Skeletal muscle respiratory uncoupling prevents diet-induced obesity and insulin resistance in mice.

To determine whether uncoupling respiration from oxidative phosphorylation in skeletal muscle is a suitable treatment for obesity and type 2 diabetes, we generated transgenic mice expressing the mitochondrial uncoupling protein (Ucp) in skeletal muscle. Skeletal muscle oxygen consumption was 98% higher in Ucp-L mice (with low expression) and 246% higher in Ucp-H mice (with high expression) than in wild-type mice. Ucp mice fed a chow diet had the same food intake as wild-type mice, but weighed less and had lower levels of glucose and triglycerides and better glucose tolerance than did control mice. Ucp-L mice were resistant to obesity induced by two different high-fat diets. Ucp-L mice fed a high-fat diet had less adiposity, lower levels of glucose, insulin and cholesterol, and an increased metabolic rate at rest and with exercise. They were also more responsive to insulin, and had enhanced glucose transport in skeletal muscle in the setting of increased muscle triglyceride content. These data suggest that manipulating respiratory uncoupling in muscle is a viable treatment for obesity and its metabolic sequelae.

Insulin unmasks a COOH-terminal Glut4 epitope and increases glucose transport across T-tubules in skeletal muscle.

An improved immunogold labeling procedure was used to examine the subcellular distribution of glucose transporters in Lowricryl HM20-embedded skeletal muscle from transgenic mice overexpressing either Glut1 or Glut4. In basal muscle, Glut4 was highly enriched in membranes of the transverse tubules and the terminal cisternae of the triadic junctions. Less than 10% of total muscle Glut4 was present in the vicinity of the sarcolemmal membrane. Insulin treatment increased the number of gold particles associated with the transverse tubules and the sarcolemma by three-fold. However, insulin also increased the total Glut4 immunogold reactivity in muscle ultrathin sections by up to 1.8-fold and dramatically increased the amount of Glut4 in muscle sections as observed by laser confocal immunofluorescence microscopy. The average diameter of transverse tubules observed in longitudinal sections increased by 50% after insulin treatment. Glut1 was highly enriched in the sarcolemma, both in the basal state and after insulin treatment. Disruption of transverse tubule morphology by in vitro glycerol shock completely abolished insulin-stimulated glucose transport in isolated rat epitrochlearis muscles. These data indicate that: (a) Glut1 and Glut4 are targeted to distinct plasma membrane domains in skeletal muscle; (b) Glut1 contributes to basal transport at the sarcolemma and the bulk of insulin-stimulated transport is mediated by Glut4 localized in the transverse tubules; (c) insulin increases the apparent surface area of transverse tubules in skeletal muscle; and (d) insulin causes the unmasking of a COOH-terminal antigenic epitope in skeletal muscle in much the same fashion as it does in rat adipocytes.

Nitrate ions: Potentiation of increased permeability to sugar associated with muscle contraction.

Nitrate ions potentiate twitch tension and enhance the increase in permeability to sugar which occurs in electrically stimulated frog sartorius muscles. However, the potentiating effect of nitrate ions on permeability is not dependent upon an increase in twitch tension. The possible relation of changes in permeability to alterations of the concentration of calcium ions in the cell is discussed.

Effects of weight changes produced by exercise, food restriction, or overeating on body composition.

