphate clearance (41, 42). In the present work, however, we found that both p85 cardiac isoforms are increased in food-restricted rats, but, concomitantly, PI 3-kinase activity and glucose uptake remain greater than the control values in basal as well as insulin-stimulated conditions. These results can be explained in the light of recent data indicating that p85{alpha} protein is not recruited to IRS-1/2 in isolated cardiomyocytes (39, 40) or cardiac muscle (40) in response to insulin or contraction; rather, an as yet unidentified 200-kDa tyrosine phosphorylated protein associates with this adapter subunit, which also seems to be the one specifically involved in GLUT-4 translocation (39, 40). Possible effects of increases in p85{alpha} or undernutrition on the content or phosphorylation of this 200-kDa protein are unknown. The p85 ß-isoform associates with IRSs in cardiac muscle and activates PI 3-kinase, but it seems to be implicated in other insulin responses (40). In fact, the precise roles of both isoforms in insulin actions are unclear (43). Therefore, the PI 3-kinase stimulation by insulin, leading to the improvement in glucose uptake, may not be under-regulated by these monomeric adapter subunits in cardiac muscle, a proposal consistent with the results obtained in the present work. Cardiac IRS-1 as well as p110{alpha} subunit of PI 3-kinase remain unchanged by undernutrition; however, Akt abundance in heart is increased in such a condition, which explains, together with the higher PI 3-kinase activity, why phosphorylation of Akt was remarkably increased after insulin treatment.

According to our data, the better insulin response derived from this up-regulation of cardiac insulin signaling in undernourished rats is mainly established regarding GLUT-1 redistribution, rather than that of GLUT-4. Both transporters probably share most of signaling mechanisms, but not necessarily all. In fact, little is known about GLUT-1 redistribution, because most of the studies have only focused on GLUT-4. Recently, a hypothesis has been raised indicating that cardiac intracellular GLUT-4 and GLUT-1 may be in not completely mixed pools, with different insulin response capacities (44). It is conceivable that these reserves could be differently affected by physiopathological conditions, and if so, undernutrition might specifically enhance the cardiac response to insulin, improving GLUT-1 translocation.

The present results do not rule out the contributions of other factors to improved heart glucose uptake associated with undernutrition, such as changes in the intrinsic activity of glucose carriers. Moreover, glucose utilization is regulated at multiple sites, which may be affected by food restriction, such as myocardial blood flow or both hexokinase activity and binding to mitochondria (45). Recent reports suggest that 5'-AMP-activated protein kinase (AMPK) plays an important role in heart energy homeostasis and substrate utilization. Hypoxia, ischemia, and heart hypertrophy activate cardiac AMPK and increase glucose uptake (46); this also occurs after pharmacological stimulation in cardiomyocytes, promoting GLUT-4 translocation (47). AMPK activation in rat heart is antagonized by insulin (48), which suggests that AMPK may be increased in hypoinsulinemic food-restricted rats, contributing to the enhancement of basal glucose uptake. We have determined cyclic nucleotide phosphodiesterase I to be a plasma membrane marker (26) and have shown that it is increased in heart by undernutrition, as previously reported in skeletal muscle (18). The physiological relevance of this result deserves further attention. The superfamily of cyclic nucleotide phosphodiesterases catalyzes the hydrolysis of cAMP and cGMP and plays an important role in regulating their intracellular levels. As recently shown, cGMP inhibits basal and insulin-stimulated glucose utilization in cardiomyocytes, interfering with GLUT-4 translocation (49). Thus, the increase in phosphodiesterase may play a role in the improvement of cardiac glucose uptake associated with food restriction.

The present study shows that undernutrition enhances the rate of heart glucose uptake and supports the proposal that it is due at least partly to an increase in GLUT-1 location at the surface membrane. This adaptation would allow cardiac myocytes to use greater amounts of glucose when food shortage is imposed. Moreover, the slight, but significant, increase in the heart weight to body weight ratio in undernourished rats suggests some degree of mass sparing. Some of the features of this cardiac adaptation are similar to those seen in hypoxia, which induces an increase in GLUT-1 (30). A different overall state occurs in diabetes, which leads to a reduction in both cardiac glucose transport and metabolism (50) and to a failure of insulin-regulated GLUT-4 recruitment (51). These alterations contribute to contractile dysfunction in diabetic patients, in part because cardiac membrane ion channels are preferentially regulated by glycolytic ATP production (52). Moreover, an increased use of free fatty acids and their toxic intermediates has been associated with diabetic cardiomyopathies (53). The type of adaptation reported in this study may be beneficial for reducing possible detrimental effects of chronic food restriction on cardiac fibers.


   Acknowledgments
 
The authors are indebted to Dr. M. Lorenzo and C. De Alvaro for help with the Northern blot analyses. We thank S. Fajardo for technical assistance.


   Footnotes
 
This work was supported in part by a Grant from Dirección General de Investigación Científica y Técnica, Ministerio de Educación y Cultura, Spain (Ref. No. BF12001-2125).

Abbreviations: AMPK, 5'-AMP-activated protein kinase; GLUT, glucose transporter; IRS-1, insulin-receptor substrate-1; Na+,K+-ATPase, Na+,K+-adenosine triphosphatase; PI 3-kinase, phosphoinositol 3-kinase; PMSF, phenylmethylsulfonylfluoride; PVDF, polyvinylidene difluoride.

Received March 4, 2002.

Accepted for publication July 9, 2002.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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