Calorie restriction increases cell surface GLUT-4 in insulin-stimulated skeletal muscle

David J. Dean1, Joseph T. Brozinick Jr.2, Samuel W. Cushman2, and Gregory D. Cartee1

1 Biodynamics Laboratory, Department of Kinesiology and Department of Nutritional Sciences, University of Wisconsin, Madison, Wisconsin 53706; and 2 Experimental Diabetes, Metabolism, and Nutrition Section, Diabetes Branch, National Institute of Diabetes and Digestive and Kidney Diseases, Bethesda, Maryland 20892

    ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Reduced calorie intake [calorie restriction (CR); 60% of ad libitum (AL)] leads to enhanced glucose transport without altering total GLUT-4 glucose transporter abundance in skeletal muscle. Therefore, we tested the hypothesis that CR (20 days) alters the subcellular distribution of GLUT-4. Cell surface GLUT-4 content was higher in insulin-stimulated epitrochlearis muscles from CR vs. AL rats. The magnitude of this increase was similar to the CR-induced increase in glucose transport, and GLUT-4 activity (glucose transport rate divided by cell surface GLUT-4) was unaffected by diet. The CR effect was specific to the insulin-mediated pathway, as evidenced by the observations that basal glucose transport and cell surface GLUT-4 content, as well as hypoxia-stimulated glucose transport, were unchanged by diet. CR did not alter insulin's stimulation of insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity. Muscle abundance of IRS-2 and p85 subunit of PI3K were unaltered by diet, but IRS-1 content was lower in CR vs. AL. These data demonstrate that, despite IRS-1-PI3K activity similar to AL, CR specifically increases insulin's activation of glucose transport by enhancing the steady-state proportion of GLUT-4 residing on the cell surface.

food restriction; insulin signaling

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

UNDERSTANDING the adaptive responses of organisms to the ebb and flow of energy availability has fundamental physiological importance. A cluster of pathophysiological outcomes (including insulin resistance, obesity, and type 2 diabetes mellitus) is intimately related to a chronic, positive energy balance, and many studies have focused on conditions of caloric overabundance. Previous research has also revealed that an energy deficit has an opposite effect on many of the same metabolic processes: e.g., moderate calorie restriction [CR; consuming ~60-75% of ad libitum (AL) intake] improves insulin action in various species including rats, rhesus monkeys, and humans (7, 18, 25, 28, 40).

Skeletal muscle accounts for most of the whole body insulin-mediated glucose clearance (16, 26), and glucose transport appears to be the rate-limiting step of glucose metabolism in skeletal muscle (45). Therefore, a pivotal adaptation to CR is the enhanced insulin-stimulated glucose transport in skeletal muscle (10, 15). This adaptation can occur rather rapidly, within 5-20 days of CR (7, 15), but very little is known about the cellular and molecular mechanisms that underlie this functional change. Therefore, the long-range goal of our research is to elucidate the mechanisms underlying the CR-induced increase in the insulin-mediated glucose transport by skeletal muscle.

GLUT-4, the predominant glucose transporter expressed by skeletal muscle, appears to account for most or all of the insulin-stimulated glucose transport in muscle. A close relationship between muscle GLUT-4 content and capacity for insulin-stimulated glucose transport has been observed (21), but we have found that CR does not alter muscle total GLUT-4 content (10). Under AL-fed conditions, the insulin-stimulated increase in glucose transport rate appears to be accounted for by an increase in the abundance of GLUT-4 residing on the cell surface (39). A reasonable hypothesis was that CR enhances insulin-stimulated glucose transport by increasing the translocation of GLUT-4 to the cell surface. Therefore, we evaluated the effect of CR on cell surface GLUT-4 content by exofacial photolabeling with 2-N-4-(1-azi-2,2,2-trifluoroethyl)-benzoyl-1,3-bis-(D-mannos-4-yloxy)-2-propylamine (ATB-BMPA) in basal and insulin-stimulated muscles.

We also evaluated several other aspects of the CR-induced increase in glucose transport. In skeletal muscle, contractile activity and in vitro hypoxia increase glucose transport via a pathway distinct from that activated by insulin. The maximal effect of insulin on glucose transport is additive with contractile activity (13) or hypoxia (8), and wortmannin [a specific phosphatidylinositol 3-kinase (PI3K) inhibitor] entirely prevents insulin-, but not contraction- or hypoxia-stimulated glucose transport (30, 43). As with insulin, contractile activity and hypoxia increase glucose transport by a mechanism involving increased appearance of GLUT-4 on the cell surface (8, 17). Thus it was possible that CR acted by a nonspecific mechanism, such as altering the susceptibility of the GLUT-4 translocation "machinery" to both insulin-dependent and insulin-independent signaling. Accordingly, we evaluated the influence of CR on glucose transport activated by in vitro hypoxia, which stimulates glucose transport by an insulin-independent pathway.

Insulin receptor substrate (IRS)-1 is an important substrate for the insulin receptor kinase (38). The tyrosine-phosphorylated IRS-1 engages PI3K (38), and PI3K activity is essential for insulin-stimulated, but not hypoxia-stimulated, glucose transport in skeletal muscle (43). Therefore, to evaluate the effect of CR on a key aspect of the insulin signaling pathway, we also measured the PI3K activity associated with IRS-1 and the abundance of three important insulin signaling proteins in skeletal muscle: IRS-1, IRS-2, and the p85 subunit of PI3K.

