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We examined skeletal and cardiac muscle responses of mature (8 mo) and old (23 mo) male Fischer 344 rats to 14 days of hindlimb suspension. Hexokinase (HK) and citrate synthase (CS) activities and GLUT-4 glucose transporter protein level, which are coregulated in many instances of altered neuromuscular activity, were analyzed in soleus (Sol), plantaris (PI), tibialis anterior (TA), extensor digitorum longus (EDL), and left ventricle. Protein content was significantly (P < 0.05) lower in all four hindlimb muscles after suspension compared with controls in both mature (21-44%) and old (17-43%) rats. Old rats exhibited significantly lower CS activities than mature rats for the Sol, Pl, and TA. HK activities were significantly lower in the old rats for the Pl (19%) and TA (33%), and GLUT-4 levels were lower in the old rats for the TA (38%) and EDL (24%) compared with the mature rats. Old age was also associated with a decrease in CS activity (12%) and an increase in HK activity (14%) in cardiac muscle. CS activities were lower in the Sol (20%) and EDL (18%) muscles from mature suspended rats and in the Sol (25%), Pl (27%), and EDL (25%) muscles from old suspended rats compared with corresponding controls. However, suspension was associated with significantly higher HK activities for all four hindlimb muscles examined, in both old (16-57%) and mature (10-43%) rats, and higher GLUT-4 concentrations in the TA muscles of the old rats (68%) but not the mature rats. These results indicate that old age is associated with decreased CS and HK activities and GLUT-4 protein concentration for several rat hindlimb muscles, and these variables are not coregulated during suspension. Finally, old rat skeletal muscle appears to respond to suspension to a similar or greater degree than mature rat muscle responds.

Skeletal muscle glucose transport can be regulated by hormonal factors such as insulin and insulin-like growth factor I (IGF-I). Although it is well established that exercise training increases insulin action on muscle glucose transport, it is currently unknown whether exercise training leads to an enhancement of IGF-I-stimulated glucose transport in skeletal muscle. Therefore, we measured glucose transport activity [by using 2-deoxy-D-glucose glucose (2-DG) uptake] in the isolated rat epitrochlearis muscle stimulated by submaximally and maximally effective concentrations of insulin (0.2 and 13.3 nM) or IGF-I (5 and 50 nM) after 1, 2, and 3 wk of voluntary wheel running (WR). After 1 wk of WR, both submaximal and maximal insulin-stimulated 2-DG uptake rates were significantly (P < 0.05) enhanced (43 and 31%) compared with those of sedentary controls, and these variables were further increased after 2 (86 and 57%) and 3 wk (71 and 70%) of WR. Submaximal and maximal IGF-I-stimulated 2-DG uptake rates were significantly enhanced after 1 wk of WR (82 and 61%, and these increases did not expand substantially after 2 (71 and 58%) and 3 wk (96 and 70%) of WR. This enhancement of hormone-stimulated 2-DG uptake in WR muscles preceded any alteration in glucose transporter (GLUT-4) protein level, which increased only after 2 (24%) and 3 wk (54%) of WR. Increases in GLUT-4 protein were significantly correlated (r = 0.844) with increases in citrate synthase. These results indicate that exercise training can enhance both insulin-stimulated and IGF-I-stimulated muscle glucose transport activity and that these improvements can develop without an increase in GLUT-4 protein.

Insulin resistance of muscle glucose metabolism is a hallmark of NIDDM. The obese Zucker (fa/fa) rat--an animal model of muscle insulin resistance--was used to test whether acute (100 mg/kg body wt for 1 h) and chronic (5-100 mg/kg for 10 days) parenteral treatments with a racemic mixture of the antioxidant alpha-lipoic acid (ALA) could improve glucose metabolism in insulin-resistant skeletal muscle. Glucose transport activity (assessed by net 2-deoxyglucose [2-DG] uptake), net glycogen synthesis, and glucose oxidation were determined in the isolated epitrochlearis muscles in the absence or presence of insulin (13.3 nmol/l). Severe insulin resistance of 2-DG uptake, glycogen synthesis, and glucose oxidation was observed in muscle from the vehicle-treated obese rats compared with muscle from vehicle-treated lean (Fa/-) rats. Acute and chronic treatments (30 mg.kg-1.day-1, a maximally effective dose) with ALA significantly (P < 0.05) improved insulin-mediated 2-DG uptake in epitrochlearis muscles from the obese rats by 62 and 64%, respectively. Chronic ALA treatment increased both insulin-stimulated glucose oxidation (33%) and glycogen synthesis (38%) and was associated with a significantly greater (21%) in vivo muscle glycogen concentration. These adaptive responses after chronic ALA administration were also associated with significantly lower (15-17%) plasma levels of insulin and free fatty acids. No significant effects on glucose transporter (GLUT4) protein level or on the activities of hexokinase and citrate synthase were observed. Collectively, these findings indicate that parenteral administration of the antioxidant ALA significantly enhances the capacity of the insulin-stimulatable glucose transport system and of both oxidative and nonoxidative pathways of glucose metabolism in insulin-resistant rat skeletal muscle.