The body weight of rats was reduced by exercise or by restriction of food intake over a period of 18 wk. Body composition was studied to determine if exercise protects against the loss of lean tissue that can occur as a result of a negative caloric balance. Rats weighing 706 +/-14 g were divided into four groups matched for weight. A baseline group was killed at the beginning of the study. An exercising group, fed ad lib., was subjected to a program of swimming. A sedentary, free-eating group was provided with food ad lib. Two sedentary, paired-weight subgroups were calorie restricted so that they lost weight at the same rate as the exercisers. The protein intake of one paired-weight subgroup was matched with that of the exercising group. The other sedentary, paired-weight animals ate the standard diet. There was no significant difference in body composition between the two sedentary, paired-weight subgroups which were, therefore, pooled for comparison with the other groups. The exercisers lost 182+/-19 g as a result of both an increase in caloric expenditure and a decrease in appetite. The sedentary, food-restricted animals lost an average of 182+/-18 g. The sedentary, free-eating animals gained 118+/-13 g. The carcasses of the exercised animals contained significantly less fat and more lean tissue than those of the sedentary, paired-weight animals, providing evidence for a fat mobilizing and protein conserving effect of exercise. The composition of the body substance lost by the exercising animals was 78% fat, 5% protein, 1% minerals, and 16% water, compared to 62% fat, 11% protein, 1% minerals, and 26% water for the sedentary, food-restricted rats. Fat accounted for 87% and water for 10% of the weight gained by the sedentary, free-eating animals.

Adaptation of muscle to exercise. Increase in levels of palmityl Coa synthetase, carnitine palmityltransferase, and palmityl Coa dehydrogenase, and in the capacity to oxidize fatty acids.

The capacity of gastrocnemius and quadriceps muscles to oxidize palmitate, oleate, linoleate, palmityl CoA, and palmityl carnitine doubled in rats subjected to a program of treadmill running. The rate of palmitate oxdation by whole homogenates of, or the mitochondrial fraction from, leg muscles was twice as great per gram wet weight of muscle in the trained as in the sedentary animals over a wide range (0.125-1.5 mM) of palmitate concentrations. The levels of activity of carnitine palmityltransferase, palmityl CoA dehydrogenase, and mitochondrial ATP-dependent palmityl CoA synthetase expressed per gram of muscle doubled in gastrocnemius and quadriceps muscles in response to the running program. The protein content of the mitochondrial fraction from these muscles was increased approximately 60%.

Glucoregulation during exercise: hypoglycemia is prevented by redundant glucoregulatory systems, sympathochromaffin activation, and changes in islet hormone secretion.

During mild or moderate nonexhausting exercise, glucose utilization increases sharply but is normally matched by increased glucose production such that hypoglycemia does not occur. To test the hypothesis that redundant glucoregulatory systems including sympathochromaffin activation and changes in pancreatic islet hormone secretion underlie this precise matching, eight young adults exercised at 55-60% of maximal oxygen consumption for 60 min on separate occasions under four conditions: (a) control study (saline infusion); (b) islet clamp study (insulin and glucagon held constant by somatostatin infusion with glucagon and insulin replacement at fixed rates before, during and after exercise with insulin doses determined individually and shown to produce normal and stable plasma glucose concentrations prior to each study); (c) adrenergic blockage study (infusions of the alpha- and beta-adrenergic antagonists phentolamine and propranolol); (d) adrenergic blockade plus islet clamp study. Glucose production matched increased glucose utilization during exercise in the control study and plasma glucose did not fall (92 +/- 1 mg/dl at base line, 90 +/- 2 mg/dl at the end of exercise). Plasma glucose also did not fall during exercise when changes in insulin and glucagon were prevented in the islet clamp study. In the adrenergic blockade study, plasma glucose declined initially during exercise because of a greater initial increase in glucose utilization, then plateaued with an end-exercise value of 74 +/- 3 mg/dl (P less than 0.01 vs. control). In contrast, in the adrenergic blockade plus islet clamp study, exercise was associated with glucose production substantially lower than control and plasma glucose fell progressively to 58 +/- 7 mg/dl (P less than 0.001); end-exercise plasma glucose concentrations ranged from 34 to 72 mg/dl. Thus, we conclude that: (a) redundant glucoregulatory systems are involved in the precise matching of increased glucose utilization and glucose production that normally prevents hypoglycemia during moderate exercise in humans. (b) Sympathochromaffin activation, perhaps sympathetic neural norepinephrine release, plays a primary glucoregulatory role by limiting glucose utilization as well as stimulating glucose production. (c) Changes in pancreatic islet hormone secretion (decrements in insulin, increments in glucagon, or both) are not normally critical but become critical when catecholamine action is deficient. (d) Glucoregulation fails, and hypoglycemia can develop, both when catecholamine action is deficient and when changes in islet hormones do not occur during exercise in humans.