Our results indicate that brief CR caused a specific increase in insulin-stimulated glucose transport rate without any effect on hypoxia-stimulated glucose transport. The increased glucose transport was largely attributable to more cell surface GLUT-4 in the insulin-stimulated muscles of CR compared with AL rats, but it was independent of a significant diet effect on glycogen concentration or IRS-1-associated PI3K activity in epitrochlearis muscles.

    MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Treatment of animals. Male Fischer 344 (F344) rats (Harlan Sprague Dawley, Indianapolis, IN) were housed singly on a 12:12-h light- (0600-1800) dark (1800-0600) cycle. After the animals had been in the Madison facility for 5-7 days, daily food consumption (NIH-31, PMI Feeds, St. Louis, MO) was monitored for 5-7 days to establish individual baseline food intake. Food consumption was determined by weighing the food provided and correcting for the amount of food not eaten, including spillage. Animals were randomly assigned to either AL or CR groups. CR animals received an allotment of food (NIH-31 supplemented with sufficient vitamins to provide equivalent intake in CR and AL rats; PMI Feeds) at 1800 that was expected to result in a food consumption equal to ~60% of AL food intake. The duration of CR was 20 days.

On the day they were killed, animals (~5.5 mo old) were weighed, and between 1300 and 1800 they were anesthetized with an intraperitoneal injection of pentobarbital sodium (60 mg/kg body wt). The order of anesthetization was randomized so that the mean time of tissue sampling did not vary between the groups. Blood, sampled by cutting the tip of the tail, was used for determination of glucose concentration. Both epitrochlearis muscles were rapidly dissected out. Blood was then sampled via cardiac puncture and subsequently used for determination of plasma insulin concentration.

Determination of blood glucose concentration. Blood glucose was determined by the glucose oxidase method with an ExacTech blood glucose sensor (Medisense, Cambridge, MA).

Determination of plasma insulin concentration. Plasma insulin levels were quantified in duplicate with a radioimmunoassay, with rat insulin as the standard (Linco, St. Louis, MO).

Incubation of isolated epitrochlearis muscles. For all incubation steps, isolated epitrochlearis muscles were placed in 25-ml Erlenmeyer flasks containing Krebs-Henseleit buffer (KHB) (29) including 0.1% bovine serum albumin (BSA) and further supplemented as we will describe. Flasks were placed in temperature-controlled shaking water baths and continuously gassed with either 95% O2-5% CO2 or 95% N2-5% CO2 (hypoxia).

Both epitrochlearis muscles were dissected from the rats, and each muscle was initially placed in Erlenmeyer flasks containing 3 ml of KHB-0.1% BSA supplemented with 2 mM sodium pyruvate and 6 mM mannitol; the flasks were shaken for 30 min in a water bath maintained at 30°C. Muscles were incubated in the absence of any stimulus (i.e., basal), in the presence of insulin (200 or 20,000 µU/ml), or under hypoxia (15 or 30 min). After the initial incubation period, muscles were transferred to a second flask for determination of 3-O-methylglucose (3-MG) accumulation or cell surface GLUT-4 quantitation (as described in Determination of muscle 3-MG transport rate and Determination of cell surface GLUT-4 content).

Determination of muscle 3-MG transport rate. After the initial incubation period, the rate of 3-MG accumulation was determined essentially as previously described (44). Briefly, muscles were incubated for 15 min with shaking at 30°C in flasks containing 2 ml of KHB supplemented with 0.1% BSA, 8 mM 3-[3H]MG (437 µCi/mmol), 2 mM [14C]mannitol (8 µCi/mmol), and the same insulin concentration as the preceding step. With the use of epitrochlearis muscles of sizes similar to those employed in this study, we have confirmed that 3-MG accumulation is linear under these experimental conditions (unpublished observations).

At the conclusion of this incubation period, muscles were rapidly blotted on filter paper dampened with ice-cold incubation medium and were frozen between aluminum clamps cooled to the temperature of liquid N2. Muscles were stored at -80°C until subsequently weighed, homogenized in 0.3 M perchloric acid (PCA), and analyzed as previously described (44).

Determination of epitrochlearis glycogen concentration. A portion of the PCA homogenate from epitrochlearis muscles incubated under basal conditions was used for measurement of glycogen concentration (32).

Determination of cell surface GLUT-4 content. After an initial 30-min incubation, muscles were transferred to 10-ml Erlenmeyer flasks containing 1 ml of KHB containing 1 mCi/ml ATB-[3H]BMPA and the same insulin concentration present in the previous incubation (i.e., 0 or 20,000 µU/ml) for 5 min at room temperature. The muscles were transferred to a Rayonet photochemical reactor (RPR100, Southern New England Ultraviolet, Branford, CT) and irradiated at 340 nm for 4 min with manual turning of the muscles after 2 min. After irradiation, muscles were frozen with aluminum tongs cooled to the temperature of liquid N2 and were stored at -80°C until further processed. The procedure for quantitation of ATB-BMPA labeling has been described previously (39). Radioactivity was expressed as count per minute per gram of wet tissue weight.

Treatment of isolated epitrochlearis muscle used for PI3K activity. Isolated epitrochlearis muscles were incubated in vitro in KHB buffer supplemented as described above while continuously gassed with 95% O2-5% CO2. During the initial 30-min incubation, muscles were incubated in the absence of insulin. Muscles were then transferred to flasks containing KHB supplemented as in the previous flask except that insulin (20,000 µU/ml) was included in some of the flasks. Insulin-treated muscles were incubated for 4, 8, 20, or 40 min; control muscles (i.e., without insulin) were incubated for 4 or 40 min. Muscles were then removed, rapidly trimmed, and immediately freeze-clamped and stored at -80°C until further processed.