Using the euglycemic-hyperinsulinemic glucose clamp and the human forearm technique, we have demonstrated that the improved glucose disposal rate observed after the administration of an angiotensin-converting enzyme (ACE) inhibitor such as captopril may be primarily due to increased muscle glucose uptake (MGU). These results are not surprising because ACE, which is identical to the bradykinin (BK)-degrading kininase II, is abundantly present in muscle tissue, and its inhibition has been observed to elicit the observed metabolic actions via elevated tissue concentrations of BK and through a BK B2 receptor site in muscle and/or endothelial tissue. These findings are supported by several previous studies. Exogenous BK applied into the brachial artery of the human forearm not only augmented muscle blood flow (MBF) but also enhanced the rate of MGU. In another investigation, during rhythmic voluntary contraction, both MBF and MGU increased in response to the higher energy expenditure, and the release of BK rose in the blood vessel, draining the working muscle tissue. Inhibition of the activity of the BK-generating protease in muscle tissue (kallikrein) with aprotinin significantly diminished these functional responses during contraction. Applying the same kallikrein inhibitor during the infusion of insulin into the brachial artery significantly reduced the effect of insulin on glucose uptake into forearm muscle. This is of interest, because in recent studies insulin has been suggested to elicit its actions on MBF and MGU via the accelerated release of endothelium-derived nitric oxide, the generation of which is also stimulated by BK in a concentration-dependent manner. This new evidence obtained from in vitro and in vivo studies sheds new light on the discussion of whether BK may play a role in energy metabolism of skeletal muscle tissue.

The calmodulin antagonist N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide (W-7) stimulates glucose transport in skeletal muscle, apparently by raising cytosolic Ca2+ (P. Palade. J. Biol. Chem. 262: 6142-6148, 1987; J.H. Youn, E.A. Gulve, and J.O. Holloszy. Am. J. Physiol. 260 (Cell Physiol. 29): C555-C561, 1991). This study was performed to describe the interactions between the effects of W-7 and those of hypoxia and of insulin on glucose transport. The effect on 3-O-methylglucose (3-MG) transport of 50 microM W-7 was additive to the effect of a maximal insulin stimulus (2,000 microU/ml) but not to the effect of maximal (60 min) hypoxic stimulus, suggesting that W-7 stimulates glucose transport via the same pathway as hypoxia, independent of the pathway activated by insulin. The effect of 50 microM W-7 was additive to that of a submaximal (20 min) hypoxia stimulus, indicating that W-7 does not interfere with the stimulation of glucose transport by hypoxia. In contrast, 50 microM W-7 had an inhibitory effect on stimulation of 3-MG transport by submaximally effective insulin levels, causing a fivefold increase in the concentration of insulin needed to produce a half-maximal stimulation of 3-MG transport, from approximately 70 to approximately 350 microU/ml (P < 0.05). Thus these data demonstrate that W-7 selectively inhibits insulin stimulation of glucose transport.(ABSTRACT TRUNCATED AT 250 WORDS)

The effects of exercise training with or without subsequent unweighting on wet weight, protein content, and in vivo fractional protein synthesis were studied in soleus and plantaris muscles of juvenile female Sprague-Dawley rats under the following four conditions: normal weight bearing (N), voluntary-activity wheel running (WR) for up to 4 weeks, mechanical unweighting for 7 days via hindlimb suspension (HS), or wheel running followed by 7 days of hindlimb suspension (WR-HS). Fractional protein synthesis was determined by the 3H-phenylalanine flooding method. Increases (P < .05) in wet weight and protein content were detected in the soleus after just 1 week of running, with no increase in fractional protein synthesis. Two weeks of running were required for an increase in protein synthesis in this muscle. Significant increases in these parameters were first observed in the plantaris after 2 weeks of running. Maximal increases occurred by 3 weeks in both muscles. Reductions (P < .05) in soleus and plantaris parameters were observed in both HS and WR-HS groups compared with N and WR groups, respectively. However, protein content and fractional synthesis were maintained at significantly higher levels in WR-HS muscles compared with HS muscles. These results indicate that (1) wheel training represents a noninvasive method for inducing rapid hypertrophy of the skeletal muscles studied, in part by increasing fractional protein synthesis; (2) unweighting decreases protein content and synthesis to the same extent whether the muscles are trained; and (3) previously hypertrophied muscles display higher protein contents and fractional protein synthesis following unweighting compared with unweighted muscles of untrained animals.

Voluntary wheel running (WR) by juvenile female rats was used as a noninterventional model of soleus muscle functional overload to study the regulation of insulin-stimulated glucose transport activity by the glucose transporter (GLUT-4 isoform) protein level and glycogen concentration. Soleus total protein content was significantly greater (+18%; P < 0.05) than in age-matched controls after 1 wk of WR, and this hypertrophic response continued in weeks 2-4 (+24-32%). GLUT-4 protein was 39% greater than in controls in 1-wk WR soleus, and this adaptation was accompanied by a similar increase in in vitro insulin-stimulated glucose transport activity (+29%). After 2 and 4 wk of WR, however, insulin-stimulated glucose transport activity had returned to control levels, despite a continued elevation (+25-28%) of GLUT-4 protein. At these two time points, glycogen concentration was significantly enhanced in WR soleus (+21-42%), which coincided with significant reductions in glycogen synthase activity ratios (-23 to -41%). These results indicate that, in this model of soleus muscle functional overload, the GLUT-4 protein level may initially regulate insulin-stimulated glucose transport activity in the absence of changes in other modifying factors. However, this regulation of glucose transport activity by GLUT-4 protein may be subsequently overridden by elevated glycogen concentration.