Effects of alkaline pH on the stimulation of glucose transport in rat skeletal muscle.

Alkaline pH has been reported to cause release of Ca2+ from skeletal muscle sarcoplasmic reticulum (SR). Elevation of sarcoplasmic Ca2+ concentration is thought to stimulate glucose transport in skeletal muscle. In this context, we examined the effect of alkaline pH (extracellular pH of 8.6) on 3-O-methylglucose transport in skeletal muscle. Incubation of rat epitrochlearis muscles at pH 8.6 for 45 min resulted in an approx. 3-fold increase in glucose transport activity, which was not affected by reducing Ca2+ concentration in the incubation medium and essentially completely blocked by 25 microM dantrolene, an inhibitor of SR Ca2+ release. In addition to stimulating glucose transport by itself, alkaline pH may partially inhibit the stimulation of sugar transport by insulin hypoxia and contractions, as the combined effect of alkaline pH and the maximal effect of insulin, contractions, or hypoxia on glucose transport are not different from the maximal effects of insulin, hypoxia, or contractions alone. The maximal effects of insulin and contractions, and of insulin and hypoxia, on glucose transport are normally additive in muscle. Alkaline pH completely prevented this additivity. In summary, our results show that alkaline pH stimulates glucose transport activity in skeletal muscle and provide evidence suggesting that this effect is mediated by Ca2+. They further show that alkaline pH blocks the additivity of the maximal effects of insulin and contractions or hypoxia suggesting that alkaline pH may partially inhibit the stimulation of glucose transport by insulin, contraction and hypoxia.

Lithium increases susceptibility of muscle glucose transport to stimulation by various agents.

Lithium is thought to have an insulin-like effect on glucose transport and metabolism in skeletal muscle and adipocytes. However, we found that lithium had only a minimal effect on basal glucose transport activity in rat epitrochlearis muscles. Instead, lithium markedly increased the sensitivity of glucose transport to insulin, so that the increase in glucose transport activity induced by 300 pM insulin was approximately 2.5-fold greater in the presence of lithium than in its absence. Lithium also caused a modest increase in insulin responsiveness. This enhancement of the susceptibility of the glucose transport process to stimulation was not limited to insulin, because lithium induced increases in the susceptibility of glucose transport to stimulation by contractile activity, hypoxia, a phorbol ester, and phospholipase C. Lithium also blunted the activation of glycogen phosphorylase by epinephrine. These effects were not mediated by inhibition of adenylate cyclase, because neither basal- nor epinephrine-stimulated muscle cAMP concentration was affected by lithium treatment. The effects of lithium on glucose transport and metabolism in skeletal muscle are strikingly similar to the persistent effects of exercise. These results support the possibility that lithium might be useful in the treatment of insulin resistance in patients with non-insulin-dependent diabetes mellitus.

Insulin resistance of muscle glucose transport in rats fed a high-fat diet: a reevaluation.