Homogenization and preparation of muscles for PI3K activity analysis. Muscles were processed essentially as described by Goodyear et al. (19). Briefly, muscles were pulverized at the temperature of liquid N2 and homogenized in ice-cold homogenization buffer (50 mM HEPES, pH 7.5, 150 mM NaCl, 10 mM NaPP, 2 mM Na3VO4, 10 mM NaF, 2 mM EDTA, 2 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 1% Nonidet P-40, and 10% glycerol). The homogenate was centrifuged at 35,000 g for 1 h at 4°C. A portion of the resultant supernatant was used for determination of protein concentration with bicinchoninic acid (BCA; Pierce, Rockford, IL); the remainder was aliquoted and stored at -80°C until used for insulin signaling analysis.

Immunoprecipitation of IRS-1 from muscle supernatant. The IRS-1-bound PI3K activity was determined essentially as previously described (19). For each sample, a 1-mg aliquot of epitrochlearis muscle supernatant was immunoprecipitated overnight at 4°C with gentle rotation in the presence of affinity-purified anti-IRS-1 antibody (Upstate Biotechnology, Lake Placid, NY). To each sample, 100 µl of protein A-sepharose were added and samples were rotated 2 h at 4°C. Samples were centrifuged (14,800 g); the resultant immunopellet was washed three times with PBS (pH = 7.5) containing 1% Nonidet P-40 and 100 µM Na3VO4, three times with 100 mM Tris (pH = 7.5) containing 500 mM LiCl2 and 100 µM Na3VO4, and twice with 10 mM Tris (pH = 7.5), 100 mM NaCl, 1 mM EDTA, and 100 µM Na3VO4.

IRS-1-associated PI3K activity. IRS-1-bound PI3K activity was measured for each sample as described previously (19). Briefly, the immunopellet was resuspended in 50 µl of the Tris · NaCl buffer containing 20 µg of phosphatidylinositol (Avanti Polar Lipids, Alabaster, AL) and 100 mM MgCl2. The reaction was initiated at room temperature by the addition of 5 µl of a phosphorylation mixture containing 880 µM ATP containing 20 µCi [gamma -32P]ATP (Amersham, Arlington Heights, IL). After 20 min with continuous vortexing, the reaction was stopped by the sequential addition of 8 N HCl and chloroform-methanol (1:1). The reaction mixture was vortexed for 5-10 min and then centrifuged at 2,000 g for 3-5 min; 40 µl of the organic phase containing the reaction products were spotted onto an aluminum-backed silica thin-layer chromatography plate pretreated with 1% potassium oxalate solution. The products were resolved in a chloroform-methanol-water-ammonium hydroxide (60:47:11.3:2) solution and visualized by autoradiography. The spots corresponding to the 3'-phosphorylated products were excised from the thin-layer chromatography plate and counted in a scintillation counter.

Determination of IRS-1, IRS-2, and PI3K p85 subunit abundance. Gastrocnemius muscles were prepared and analyzed essentially as described by Goodyear et al. (19). A portion of the supernatant was analyzed for protein content with the BCA assay. The remaining supernatant was combined with Laemmli buffer and boiled for 3-5 min. Duplicate aliquots (20 µg protein) from each sample were subjected to SDS-PAGE with a 7.5% resolving gel. Proteins were electrophoretically transferred to 0.45-µm Immobilon-P membranes (Millipore, Bedford, MA). Membranes were blocked for 1 h at room temperature with phosphate-buffered saline (PBST; 80 mM Na2HPO4, 100 mM NaCl, and 0.1% Tween-20, pH 7.5) containing 5% nonfat dry milk. Membranes were rinsed three times with PBST at room temperature and then incubated overnight at 4°C in PBST containing 1% milk, 0.02% NaN3, and the appropriate primary antibody (IRS-1: 1,000-fold dilution of rabbit polyclonal antibody against 14 amino acid carboxy terminus, Upstate Biotechnology; PI3K: 1,667-fold dilution of antibody against the entire p85 subunit and SH2 region of the p85 subunit, Upstate Biotechnology; IRS-2: 500-fold dilution of antibody against a glutathione S-transferase fusion protein consisting of amino acids 976-1094 of IRS-2, kindly provided by Dr. M. F. White). Membranes were then rinsed three times at room temperature with PBST followed by PBST with 1% nonfat dry milk and a 5,000-fold dilution of horseradish peroxidase-conjugated goat anti-rabbit antibody (Jackson Immunoresearch Laboratories, West Grove, PA) for 1 h at room temperature. The membranes were then rinsed five times in PBST and incubated in enhanced chemiluminescence (ECL; Amersham, Arlington Heights, IL) for 1 min at room temperature. Blots were exposed to Hyperfilm (Amersham), and the IRS-1, PI3K, and IRS-2 were quantified with a Bio-Rad (Hercules, CA) scanning densitometer. For each protein, the amount of sample loaded on the gel was linearly related (r >=  0.94) to the signal determined by densitometry. Values are expressed relative to the mean value for the AL group (mean AL = 100%).

Statistics. Unless otherwise stated, comparisons between AL and CR groups were made with a two-tailed t-test; differences were accepted as statistically significant at the P <=  0.05 level.

    RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Food intake and body weight. The CR rats ate 56% as much as their AL cohorts (Table 1). The body weight of CR animals was 81% of AL animals.

                              
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Table 1.   Body weight, food intake, blood glucose, and plasma insulin

Blood glucose and plasma insulin. CR was associated with a reduction in blood glucose of 12% (Table 1). Plasma insulin concentration was 69% lower in CR vs. AL.