The perfusion of rat hindlimb muscles and the isolated in vitro muscle preparation are usually the preferred methods for investigating muscle metabolism. In light of recent concerns about the incubated muscle preparation, we have examined the problems, the advantages, and the viability of these two experimental techniques, with focus on glucose metabolism. A major advantage of the hindlimb perfusion system is that it maintains its metabolic viability very well, and perfusions in resting muscles can be achieved successfully with cell-free media. However, variations in the perfused rat hindlimb procedures result in considerable differences in perfusate flow among muscles, making quantitative comparisons among perfusion procedures difficult. Metabolic viability has been identified as a problem in some isolated in vitro muscle preparations. We have provided criteria to avoid muscle hypoxia. Minimum levels of insulin seem to be a key requirement to maintaining the muscle's viability, and essential amino acids are required to retard an increase in the basal rate of glucose and amino acid uptake. Under such conditions metabolic viability can be maintained during prolonged incubations (9-30 h). Both the isolated in vitro muscle preparation and the hindlimb perfusion preparation are viable models for the study of muscle metabolism.

We investigated the effects of purified histone H4 on glucose transport activity in rat soleus and flexor digitorum brevis muscles. Histone H4, at concentrations up to 11.8 microM, increased 2-deoxyglucose (2-DG) uptake in a dose-dependent fashion. However, at concentrations higher than 11.8 microM, H4 caused a decrease in 2-DG uptake from the maximum, suggesting a secondary inhibitory action of this compound. The maximal effect of H4 on 2-DG uptake was not additive to the maximal effect of insulin. Moreover, 2-DG uptake in the presence of both H4 and insulin was significantly lower than the 2-DG uptake in the presence of insulin alone. The maximal effect of H4 on stimulation of 2-DG uptake was neither additive nor inhibitory to the maximal effects of the intracellularly acting insulin mimetics sodium vanadate or H2O2. It was, on the other hand, additive to the maximal effects of muscle contractions. Also, in contrast with the effects of H4 on insulin-stimulated 2-DG uptake, H4 did not inhibit insulin-like growth factor-I (IGF-I)-stimulated 2-DG uptake, as the maximal effects of H4 and IGF-I were additive. Scatchard analysis of the binding of 125I-insulin in the absence or presence of histone H4 revealed that H4 increased the specific binding of insulin without affecting receptor affinity. These data suggest that H4 interacts with the insulin, rather than the hypoxia/contraction, pathway for activation of glucose transport in muscle tissue, and that H4 acts either directly or indirectly to increase the number of insulin receptors at the surface of the muscle cell. This interaction does not appear to occur with the similar, although distinct, IGF-I receptor. These studies may provide additional insight into the complex signal-transduction systems of insulin action.

Recent evidence by Kandarian et al. (J. Appl. Physiol. 71: 910-914, 1991) indicates that prolonged (28 days) unweighting of the rat soleus by hindlimb suspension results in a substantial increase in interstitial fluid volume (IFV), as defined by the inulin space. The lack of any significant difference in absolute IFV values between unweighted and control groups suggested that this elevated IFV was a consequence of muscle atrophy. Using young female rats, we directly tested this hypothesis by comparing the early responses of soleus muscle weight and IFV with unweighting by tail-cast suspension or actual exposure to microgravity during spaceflight. Significant differences from control were first observed after 3 days of suspension unweighting for soleus wet weight (-14%; P < 0.01) and IFV (+35%; P < 0.01) and increased further after 6 days (-32% and +53%, respectively; both P < 0.001). After 5.4 days of spaceflight, soleus wet weight was 38% less and IFV was 52% greater than control (both P < 0.001). A highly significant negative correlation between soleus wet weight and IFV for all groups was observed (r = -0.70, P < 0.001). These data indicate that elevated soleus IFV develops at an early time point during unweighting and that there is a direct relationship between the magnitude of this increase in IFV and the extent of muscle atrophy. This relationship also exists in soleus muscles unweighted by exposure to a microgravity environment.

We have previously demonstrated that mechanical unweighting of the soleus muscle by hindlimb suspension leads to increases in insulin receptor binding, muscle/fat-specific glucose transporter (GLUT-4) protein levels, and insulin-stimulated glucose transport activity. The present study used a novel approach to further evaluate the potential role of postreceptor binding mechanisms in this enhanced insulin effect after unweighting. Insulin-like growth factor I (IGF-I), vanadate, and phospholipase C were used to stimulate glucose transport activity independently of insulin receptor binding. Soleus glucose transport activity (assessed by 2-deoxyglucose uptake) was evaluated in vitro with soleus strips (approximately 18 mg). Progressively increased responses to maximally effective doses of insulin or IGF-I were observed after 3 and 6 days of unweighting compared with weight-matched control strips. Enhanced maximal responses to vanadate (6 days only) and phospholipase C (3 and 6 days) for 2-deoxyglucose uptake were also observed. The results of this study 1) provide evidence that post-insulin receptor binding mechanisms also play a role in the enhanced response of the insulin-dependent pathway for stimulation of glucose transport in unweighted skeletal muscle and 2) indicate that IGF-I action on glucose transport is included in this enhanced response in unweighted muscle.