Rats fed a high-fat diet develop skeletal muscle insulin resistance. There is disagreement regarding whether a decrease in the GLUT4 isoform of the glucose transporter is responsible. We found that feeding rats a high-fat diet that reduced the responsiveness of glucose transport to insulin in skeletal muscles by approximately 25-45% in 4 weeks, had no significant effect on muscle GLUT4 content. There is also controversy regarding whether the contraction/anoxia activated pathway of glucose transport stimulation is affected by fat feeding. We found that stimulation of muscle glucose transport by either swimming, in situ contractions, or anoxia was depressed to a similar extent as insulin responsiveness in high-fat-fed rats. It has been suggested that the muscle insulin resistance caused by a high-fat diet is due to increased fat oxidation and glucose-fatty acid cycle activity. However, we found that insulin-stimulated glucose transport was reduced by approximately 40% when muscles of fat-fed rats were incubated under anoxic conditions under which fatty acid oxidation should not occur. Rats maintained on the high-fat diet up to 32 weeks developed the characteristics of the abdominal obesity syndrome, including insulin resistance, hyperinsulinemia, hyperglycemia, elevated LDL cholesterol and VLDL triglycerides, and marked visceral obesity. We conclude that 1) in rats fed a high-fat diet the muscle insulin resistance is not due to a decrease in total GLUT4 content or to increased fat oxidation, 2) fat feeding also results in resistance of muscle glucose transport to stimulation via the contraction/anoxia pathway, and 3) rats fed a high-fat diet may be a useful model of the abdominal obesity syndrome.

Removal of adenosine decreases the responsiveness of muscle glucose transport to insulin and contractions.

Adenosine in the extracellular space modulates stimulated glucose transport in striated muscle. In the heart and in adipocytes, adenosine potentiates insulin-stimulated glucose transport. There is controversy regarding the effect of adenosine in skeletal muscle, with reports of both an inhibitory effect and no effect, on insulin-stimulated glucose transport. We found that, in rat epitrochlearis and soleus muscles, removing adenosine with adenosine deaminase or blocking its action with the adenosine receptor blocker CPDPX markedly reduces the responsiveness of glucose transport to stimulation by 1) insulin alone, 2) contractions alone, and 3) insulin and contractions in combination. Measurement of the increase in GLUT4 at the cell surface in response to a maximally effective insulin stimulus in the epitrochlearis muscle, using the exofacial label ATB-[3H]BMPA, showed that adenosine deaminase treatment markedly reduces cell-surface GLUT4 labeling. The reduction in cell-surface GLUT4 labeling was similar in magnitude to the decrease in maximally insulin-stimulated glucose transport activity in adenosine deaminase-treated muscles. These results show that adenosine potentiates insulin- and contraction-stimulated glucose transport in skeletal muscle by enhancing the increase in GLUT4 at the cell surface and raise the possibility that decreased adenosine production or action could play a causative role in insulin resistance.

Effects of exercise training on insulin-regulatable glucose-transporter protein levels in rat skeletal muscle.

Exercise training has been shown to enhance the ability of insulin to stimulate glucose uptake in responsive tissues. The purpose of this study was to determine the effects of exercise training on the levels of the insulin-regulatable glucose transporter (IRGT) in rat skeletal muscle. After 6 wk of voluntary running in exercise-wheel cages, male Sprague-Dawley rats were rested for approximately 27 h and fasted overnight before removal of plantaris and soleus muscles. The concentration of glucose transporters per unit of muscle protein or DNA was quantitated by immunoblotting with an anti-IRGT polyclonal antibody raised against a synthetic peptide. The IRGT protein was increased by 60% (141 +/- 14 vs. 229 +/- 24 counts/min [cpm]/25 micrograms protein, P less than 0.01) in plantaris muscle from exercise-trained rats compared with controls. Total protein yield, DNA content, and 5'-nucleotidase activity were not different in plantaris muscle from control and exercise-trained rats. In contrast, there was no significant increase in the IRGT protein in soleus muscle after training when data were expressed per unit of muscle protein (292 +/- 22 vs. 346 +/- 16 cpm/25 micrograms protein). These data indicate that the increase in the IRGT in plantaris muscle is a selective response to exercise training that does not reflect an overall increase in muscle protein. The changes in IRGT for these muscles with exercise training parallel changes observed in insulin-mediated glucose uptake. We propose that this increase in the total number of glucose transporters may be a major component of the increase in insulin-mediated glucose uptake that is observed with exercise training.

Coordinate reduction of rat pancreatic islet glucokinase and proinsulin mRNA by exercise training.