Basal and insulin- and hypoxia-stimulated 3-MG transport rates. Basal 3-MG transport was not significantly altered by CR (Table 2). CR led to marked differences in glucose transport compared with AL values with 200 µU/ml (Table 2) or 20,000 µU/ml (Fig. 1A) insulin. In contrast, hypoxia-stimulated glucose transport rates were similar between muscles from CR and AL animals (Table 2).

                              
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Table 2.   3-O-Methylglucose transport rate in isolated epitrochlearis muscles


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Fig. 1.   A: basal and insulin-stimulated (20,000 µU/ml) 3-O-methylglucose (3-MG) transport rate. * P < 0.05 vs. ad libitum (AL) for insulin-stimulated 3-MG transport. B: cell surface GLUT-4 content in isolated epitrochlearis muscles from AL and calorie-restricted (CR) rats. * P < 0.05 CR vs. AL for insulin-stimulated cell surface GLUT-4 with a one-tailed t-test. C: GLUT-4 transporter activity (3-MG transport rate / cell surface GLUT-4) in insulin-stimulated muscles. One muscle from each rat was used to measure 3-MG transport, whereas contralateral muscle was used to assess cell surface GLUT-4 content. Values are means ± SE for 10 muscles. cpm, Counts per minute.

Muscle glycogen concentration. AL and CR epitrochlearis muscle glycogen concentrations were not significantly different (21.1 ± 2.4 and 18.2 ± 1.8 µmol/g, respectively).

Cell surface GLUT-4 content and glucose transport rate. The basal 3-MG transport rate (Fig. 1A) and basal cell surface GLUT-4 content (Fig. 1B) were not significantly altered by diet. Consistent with the results of earlier studies, insulin-stimulated 3-MG transport was significantly increased for muscles from CR vs. AL rats (Fig. 1A). Because we had demonstrated this CR-induced increase in the insulin-stimulated 3-MG transport before processing the contralateral muscles for cell surface GLUT-4 content, we used a one-tailed t-test to compare the AL and CR groups for the amount of cell surface GLUT-4 in insulin-stimulated muscles. CR muscles incubated in the presence of 20,000 µU/ml insulin had significantly more GLUT-4 at the cell surface compared with muscles from the AL group (Fig. 1B). Because we measured cell surface GLUT-4 content and 3-MG transport rate in paired muscles from the same rats, we were able to evaluate GLUT-4 glucose transporter activity for each muscle pair: the 3-MG transport rate (nmol · g-1 · 15 min-1) determined for each muscle was divided by the cell surface GLUT-4 (counts · min-1 · g-1) determined for the contralateral muscle. GLUT-4 glucose transporter activity was not significantly different for insulin-stimulated muscles from CR vs. AL rats (Fig. 1C). There was also no significant difference between AL and CR groups for GLUT-4 activity determined in basal muscles or for the insulin-stimulation above basal (determined by subtracting the mean value of the group for 3-MG transport and cell surface GLUT-4 under basal conditions, respectively, from the corresponding values for individual insulin-stimulated muscles, and calculating the 3-MG-to-GLUT-4 ratio with these Delta  values; results not shown).

IRS-1-associated PI3K activity. IRS-1-associated PI3K activity in muscles incubated without added insulin did not differ between AL and CR groups (Fig. 2). As expected, muscles incubated with insulin had a higher IRS-1-associated PI3K activity compared with the basal controls. The time course for this increase was quite similar between the diet groups. A nonsignificant trend was observed for somewhat higher values (~30%) for insulin-stimulated muscles from the CR vs. AL rats at 8, 20, and 40 min, but at no time point was the difference significant. If the values for these three time points are pooled (n = 17-18 per diet group), the trend still does not attain statistical significance.


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Fig. 2.   Basal and insulin-stimulated (20,000 µU/ml) insulin receptor substrate (IRS)-1-associated phosphatidylinositol 3-kinase (PI3K) activity in isolated epitrochlearis muscles from AL and CR rats. Values are means ± SE for 4-6 muscles.

IRS-1, IRS-2, and PI3K p85 subunit abundance. In CR compared with AL rats, IRS-1 abundance was 50% lower in gastrocnemius muscles (P < 0.05; Fig. 3A). Muscle levels of IRS-2 (Fig. 3B) and the p85 subunit of PI3K (Fig. 3C) did not differ between the diet groups.


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Fig. 3.   Effect of diet (AL vs. CR) on skeletal muscle IRS-1 (A), IRS-2 (B), and PI3K p85 subunit (C) abundance. Representative immunoblots are shown above values (means ± SE) for 6-10 muscles.

    DISCUSSION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References

Altered energy availability is a matter of fundamental biological importance, and reduced calorie intake has profound effects on glucose homeostasis and insulin action. The results of this experiment provide important new insights regarding the mechanisms by which CR amplifies insulin stimulation of glucose transport. We have previously demonstrated that CR does not alter total GLUT-4 levels in epitrochlearis or gastrocnemius muscles (10). This observation suggested that CR enhances the ability of insulin to increase the number of glucose transporters at the cell surface and/or the activity of those glucose transporters on the cell surface (i.e., glucose flux per transporter at the surface). Our results indicate that the CR-induced enhancement in insulin-stimulated glucose transport is the result of more GLUT-4 transporters at the cell surface rather than enhanced GLUT-4 activity: the abundance of cell surface GLUT-4 content was increased in CR vs. AL muscles, and GLUT-4 activity (glucose transport rate divided by cell surface GLUT-4) was similar for CR compared with AL muscles. Insulin increases the glucose transport rate of adipocytes and myocytes by activating the redistribution of GLUT-4 glucose transporters from intracellular vesicles to the cell surface. In adipocytes, the insulin-mediated redistribution appears to be primarily the result of enhanced exocytosis of GLUT-4-containing vesicles (35). Our data indicate that the steady-state proportion of GLUT-4 residing in the cell surface of insulin-stimulated muscles is greater in CR vs. AL animals.