Our knowledge of the effects of unweighting on skeletal muscle of juvenile rapidly growing rats has been obtained entirely by using hindlimb-suspension models. No spaceflight data on juvenile animals are available to validate these models of simulated weightlessness. Therefore, eight 26-day-old female Sprague-Dawley albino rats were exposed to 5.4 days of weightlessness aboard the space shuttle Discovery (mission STS-48, September 1991). An asynchronous ground control experiment mimicked the flight cage condition, ambient shuttle temperatures, and mission duration for a second group of rats. A third group of animals underwent hindlimb suspension for 5.4 days at ambient temperatures. Although all groups consumed food at a similar rate, flight animals gained a greater percentage of body mass per day (P < 0.05). Mass and protein data showed weight-bearing hindlimb muscles were most affected, with atrophy of the soleus and reduced growth of the plantaris and gastrocnemius in both the flight and suspended animals. In contrast, the non-weight-bearing extensor digitorum longus and tibialis anterior muscles grew normally. Earlier suspension studies showed that the soleus develops an increased sensitivity to insulin during unweighting atrophy, particularly for the uptake of 2-[1,2-3H]deoxyglucose. Therefore, this characteristic was studied in isolated muscles within 2 h after cessation of spaceflight or suspension. Insulin increased uptake 2.5- and 2.7-fold in soleus of flight and suspended animals, respectively, whereas it increased only 1.6-fold in control animals. In contrast, the effect of insulin was similar among the three groups for the extensor digitorum longus, which provides a control for potential systemic differences in the animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Functional vicinal sulfhydryls are essential for insulin-stimulated system A neutral amino acid uptake in the rat epitrochlearis muscle. In skeletal muscle, system A uptake is also activated by contractile activity. Therefore, the purposes of this study were to characterize the stimulation of system A activity by contractions induced by electrical stimulation in vitro, and to assess the role of vicinal sulfhydryls in this process. System A activity in the isolated epitrochlearis muscle was measured using the nonmetabolizable analogue alpha-(methylamino)isobutyric acid (MeAIB). Contractions increased MeAIB uptake by increasing the apparent maximal velocity (Vmax), with no alteration in the apparent Km. The maximal stimulatory effects of insulin and contractions on MeAIB uptake were completely additive, demonstrating that these two stimuli exert their effects via different mechanisms. Phenylarsine oxide (PAO), a vicinal sulfhydryl antagonist, at greater than 20 mumol/L inhibited basal and contraction-stimulated MeAIB uptake by approximately 50% and 70%, respectively, by decreasing Vmax, with no change in Km. Both inhibitory effects were completely prevented by cotreatment with the vicinal dithiol dimercaptopropanol (DMP), indicating the effects were mediated by interactions with vicinal sulfhydryls. Contraction-stimulated MeAIB uptake was rapidly (half-time, approximately 7 minutes) reversed by the addition of PAO. These results (1) define conditions under which contraction-stimulated system A amino acid uptake can be studied in an isolated mammalian skeletal muscle preparation, and (2) indicate that vicinal sulfhydryls are essential for stimulation of system A activity by muscle contractions.

Unweighting of the rat soleus by tail-cast suspension results in increased insulin action on stimulation of glucose transport, which can be explained, at least in part, by increased insulin binding and enhanced glucose transporter protein levels. Glucose transport is also activated by an insulin-independent mechanism stimulated by in vitro muscle contractions or hypoxia. Therefore, the purpose of this study was to determine if soleus unweighting leads to an enhanced response of the insulin-independent pathway for stimulation of glucose transport. The hindlimbs of juvenile male Wistar rats were suspended by a tail-cast system for 3 or 6 days. Glucose transport activity in isolated soleus strips (approximately 18 mg) was then assessed by using 2-deoxy-[1,2-3H]glucose (2-DG) uptake. Insulin (2 mU/ml) had a progressively enhanced effect on 2-DG uptake after 3 and 6 days of unweighting (+44 and +72% vs. control, respectively; both P < 0.001). At these same times, there was no difference between groups for activation of 2-DG uptake by maximally effective treatments with contractions (10 tetanuses), hypoxia (60 min), or caffeine (5 mM). These results indicate that the enhanced capacity for stimulation of glucose transport after soleus unweighting is restricted to the insulin pathway, with no apparent enhancement of the insulin-independent pathway.