Exercise training results not only in enhanced insulin sensitivity but also in a reduction in insulin secretion. In this study, we examined the effects of exercise training on the expression of genes potentially related to insulin synthesis and glucose-stimulated insulin release by measuring pancreatic islet proinsulin, glucose-transporter (GLUT2), and glucokinase mRNAs. Female Wistar rats were subjected to 100 min of running at 25 m.min-1 up a 15% incline for 90 min/day for 6 days/wk for 3 wk. Pancreatic mRNA was evaluated by Northern- and dot-blot analysis with [32P]cRNA probes. We found no change in the pancreatic content of GLUT2 mRNA but found marked decreases in the content of proinsulin mRNA (78%, P less than 0.005) and glucokinase mRNA (65%, P less than 0.001). These results suggest that exercise modulates both islet glucose metabolism and insulin synthesis at the level of gene expression. Furthermore, there was a significant correlation between the decreases in glucokinase and proinsulin mRNA concentrations (r = 0.95, P less than 0.001), suggesting that expression of these genes is regulated in parallel.

The HIV protease inhibitor indinavir decreases insulin- and contraction-stimulated glucose transport in skeletal muscle.

In many patients with human immunodeficiency virus (HIV) treated with HIV protease inhibitors, a complication develops that resembles abdominal obesity syndrome, with insulin resistance and glucose intolerance that, in some cases, progresses to diabetes. In this study, we tested the hypothesis that indinavir, an HIV-protease inhibitor, directly induces insulin resistance of glucose transport in skeletal muscle. Rat epitrochlearis muscles were incubated with a maximally effective insulin concentration (12 nmol/l) and 0, 1, 5, 20, or 40 micromol/l indinavir for 4 h. In control muscles, insulin increased 3-O-[(3)H]methyl-D-glucose (3MG) transport from 0.15 +/- 0.03 to 1.10 +/- 0.05 micromol. ml(-)(1). 10 min(-)(1). Incubation of muscles with 5 micromol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 40%, whereas 20 micromol/l indinavir reduced the insulin-stimulated increase in 3MG transport by 58%. Indinavir induced a similar reduction in maximally insulin-stimulated 3MG transport in the soleus muscle. The increase in glucose transport activity induced by stimulating epitrochlearis muscles to contract was also markedly reduced by indinavir. The insulin-stimulated increase in cell-surface GLUT4, assessed using the 2-N-4-(1-azi-2,2,2-trifluoroethyl)benzoyl-1,3-bis-[2-(3)H] (D-mannose-4-yloxy)-2-propylamine exofacial photolabeling technique, was reduced by approximately 70% in the presence of 20 micromol/l indinavir. Insulin stimulation of phosphatidylinositol 3-kinase activity and phosphorylation of protein kinase B were not decreased by indinavir. These results provide evidence that indinavir inhibits the translocation or intrinsic activity of GLUT4 rather than insulin signaling.

Insulin resistance in aging is related to abdominal obesity.

Studies have shown that insulin resistance increases with age, independent of changes in total adiposity. However, there is growing evidence that the development of insulin resistance may be more closely related to abdominal adiposity. To evaluate the independent effects of aging and regional and total adiposity on insulin resistance, we performed hyperinsulinemic euglycemic clamps on 17 young (21-33 yr) and 67 older (60-72 yr) men and women. We assessed FFM and total and regional adiposity by hydrodensitometry and anthropometry. Insulin-stimulated GDRs at a plasma insulin concentration of approximately 450 pM averaged 45.6 +/- 3.3 mumol.kg FFM-1 x min-1 (mean +/- SE) in the young subjects, 45.6 +/- 10.0 mumol.kg FFM-1 x min-1 in 24 older subjects who were insulin sensitive, and 23.9 +/- 11.7 mumol.kg FFM-1 x min-1 in 43 older subjects who were insulin resistant. Few significant differences were apparent in skin-fold and circumference measurements between young and insulin-sensitive older subjects, but measurements at most central body sites were significantly larger in the insulin-resistant older subjects. Waist girth accounted for > 40% of the variance in insulin action, whereas age explained only 10-20% of the total variance and < 2% of the variance when the effects of waist circumference were statistically controlled. These results suggest that insulin resistance is more closely associated with abdominal adiposity than with age.