GLUT-4 is the predominant glucose transporter expressed by skeletal muscle, but a small amount of GLUT-1 is also present, primarily in the perineural sheaths (20). Therefore, total glucose transport equals the sum of GLUT-1-mediated and GLUT-4-mediated glucose transport. Because insulin induces little or no detectable translocation of GLUT-1 in skeletal muscle (17, 31), GLUT-1 is more important for basal than insulin-stimulated glucose transport rate. Basal glucose transport and cell surface GLUT-4 content were virtually identical between the diet groups, suggesting little or no diet-related change in GLUT-1 function. In insulin-stimulated muscle, the abundance of GLUT-4 is more than fivefold that of GLUT-1 (31), and intrinsic activity of GLUT-4 is about threefold that of GLUT-1 (22). In this context, our calculated values of GLUT-4 activity (which were determined with total glucose transport rate) are probably slight overestimates, but this would be minimized by the increase in cell surface GLUT-4 in insulin-stimulated muscles, and it seems unlikely that GLUT-1 is quantitatively important for the CR-induced increase in insulin-stimulated glucose transport rate.

In light of the reduced concentrations of circulating glucose and insulin with CR, it was reasonable to suspect that muscle glycogen concentration would also be reduced. A reciprocal relationship between glucose transport rate and muscle glycogen concentration has been noted under some conditions (e.g., after exercise), so a decline in glycogen stores might be important for the CR-induced increase in glucose transport. However, glycogen concentration did not differ between the diet groups, demonstrating that the CR effect on glucose transport was not secondary to muscle glycogen depletion. The preservation of muscle glycogen in CR rats, despite moderately lower glycemia and markedly reduced insulinemia, was presumably due, at least in part, to an enhanced capacity for insulin-stimulated glucose transport.

In skeletal muscle, hypoxia stimulates glucose transport via a pathway that is distinct from that activated by insulin, as evidenced by the observations that the maximal effects of insulin and hypoxia on glucose transport are additive (8), and low concentrations of wortmannin, which can entirely prevent insulin-stimulated glucose transport, do not affect hypoxia-stimulated glucose transport (30, 43). We evaluated hypoxia-stimulated glucose transport because it seemed possible that CR might induce a nonspecific alteration (e.g., membrane fluidity) that would lead to enhanced activation of glucose transport by various stimuli. Our finding that CR enhances insulin-mediated, but not basal or hypoxia-stimulated, glucose transport indicates that CR acts specifically on the insulin-stimulated pathway. Type 2 diabetes also has a specific effect on the insulin-stimulated pathway but in the converse direction: muscle strips from people with type 2 diabetes were profoundly insulin resistant yet retained a robust stimulation of glucose transport by in vitro hypoxia (4).

In this context, it is interesting that a moderate reduction in calorie intake and/or increased physical activity is commonly recommended for treating individuals with insulin resistance (1, 2), and the benefits of these interventions are due, at least in part, to an increase in skeletal muscle glucose transport (7, 11). Our current results, taken together with the results of earlier studies of exercise, reveal the distinctive characteristics of the two interventions. Muscle glycogen depletion during vigorous exercise appears to be crucial for the insulin-independent, postexercise increase in glucose transport (24). By contrast, 20 days of CR did not elicit alterations in glycogen concentration or basal glucose transport. The specific enhancement in the activation of glucose transport via the insulin pathway after CR also differs from the postexercise condition, which is characterized by a nonspecific increase in susceptibility to activation by various stimuli, including in vitro hypoxia (9). Exercise and CR appear to enhance glucose transport by separate mechanisms, indicating that the combination of the two interventions might result in an additive increase in glucose disposal.

The specificity of the effect of CR on the insulin pathway suggested that CR influences the insulin signal transduction system. Brief CR does not alter the number, binding affinity, or tyrosine kinase activity of insulin receptors from rat skeletal muscle (5, 12). Accordingly, we focused on postreceptor insulin signaling (IRS-1-associated PI3K activity) and, consistent with other studies (19, 42, 46), found that insulin led to a substantial increase above basal values. Our novel findings were that CR altered neither the magnitude nor the time course for insulin's stimulation of IRS-1-associated PI3K activity. Possible explanations for the CR-induced increase in insulin-stimulated glucose transport include the observations that 1) CR acts on a pathway that does not involve IRS-1-associated PI3K (e.g., IRS-2-associated PI3K), 2) CR amplifies steps distal to IRS-1-associated PI3K, or 3) CR alters the subcellular localization of a portion of the IRS-1-associated PI3K. Regarding the third possibility, there is evidence supporting the concept that the subcellular localization of the PI3K activity is functionally important for insulin signal transduction and action (23, 27). We focused on a maximally effective insulin concentration in this study, so our results do not address the possible influence of CR on insulin signaling with submaximally effective concentrations.