It is widely thought that aging results in development of insulin resistance in skeletal muscle. In this study, we examined the effects of growth and aging on the concentration of the GLUT-4 glucose transporter and on glucose transport activity in skeletal muscles of female Long-Evans rats. Relative amounts of immunoreactive GLUT-4 protein were measured in muscle homogenates of 1-, 10-, and 25-mo-old rats by immunoblotting with a polyclonal antibody directed against GLUT-4. In the epitrochlearis, plantaris, and the red and white regions of the quadriceps muscles, GLUT-4 immunoreactivity decreased by 14-33% between 1 and 10 mo of age and thereafter remained constant. In flexor digitorum brevis (FDB) and soleus muscles, GLUT-4 concentration was similar at all three ages studied. Glucose transport activity was assessed in epitrochlearis and FDB muscles by incubation with 2-deoxyglucose under the following conditions: basal, submaximal insulin, and either maximal insulin or maximal insulin combined with contractile activity. Glucose transport in the epitrochlearis muscle decreased by approximately 60% between 1 and 4 mo of age and then did not decline further between 4 and 25 mo of age. Transport activity in the FDB assessed with a maximally effective insulin concentration decreased only slightly (< 20%) between 1 and 7 mo of age. Aging, i.e., the transition from young adulthood to old age, was not associated with a decrease in glucose transport activity in either the epitrochlearis or the FDB.(ABSTRACT TRUNCATED AT 250 WORDS)

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.

We examined the effects of voluntary exercise on glucose transporter concentration in skeletal muscle from young adult and old female Long-Evans rats. Rats had free access to voluntary running wheels beginning at 4 months of age or remained sedentary. Exercising rats ran approximately 7.5, 6.2, 5.6 and 5.3 km/day during their 6th, 8th, 9th and 10th month of age, respectively. During the 23rd, 24th and 25th month of age running distance averaged 3.0, 2.8 and 2.4 km/day, respectively. At 10 and 25 months of age, glucose transporter protein concentration was assessed in epitrochlearis and flexor digitorum brevis muscles with a polyclonal antibody directed against the GLUT4 transporter isoform. GLUT4 protein concentration was not altered by the aging process (i.e., comparing 10- and 25-month-old rats) in either muscle type. Wheel running increased GLUT4 protein concentration by 45% in epitrochlearis muscles of 10-month-old rats relative to age-matched sedentary controls. The training-induced adaptation in GLUT4 protein was no longer present at age 25 months, probably because the running distance had declined by 50%. In the flexor digitorum brevis, exercise did not alter GLUT4 concentration at either 10 or 25 months, presumably due to insufficient recruitment of this muscle during wheel running as assessed by measurement of citrate synthase and hexokinase enzyme activities. Wheel running induced cardiac and soleus muscle hypertrophy in 10- and 25-month-old rats. In summary, voluntary wheel running can induce an increase in skeletal muscle GLUT4 protein concentration in adult rats. Older rats that run less exhibit cardiac and soleus muscle hypertrophy, but do not maintain an elevated GLUT4 protein concentration in the epitrochlearis muscle. Aging does not alter GLUT4 protein concentration in the epitrochlearis or FDB muscles.

Voluntary wheel running induces an increase in the concentration of the regulatable glucose transporter (GLUT4) in rat plantaris muscle but not in soleus muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). Wheel running also causes hypertrophy of the soleus in rats. This study was undertaken to ascertain whether endurance training that induces enzymatic adaptations but no hypertrophy results in an increase in the concentration of GLUT4 protein in rat soleus (slow-twitch red) muscle and, if it does, to determine whether there is a concomitant increase in maximal glucose transport activity. Female rats were trained by treadmill running at 25 m/min up a 15% grade, 90 min/day, 6 days/wk for 3 wk. This training program induced increases of 52% in citrate synthase activity, 66% in hexokinase activity, and 47% in immunoreactive GLUT4 protein concentration in soleus muscles without causing hypertrophy. Glucose transport activity stimulated maximally with insulin plus contractile activity was increased to roughly the same extent (44%) as GLUT4 protein content in soleus muscle by the treadmill exercise training. In a second set of experiments, we examined whether a swim-training program increases glucose transport activity in the soleus in the presence of a maximally effective concentration of insulin. The swimming program induced a 44% increase in immunoreactive GLUT4 protein concentration. Glucose transport activity maximally stimulated with insulin was 62% greater in soleus muscle of the swimmers than in untrained controls. Training did not alter the basal rate of 2-deoxyglucose uptake.(ABSTRACT TRUNCATED AT 250 WORDS)

Prior exercise increases insulin sensitivity for glucose and system A neutral amino acid transport activities in skeletal muscle. Insulin-like growth factor I (IGF-I) also activates these transport processes in resting muscle. It is not known, however, whether prior exercise increases IGF-I action in muscle. Therefore we determined the effect of a single exhausting bout of swim exercise on IGF-I-stimulated glucose transport activity [assessed by 2-deoxy-D-glucose (2-DG) uptake] and system A activity [assessed by alpha-(methylamino)isobutyric acid (MeAIB) uptake] in the isolated rat epitrochlearis muscle. When measured 3.5 h after exercise, the responses to a submaximal concentration (0.2 nM), but not a maximal concentration (13.3 nM), of insulin for activation of 2-DG uptake and MeAIB uptake were enhanced. In contrast, prior exercise increased markedly both the submaximal (5 nM) and maximal (20 nM) responses to IGF-I for activation of 2-DG uptake, whereas only the submaximal response to IGF-I (3 nM) for MeAIB uptake was enhanced after exercise. We conclude that 1) prior exercise significantly enhances the response to a submaximal concentration of IGF-I for activation of the glucose transport and system A neutral amino acid transport systems in skeletal muscle and 2) the enhanced maximal response for IGF-I action after exercise is restricted to the signaling pathway for activation of the glucose transport system.