Short-term exposure to tumor necrosis factor-alpha does not affect insulin-stimulated glucose uptake in skeletal muscle.

It has been hypothesized that increased production of tumor necrosis factor-alpha (TNF-alpha) plays a role in causing the insulin resistance associated with obesity. Obesity with insulin resistance is associated with increased production of TNF-alpha by fat cells. Exposure of 3T3-L1 adipocytes to TNF-alpha for 3-4 days makes them insulin resistant. TNF-alpha has also been reported to rapidly (15-60 min) cause insulin resistance, with a decrease in insulin-stimulated tyrosine phosphorylation, in a number of cultured cell lines. Because skeletal muscle is the major tissue responsible for insulin-stimulated glucose disposal, we performed the present study to determine if acute exposure to TNF-alpha causes insulin resistance in muscle. We found that exposure of soleus muscles to 6 nmol/l TNF-alpha for 45 min in vitro had no inhibitory effect on insulin-stimulated tyrosine phosphorylation of the insulin receptor or insulin receptor substrate 1 (IRS-1) or on phosphatidylinositol 3-kinase association with IRS-1. Incubation of epitrochlearis and soleus muscles with 6 nmol/l TNF-alpha for 45 min or 4 h had no effect on insulin-stimulated 2-deoxyglucose (2-DG) uptake. Treatment of epitrochlearis muscles with 2 nmol/l TNF-alpha for 8 h also had no effect on insulin-stimulated 2-DG uptake. We conclude that in contrast to Fao hepatoma cells and 3T3-L1 fibroblasts, skeletal muscle does not become insulin resistant in response to short-term exposure to TNF-alpha.

Enhanced permeability to sugar associated with muscle contraction. Studies of the role of Ca++.

When contractures were induced in isolated frog sartorius muscles with 4 mM caffeine, there was an increase in permeability of the muscle cells to 3-methylglucose. This observation suggests that the changes in permeability to sugar that are known to occur in electrically stimulated muscles may not be intimately related to the depolarization phase of the tissue response. Contractures that were elicited by exposing the muscles to a high concentration of K+ were also associated with an increased permeability to sugar. As the concentration of 45Ca in the medium was raised, more 45Ca entered the muscles during potassium contractures, and the contractures lasted longer, in agreement with the observations of other investigators. There was also a greater change in permeability to sugar when potassium contractures were elicited in the presence of higher concentrations of Ca++. The possibility that the enhanced permeability to sugar may be related to changes in the intracellular concentration of Ca++ is discussed.

The effect of 7 years of intense exercise training on patients with coronary artery disease.

The purpose of this study was to evaluate the effects of long-term exercise training on maximal aerobic exercise capacity, evidence of myocardial ischemia and plasma lipid-lipoprotein concentrations in patients with coronary artery disease. Nine men with coronary artery disease, aged 57 +/- 2 years, who had completed 12 months of supervised intense exercise training were restudied after 6 additional years during which they continued to exercise. The first 12 months of training resulted in a 44% increase in maximal oxygen consumption (VO2max) from 25.0 +/- 1.3 to 35.9 +/- 1.5 ml X kg-1 X min-1 (p less than 0.001). The VO2max after 6 additional years (total 7 years) of intense training was 36.8 +/- 2.4 ml X kg-1 X min-1. Plasma high density lipoprotein (HDL)-cholesterol concentration increased from 38 +/- 3 to 45 +/- 4 mg X dl-1 at 12 months and rose further to 53 +/- 5 mg X dl-1 at 6 years of follow-up (p less than 0.05). The atherogenic index (total cholesterol/HDL-cholesterol ratio) decreased from 5.8 +/- 0.4 to 4.9 +/- 0.4 by 12 months (p less than 0.01) and to 4.1 +/- 0.4 after 6 additional years of training (p less than 0.05). Although the maximal heart rate-pressure product was 14% higher after 12 months of training, maximal ST segment depression was significantly less, 0.27 +/- 0.06 versus 0.19 +/- 0.04 mV (p less than 0.05); this improvement was maintained after 6 years of additional training. These data provide evidence that the beneficial effects of a program of intense exercise training can be maintained for long periods in some motivated patients with coronary artery disease who continue to exercise.