We also measured the abundance of three proteins that are instrumental for insulin signaling (IRS-1, IRS-2, and p85 PI3K). As in the epitrochlearis, insulin-stimulated glucose uptake by the gastrocnemius is increased above AL levels after brief CR (14), and we previously demonstrated that CR does not alter the GLUT-4 protein expression in either the gastrocnemius or epitrochlearis (10). The fiber-type composition of the gastrocnemius (74% fast glycolytic, FG; 20% fast oxidative glycolytic, FOG; 6% slow oxidative, SO) is almost identical to the epitrochlearis (75% FG; 17% FOG; 8% SO), and each muscle corresponds quite closely to the average fiber-type composition (76% FG; 19% FOG; 5% SO) of all the muscles in the rat hindlimb (3, 37). The CR-induced increase in glucose uptake by the gastrocnemius (14) also corresponds with the effect of CR on a large portion of the total muscle mass, as evidenced by the induction of substantial increases in whole body glucose clearance (34) and glucose uptake by the perfused hindlimb (25). Taken together, these results indicate that the gastrocnemius and epitrochlearis are representative muscles for evaluating the effects of CR. There was no effect of diet on muscle concentration of the p85 subunit of PI3K, but the abundance of IRS-1 protein was substantially lower in muscles from CR compared with AL rats. It seems unlikely that a reduction in IRS-1 per se leads to increased insulin action; e.g., mice heterozygous for the IRS-1 null allele have an ~50% reduction in muscle IRS-1 (comparable to the difference between AL and CR), but the heterozygous (IRS-1 +/-) mice are not more insulin sensitive than wild-type (IRS-1 +/+) littermates (6).

Recent research has identified a family of IRS proteins, including IRS-1, IRS-2, IRS-3, and IRS-4. Information about the expression of IRS-4 by skeletal muscle has apparently not been published, but IRS-3 is reportedly not expressed by skeletal muscle (36). IRS-1 and IRS-2 are members of the IRS family known to be expressed by skeletal muscle (33). IRS-2 is relevant to glucoregulation, as evidenced by the finding that IRS-2 null mice become diabetic and insulin resistant (41). Accordingly, we measured the abundance of IRS-2 in the gastrocnemius (which has a representative fiber-type composition and which exhibits increased insulin-stimulated glucose uptake with brief CR) and found that the quantity of IRS-2 was similar in muscles from AL and CR rats, arguing against the possibility that altered IRS-2 expression accounts for the increased insulin action. It remains possible that a qualitative change in IRS-2 (e.g., phosphorylation status, subcellular distribution, and/or association with PI3K) plays a role in the increased glucose transport.

In conclusion, the CR-induced enhancement of insulin action in skeletal muscle is well-known, but few studies have addressed the cellular and molecular processes underlying this fundamentally important aspect of physiology. Our data provide new insights into the mechanism for increased glucose transport in skeletal muscle of CR animals, including that this effect is independent of 1) a change in glycogen concentration; 2) an altered magnitude or time course of IRS-1-PI3K activity; 3) an increased expression of IRS-1, IRS-2, or p85 PI3K proteins; and 4) an increased GLUT-4 activity. Furthermore, our data demonstrated that the CR effect on glucose transport is specific to the insulin pathway and is attributable to an increase in the amount of GLUT-4 translocated to the cell surface in response to insulin stimulation.

    ACKNOWLEDGEMENTS

We thank Dr. Morris F. White for generously providing antibody against IRS-2, and Dr. Laurie J. Goodyear for advice regarding the measurement of IRS-1-associated PI3K activity.

    FOOTNOTES

This research was supported by National Institute on Aging Grant AG-10026 to G. D. Cartee. D. J. Dean received support from the Gatorade Sports Science Institute Student Research Grant Program.

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. §1734 solely to indicate this fact.

Address for reprint requests: G. D. Cartee, Biodynamics Laboratory, University of Wisconsin-Madison, 2000 Observatory Dr., Madison, WI 53706.

Received 20 May 1998; accepted in final form 13 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.  American Diabetes Association. Nutrition recommendations and principles for people with diabetes mellitus. Diabetes Care 21, Suppl.: S32-S39, 1998.

2.  American Diabetes Association. Diabetes mellitus and exercise. Diabetes Care 21, Suppl.: S40-S44, 1998.

3.   Armstrong, R. B., and R. O. Phelps. Muscle fiber type composition of the rat hindlimb. Am. J. Anat. 171: 259-272, 1984[Medline].

4.   Azevedo, J. L., J. O. Carey, W. J. Pories, P. G. Morris, and G. L. Dohm. Hypoxia stimulates glucose transport in insulin-resistant human muscle. Diabetes 44: 695-698, 1995[Abstract].

5.   Balage, M., J. Grizard, and M. Manin. Effect of calorie restriction on skeletal muscle and liver insulin binding in growing rat. Horm. Metab. Res. 22: 207-214, 1990[Medline].

6.   Bruning, J. C., J. Winnay, D. Accili, and C. R. Kahn. Development of a novel polygenic model of NIDDM in mice heterozygous for IR and IRS-1 null alleles. Cell 88: 561-572, 1997[Medline].

7.   Cartee, G. D., and D. J. Dean. Glucose transport with brief dietary restriction: heterogenous responses in muscles. Am. J. Physiol. 266 (Endocrinol. Metab. 29): E946-E952, 1994[Abstract/Free Full Text].

8.   Cartee, G. D., A. G. Douen, T. Ramlal, A. Klip, and J. O. Holloszy. Stimulation of glucose transport in skeletal muscle by hypoxia. J. Appl. Physiol. 70: 1593-1600, 1991[Abstract/Free Full Text].