It was previously found that voluntary wheel running induces an increase in the insulin-sensitive glucose transporter, i.e., the GLUT4 isoform, in rat plantaris muscle (K. J. Rodnick, J. O. Holloszy, C. E. Mondon, and D. E. James. Diabetes 39: 1425-1429, 1990). The present study was undertaken to determine whether 1) the increase in muscle GLUT4 protein is associated with an increase in maximally stimulated glucose transport activity, 2) a conversion of type IIb to type IIa or type I muscle fibers plays a role in the increase in GLUT4 protein, and 3) an increase in the GLUT1 isoform is a component of the adaptation of muscle to endurance exercise. Five weeks of voluntary wheel running that resulted in a 33% increase in citrate synthase activity induced a 50% increase in GLUT4 protein in epitrochlearis muscles of female Sprague-Dawley rats. The rate of 2-deoxy-glucose transport maximally stimulated with insulin or insulin plus contractions was increased approximately 40% (P less than 0.05). There was no change in muscle fiber type composition, evaluated by myosin ATPase staining, in the epitrochlearis. There was also no change in GLUT1 protein concentration. We conclude that an increase in GLUT4, but not of GLUT1 protein, is a component of the adaptive response of muscle to endurance exercise and that the increase in GLUT4 protein is associated with an increased capacity for glucose transport.

The relationships between muscle size, diffusion distance, and glucose uptake were studied using the Type IIb epitrochlearis (13 +/- 1 mg intact), Type I soleus (25 +/- 1 mg), and mixed Type IIa/IIb extensor digitorum longus (25 +/- 1 mg) from 60-70 g rats. Using intact muscles, the relative rates of 3-O-methyl-glucose uptake in response to 2 mUml-1 insulin were soleus = epitrochlearis greater than extensor digitorum longus, a finding inconsistent with the fibre-type compositions and the relative GLUT-4 protein levels (soleus greater than extensor digitorum longus greater than epitrochlearis). To test whether these results were influenced by substrate diffusion limitations in the tubular muscles, soleus and extensor digitorum longus were split longitudinally from tendon to tendon into strips of comparable size (13 +/- 1 mg) to the epitrochlearis. Insulin-stimulated rates of 3-O-methyl-glucose uptake were significantly enhanced in the split soleus (+120%) and split extensor digitorum longus (+200%), but not in the epitrochlearis, with the relative rates being soleus greater than extensor digitorum longus greater than epitrochlearis. Diffusion distances of the split soleus and extensor digitorum longus, as reflected by [14C]mannitol space equilibration time, were markedly enhanced (by at least 50%) relative to the intact muscles, and were comparable to that of the epitrochlearis. These results indicate that when muscles of different size and/or shape are used for in vitro measurement of glucose transport, the muscle preparations used must have similar diffusion distances for physiologically meaningful comparisons to be made.

The role of vicinal sulfhydryls in the stimulation by insulin of system A amino acid uptake in mammalian skeletal muscle was investigated. Neutral amino acid uptake via system A carriers was assessed using the nonmetabolizable analogue alpha-(methylamino)isobutyric acid (MeAIB). Phenylarsine oxide (PAO), a trivalent arsenical that interacts with vicinal sulfhydryls, at 40 microM inhibited basal and insulin-stimulated (2 mU/ml) MeAIB uptake in rat epitrochlearis muscles by approximately 50% and approximately 80%, respectively. No significant changes in the ATP level or in the lactate-to-pyruvate ratio were observed. Both inhibitory effects were completely preventable by coincubation with dimercaptopropanol, a vicinal dithiol, indicating the effects were mediated specifically by interactions with vicinal sulfhydryls. Stimulation of MeAIB uptake by the insulin-mimicker vanadate (10 mM) or by insulin-like growth factor I (IGF-I, 20 nM) was also inhibited by 80-90% by PAO. Kinetic analysis showed that PAO decreased the apparent Vmax for basal and insulin-stimulated MeAIB uptake without altering the apparent Km. MeAIB uptake already maximally stimulated by insulin was rapidly (half-time = approximately 10 min) reversed by the addition of PAO so that the rate of MeAIB uptake was the same as in muscles incubated throughout with insulin and PAO. These results implicate a major role for vicinal sulfhydryls in the stimulation by insulin of amino acid uptake via system A carriers in skeletal muscle and suggest that the site of action of PAO on this system is distal to the insulin receptor, possibly at the carrier molecule itself.