Improvement in glucose tolerance after 1 wk of exercise in patients with mild NIDDM.

We investigated the effects of 1 wk of intense exercise on glucose tolerance in 10 men with abnormal glucose tolerance [7 had mild non-insulin-dependent diabetes mellitus (NIDDM), and 3 had impaired glucose tolerance]. The 7 days of exercise did not result in significant changes in body weight or maximal oxygen uptake. Plasma glucose concentration at 120 min averaged 227 +/- 23 mg/dl in an oral glucose tolerance test (OGTT) before and 170 +/- 18 mg/dl after the 7 days of exercise (P less than .001). There was a 36% reduction in the area under the glucose tolerance curve. Plasma insulin concentration at 120 min of the OGTT averaged 172 +/- 27 microU/ml before and 106 +/- 13 microU/ml after 7 days of exercise (P less than .001); the area under the insulin curve was decreased by 32%. In contrast to the response to 7 days of exercise, one bout of exercise did not result in an improvement in glucose tolerance. These results provide evidence that regularly performed, vigorous exercise can be effective in decreasing insulin resistance and improving glucose tolerance within 7 days in some patients with mild NIDDM.

Exercise-induced increase in glucose transporters in plasma membranes of rat skeletal muscle.

A previously developed technique for the isolation of plasma and intracellular membrane fractions from rat skeletal muscle was used to investigate transporter migration after insulin treatment or a bout of exercise (45 min of treadmill). Glucose-inhibitable cytochalasin-B binding was used to estimate the number of glucose transporters. Insulin and exercise caused increases in glucose uptake into the hindlimb muscles of 5- and 3-fold, respectively. Each stimulus also caused a 2-fold increase in the number of glucose transporters in plasma membranes prepared from hindlimb muscles. The insulin-induced increase in plasma membrane transporters was accompanied by a concomitant decrease in transporters from the intracellular pool. In contrast to insulin, there was no concomitant decrease in the number of cytochalasin-B-binding sites in the intracellular membrane fraction from exercised muscles. The ability of both insulin and exercise to increase the number of transporters in the plasma membrane is in accordance with recruitment of transporters as one cause of increased transport activity. However, the inability of exercise to decrease the number of transporters in the insulin-sensitive intracellular pool suggests the existence of either a second recruitable transporter pool or masked glucose transporters in the plasma membrane that are unmasked by the muscle contractile activity.

Wortmannin inhibits insulin-stimulated but not contraction-stimulated glucose transport activity in skeletal muscle.

In skeletal muscle, glucose transport is stimulated by insulin, contractions and hypoxia. In this study, we used the phosphatidylinositol 3-kinase (PI 3-kinase) inhibitor wortmannin to examine whether (i) PI 3-kinase activity is necessary for stimulation of glucose transport by insulin in muscle, and (ii) PI 3-kinase mediates a step in the pathway by which contractions/hypoxia stimulate glucose transport. Wortmannin completely blocked insulin- and insulin-like growth factor-1-stimulated glucose transport in muscle. In contrast, wortmannin had no effect on the stimulation of glucose transport by contractions or hypoxia, providing evidence that PI 3-kinase activity is not involved in the activation of glucose transport by these stimuli.