9.   Cartee, G. D., and J. O. Holloszy. Exercise increases susceptibility of muscle glucose transport to activation by various stimuli. Am. J. Physiol. 258 (Endocrinol. Metab. 21): E390-E393, 1990[Abstract/Free Full Text].

10.   Cartee, G. D., E. W. Kietzke, and C. Briggs-Tung. Adaptation of muscle glucose transport with caloric restriction in adult, middle-aged, and old rats. Am. J. Physiol. 266 (Regulatory Integrative Comp. Physiol. 35): R1443-R1447, 1994[Abstract/Free Full Text].

11.   Cartee, G. D., D. A. Young, M. D. Sleeper, J. Zierath, H. Wallberg-Henriksson, and J. O. Holloszy. Prolonged increase in insulin-stimulated glucose transport in muscle after exercise. Am. J. Physiol. 256 (Endocrinol. Metab. 19): E494-E499, 1989[Abstract/Free Full Text].

12.   Cecchin, F., O. Ittoop, M. K. Sinha, and J. F. Caro. Insulin resistance in uremia: insulin receptor kinase activity in liver and muscle from chronic uremic rats. Am. J. Physiol. 254 (Endocrinol. Metab. 17): E394-E401, 1988[Abstract/Free Full Text].

13.   Constable, S. H., R. J. Favier, G. D. Cartee, D. A. Young, and J. O. Holloszy. Muscle glucose transport: interactions of in vitro contractions, insulin, and exercise. J. Appl. Physiol. 64: 2329-2332, 1988[Abstract/Free Full Text].

14.   Cusin, I., K. E. Zakrzewska, O. Boss, P. Muzzin, J.-P. Giacobino, D. Ricquier, B. Jeanrenaud, and F. Rohner-Jeanrenaud. Chronic central leptin infusion enhances insulin-stimulated glucose metabolism and favors the expression of uncoupling proteins. Diabetes 47: 1014-1019, 1998[Abstract].

15.   Dean, D. J., and G. D. Cartee. Brief dietary restriction increases skeletal muscle glucose transport in old Fischer 344 rats. J. Gerontol. A Biol. Sci. Med. Sci. 51: B208-B213, 1996[Abstract].

16.   DeFronzo, R. A., E. Jacot, E. Jequier, E. Maeder, J. Wahren, and J. P. Felber. The effect of insulin on the disposal of intravenous glucose. Diabetes 30: 1000-1007, 1981[Medline].

17.   Douen, A. G., T. Ramlal, S. Rastogi, P. J. Bilan, G. D. Cartee, M. Vranic, J. O. Holloszy, and A. Klip. Exercise induces recruitment of the "insulin-responsive glucose transporter": evidence for distinct intracellular insulin- and exercise-recruitable transporter pools in skeletal muscle. J. Biol. Chem. 265: 13427-13430, 1990[Abstract/Free Full Text].

18.   Friedman, J. E., G. L. Dohm, N. Legget-Frazier, C. W. Elton, E. B. Tapscott, W. P. Pories, and J. F. Caro. Restoration of insulin responsiveness in skeletal muscle of morbidly obese patients after weight loss. J. Clin. Invest. 89: 701-705, 1992[Medline].

19.   Goodyear, L. J., F. Giorgino, L. A. Sherman, J. Carey, R. J. Smith, and G. L. Dohm. Insulin receptor phosphorylation, and phosphatidylinositol 3-kinase activity are decreased in intact skeletal muscle strips from obese subjects. J. Clin. Invest. 95: 2195-2204, 1995[Medline].

20.   Handberg, A., L. Kayser, P. E. Hoyer, and J. Vinten. A substantial part of GLUT-1 in crude membranes from muscle originates from perineurial sheaths. Am. J. Physiol. 262 (Endocrinol. Metab. 25): E721-E727, 1992[Abstract/Free Full Text].

21.   Henriksen, E., R. E. Bourey, K. J. Rodnick, L. Koranyi, M. A. Permutt, and J. O. Holloszy. Glucose transporter protein content and glucose transport capacity in rat skeletal muscles. Am. J. Physiol. 259 (Endocrinol. Metab. 22): E593-E598, 1990[Abstract/Free Full Text].

22.   Holman, G. D., I. J. Kozka, A. E. Clark, C. Flower, J. Saltis, A. D. Habberfield, I. A. Simpson, and S. W. Cushman. Cell surface labeling of glucose transporter isoform GLUT-4 by bis-mannose photolabel. Correlations with stimulation of glucose transport in rat adipose cells by insulin and phorbol ester. J. Biol. Chem. 265: 18172-18179, 1990[Abstract/Free Full Text].

23.   Inoue, G., B. Cheatham, R. Emkey, and C. R. Kahn. Dynamics of insulin signaling in 3T3-L1 adipocytes. Differential compartmentalization and trafficking of insulin receptor substrate (IRS)-1 and IRS-2. J. Biol. Chem. 273: 11548-11555, 1998[Abstract/Free Full Text].

24.   Ivy, J. L., and C.-H. Kuo. Regulation of GLUT4 protein and glycogen synthase during muscle glycogen synthesis after exercise. Acta Physiol. Scand. 162: 295-304, 1998[Medline].

25.   Ivy, J. L., J. C. Young, B. W. Craig, W. M. Kohrt, and J. O. Holloszy. Ageing, exercise and food restriction: effects on skeletal muscle glucose uptake. Mech. Ageing Dev. 61: 123-133, 1991[Medline].

26.   James, D. E., K. M. Burleigh, and E. W. Kraegen. In vivo glucose metabolism in individual tissues of the rat. J. Biol. Chem. 261: 6366-6374, 1986[Abstract/Free Full Text].