The purpose of this study was to test the hypothesis that the decreased capacity for glucose transport in the denervated rat soleus and the increased capacity for glucose transport in the unweighted rat soleus are related to changes in the expression of the regulatable glucose transporter protein in skeletal muscle (GLUT-4). One day after sciatic nerve sectioning, when decreases in the stimulation of soleus 2-deoxyglucose (2-DG) uptake by insulin (-51%, P less than 0.001), contractions (-29%, P less than 0.05), or insulin and contractions in combination (-40%, P less than 0.001) were observed, there was a slight (-18%, NS) decrease in GLUT-4 protein. By day 3 of denervation, stimulation of 2-DG uptake by insulin (-74%, P less than 0.001), contractions (-31%, P less than 0.001), or the two stimuli in combination (-59%, P less than 0.001), as well as GLUT-4 protein (-52%, P less than 0.001), was further reduced. Soleus muscle from hindlimb-suspended rats, which develops an enhanced capacity for insulin-stimulated glucose transport, showed muscle atrophy similar to denervated soleus but, in contrast, displayed substantial increases in GLUT-4 protein after 3 (+35%, P less than 0.05) and 7 days (+107%, P less than 0.001). These results indicate that altered GLUT-4 expression may be a major contributor to the changes in insulin-stimulated glucose transport that are observed with denervation and unweighting. We conclude that muscle activity is an important factor in the regulation of GLUT-4 expression in skeletal muscle.

The relationships among fiber type, glucose transporter (GLUT-4) protein content, and glucose transport activity stimulated maximally with insulin and/or contractile activity were studied by use of the rat epitrochlearis (15% type I-20% type II2a-65% type IIb), soleus (84-16-0%), extensor digitorum longus (EDL, 3-57-40%), and flexor digitorum brevis (FDB, 7-92-1%) muscles. Insulin-stimulated 2-deoxy-D-glucose (2-DG) uptake was greatest in the soleus, followed (in order) by the FDB, EDL, and epitrochlearis. On the other hand, contractile activity induced the greatest increase in 2-DG uptake in the FDB, followed by the EDL, soleus, and epitrochlearis. The effects of insulin and contractile activity on 2-DG uptake were additive in all the muscle preparations, with the relative rates being FDB greater than soleus greater than EDL greater than epitrochlearis. Quantitation of the GLUT-4 protein content with the antiserum R820 showed the following pattern: FDB greater than soleus greater than EDL greater than epitrochlearis. Linear regression analysis showed that whereas a relatively low and nonsignificant correlation existed between GLUT-4 protein content and 2-DG uptake stimulated by insulin alone, significant correlations existed between GLUT-4 protein content and 2-DG uptake stimulated either by contractions alone (r = 0.950) or by insulin and contractions in combination (r = 0.992). These results suggest that the differences in maximally stimulated glucose transport activity among the three fiber types may be related to differences in their content of GLUT-4 protein.

Previous reports indicated that glucose uptake in denervated muscle is resistant to insulin, while in non-weight-bearing (unweighted) muscle this effect of insulin is enhanced. To extend the comparison of these differences, insulin effects on amino acid uptake and protein metabolism were studied in soleus muscles subjected to denervation or unweighting. Denervated muscle showed insulin resistance of both 2-deoxy[1,2-3H]glucose and alpha-[methyl-3H]aminoisobutyric acid uptake whereas unweighted muscle showed an increased or normal response, respectively. Atrophy was greater in denervated than in unweighted muscle, apparently due to faster protein degradation. The stimulation of protein synthesis and the inhibition of protein degradation by insulin was generally less in denervated than in unweighted muscle. Since metabolic measurements in denervated-unweighted muscles did not differ from those in denervated-weight-bearing muscles, effects of denervation must be independent of leg posture.

Mechanisms of accelerated proteolysis were compared in denervated and unweighted (by tail-cast suspension) soleus muscles. In vitro and in vivo proteolysis were more rapid and lysosomal latency was lower in denervated than in unweighted muscle. In vitro, lysosomotropic agents (eg, chloroquine, methylamine) did not lessen the increase in proteolysis caused by unweighting, but abolished the difference in proteolysis between denervated and unweighted muscle. Leucine methylester, an indicator of lysosome fragility, lowered latency more in denervated than in unweighted muscle. 3-Methyladenine, which inhibits phagosome formation, increased latency similarly in all muscles tested. Mersalyl, a thiol protease inhibitor, and 8-(diethylamino)octyl-3,4,5-trimethoxybenzoate hydrochloride (TMB-8), which antagonizes sarcoplasmic reticulum release of Ca2+, reduced accelerated proteolysis caused by unweighting without diminishing the faster proteolysis due to denervation. Calcium ionophore (A23187) increased proteolysis more so in unweighted than control muscles whether or not Ca2+ was present. Different mechanisms of accelerated proteolysis were studied further by treating muscles in vivo for 24 hours with chloroquine or mersalyl. Chloroquine diminished atrophy of the denervated but not the unweighted muscle, whereas mersalyl prevented atrophy of the unweighted but not of the denervated muscle, both by inhibiting in vivo proteolysis. These results suggest that (1) atrophy of denervated, but not of unweighted, soleus muscle involves increased lysosomal proteolysis, possibly caused by greater permeability of the lysosome, and (2) cytosolic proteolysis is important in unweighting atrophy, involving some role of Ca2(+)-dependent proteolysis and/or thiol proteases.