27.   Kelly, K. L., and N. B. Ruderman. Insulin-stimulated phosphatidylinositol 3-kinase. Association with a 185-kDa tyrosine-phosphorylated protein (IRS-1) and localization in a low density membrane vesicle. J. Biol. Chem. 268: 4391-4398, 1993[Abstract/Free Full Text].

28.   Kemnitz, J. W., R. Weindruch, E. B. Roecker, K. Crawford, P. L. Kaufman, and W. B. Ershler. Dietary restriction of adult male rhesus monkeys: design, methodology, and preliminary findings from the first year of study. J. Gerontol. B Psychol. Sci. Soc. Sci. 48: B17-B26, 1993.

29.   Krebs, H. A., and K. Henseleit. Untersuchungen uber die Harnstoffbildung im Tierkorper. Hoppe Seylers Z. Physiol. Chem. 210: 33-36, 1932.

30.   Lee, A. D., P. A. Hansen, and J. O. Holloszy. Wortmannin inhibits insulin-stimulated but not contraction-stimulated glucose transport activity in skeletal muscle. FEBS Lett. 361: 51-54, 1995[Medline].

31.   Lund, S., A. Flyvbjerg, G. D. Holman, F. S. Larsen, O. Pedersen, and O. Schmitz. Comparative effects of IGF-I and insulin on the glucose transporter system in rat muscle. Am. J. Physiol. 267 (Endocrinol. Metab. 30): E461-E466, 1994[Abstract/Free Full Text].

32.   Passonneau, J. V., and V. R. Lauderdale. A comparison of three methods of glycogen measurement in tissues. Anal. Biochem. 60: 405-412, 1974[Medline].

33.   Patti, M.-E., X.-J. Sun, J. C. Bruening, E. Araki, M. A. Lipes, M. F. White, and C. R. Kahn. 4PS/Insulin receptor substrate (IRS)-2 is the alternative substrate of the insulin receptor in IRS-1-deficient mice. J. Biol. Chem. 270: 24670-24673, 1995[Abstract/Free Full Text].

34.   Reaven, E., D. Wright, C. E. Mondon, R. Solomon, H. Ho, and G. M. Reaven. Effect of age and diet on insulin secretion and insulin action in the rat. Diabetes 32: 175-178, 1983[Abstract].

35.   Satoh, S., H. Nishimura, A. E. Clark, I. J. Kozka, S. J. Vannucci, I. A. Simpson, M. J. Quon, S. W. Cushman, and G. D. Holman. Use of bismannose photolabel to elucidate insulin-regulated GLUT4 subcellular trafficking kinetics in rat adipose cells. Evidence that exocytosis is a critical site of hormone action. J. Biol. Chem. 268: 17820-17829, 1993[Abstract/Free Full Text].

36.   Schiachito, S., and S. I. Taylor. Cloning, tissue expression, and chromosomal localization of the mouse IRS-3 gene. Endocrinology 138: 4931-4940, 1997[Abstract/Free Full Text].

37.  Wallberg-Henriksson, H. Glucose transport into skeletal muscle. Acta Physiol. Scand. 564, Suppl.: 1-80, 1987.

38.   White, M. F., and C. R. Kahn. The insulin signalling system. J. Biol. Chem. 269: 1-4, 1994[Free Full Text].

39.   Wilson, C. M., and S. W. Cushman. Insulin stimulation of glucose transport activity in rat skeletal muscle: increase in cell surface GLUT4 as assessed by photolabelling. Biochem. J. 299: 755-759, 1994[Medline].

40.   Wing, R. R., E. H. Blair, P. Bononi, M. D. Marcus, R. Watanabe, and R. N. Bergman. Caloric restriction per se is a significant factor in improvements in glycemic control and insulin sensitivity during weight loss in obese NIDDM patients. Diabetes Care 17: 30-36, 1994[Abstract].

41.   Wither, D. J., J. S. Gutierrez, H. Towery, D. J. Burks, J.-M. Ren, S. Previs, Y. Zhang, D. Bernal, S. Pons, G. I. Shulman, G. I. Bonner-Weir, and M. F. White. Disruption of IRS-2 causes type 2 diabetes in mice. Nature 391: 900-904, 1998[Medline].

42.   Wojtaszewski, J. F. P., B. F. Hansen, B. Kiens, and E. A. Richter. Insulin signaling in human skeletal muscle: time course and effect of exercise. Diabetes 46: 1775-1781, 1997[Abstract].

43.   Yeh, J.-I., E. A. Gulve, L. Rameh, and M. J. Birnbaum. The effects of wortmannin on rat skeletal muscle. J. Biol. Chem. 270: 2107-2111, 1995[Abstract/Free Full Text].

44.   Young, D. A., J. J. Uhl, G. D. Cartee, and J. O. Holloszy. Activation of glucose transport in muscle by prolonged exposure to insulin. J. Biol. Chem. 261: 16049-16053, 1986[Abstract/Free Full Text].

45.   Ziel, F. H., N. Venkatesan, and M. B. Davidson. Glucose transport is rate limiting for skeletal muscle metabolism in normal and STZ-induced diabetic rats. Diabetes 37: 885-890, 1988[Abstract].

46.   Zierath, J. R., K. L. Houseknecht, L. Gnudi, and B. B. Kahn. High-fat feeding impairs insulin-stimulated GLUT4 recruitment via an early insulin-signaling defect. Diabetes 46: 215-223, 1997[Abstract].


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