The trivalent arsenical phenylarsine oxide (PAO) inhibits insulin-stimulated glucose transport in adipocytes and skeletal muscle through direct interactions with vicinal sulfhydryls. In muscle, glucose transport is also activated by contractile activity and hypoxia. It was therefore the purpose of the present study to investigate whether vicinal sulfhydryls are involved in the stimulation of glucose transport activity in the isolated rat epitrochlearis muscle by hypoxia or contractions. PAO (greater than 5 microM) caused a twofold increase in rate of transport of the nonmetabolizable glucose analogue 3-O-methylglucose (3-MG) that was completely prevented by cytochalasin B, the vicinal dithiol dimercaptopropanol, dantrolene, or 9-aminoacridine, both inhibitors of sarcoplasmic reticulum Ca2+ release, or omission of extracellular Ca2+. Although PAO treatment (greater than or equal to 20 microM) prevented approximately 80% of the increase in 3-MG transport caused by insulin, it resulted in only a approximately 50% inhibition of the stimulation of 3-MG transport by either hypoxia or contractile activity. PAO treatment (40 microM) of muscles already maximally stimulated by insulin, contractile activity, or hypoxia did not reverse the enhanced rate of 3-MG transport. These data suggest that vicinal sulfhydryls play a greater role in the activation of glucose transport by insulin than by muscle contractions or hypoxia. The finding that PAO inhibits the stimulation of glucose transport, but does not affect glucose transport after it has been stimulated, provides evidence that vicinal sulfhydryls are involved in the pathways for glucose transport activation in muscle, but not in the glucose transport mechanism itself.

Effects of stretching on muscle amino acids were tested in unloaded soleus by casting the foot in dorsiflexion on one limb of tail-casted, hindquarter-suspended rats. For comparison with unloading, amino acids also were measured in shortened extensor digitorum longus (EDL) in the same casted limb and in denervated leg muscles. Concentrations of tyrosine and glutamate were lower, while aspartate, ammonia, and the ratio of glutamine to glutamate were greater in the stretched than in the freely moving, unloaded soleus, but stretched did not differ from weight-bearing, control muscle. Therefore, stretching the soleus muscle prevented changes in certain amino acids due to unloading. Aspartate, ammonia, glutamine, and the ratio of glutamine to glutamate were lower in the shortened EDL than in the freely moving muscle of the contralateral limb, or in the control muscle. When denervated, these leg muscles also showed lower aspartate, ammonia, and ratio of glutamine to glutamate relative to innervated muscles. Since muscle shortening or denervation produced amino acid changes that mimicked the effects of unloading on the soleus, these responses must reflect the effect of muscle disuse. These data suggested that lower ammonia might cause the lower ratio of glutamine to glutamate with disuse. Because the fresh muscle energy charge, one factor which controls AMP deaminase, generally was not affected by disuse, altered deamination of glutamate via glutamate dehydrogenase may explain the variations in muscle ammonia.(ABSTRACT TRUNCATED AT 250 WORDS)

The time course of glycogen changes in soleus muscle recovering from 3 days of nonweight bearing by hindlimb suspension was investigated. Within 15 min and up to 2 h, muscle glycogen decreased. Coincidentally, muscle glucose 6-phosphate and the fractional activity of glycogen phosphorylase, measured at the fresh muscle concentrations of AMP, increased. Increased fractional activity of glycogen synthase during this time was likely the result of greater glucose 6-phosphate and decreased glycogen. From 2 to 4 h, when the synthase activity remained elevated and the phosphorylase activity declined, glycogen levels increased (glycogen supercompensation). A further increase of glycogen up to 24 h did not correlate with the enzyme activities. Between 24 and 72 h, glycogen decreased to control values, possibly initiated by high phosphorylase activity at 24 h. At 12 and 24 h, the inverse relationship between glycogen concentration and the synthase activity ratio was lost, indicating that reloading transiently uncoupled glycogen control of this enzyme. These data suggest that the activities of glycogen synthase and phosphorylase, when measured at physiological effector levels, likely provide the closest approximation to the actual enzyme activities in vivo. Measurements made in this way effectively explained the majority of the changes in the soleus glycogen content during recovery from nonweight bearing.

The effect of acutely reduced weight bearing (unloading) on the in vitro uptake of 2-[1,2-3H]deoxy-D-glucose was studied in the soleus muscle by tail casting and suspending rats. After just 4 h, the uptake of 2-deoxy-D-glucose fell (-19%, P less than 0.01) and declined further after an additional 20 h of unloading. This diminution at 24 h was associated with slower oxidation of [14C]glucose and incorporation of [14C]glucose into glycogen. Unlike after 1 day, at 3 days of unloading basal uptake of 2-deoxy-D-glucose did not differ from control. Reloading of the soleus after 1 or 3 days of unloading increased uptake of 2-deoxy-D-glucose above control and returned it to normal within 6 h and 4 days, respectively. These effects of unloading and recovery were caused by local changes in the soleus, because the extensor digitorum longus from the same hindlimbs did not display any alterations in uptake of 2-deoxy-D-glucose or metabolism of glucose. This study demonstrates that alterations in contractile activity, brought about by unloading or recovery from unloading, can influence the regulation of glucose transport in the soleus.

The time course of the response of carbohydrate metabolism to unloading was studied in the soleus muscle of rats subjected to tail-cast suspension. In the fresh soleus, just 12 hours of unloading led to higher concentrations of glycogen and lower activity ratios of both glycogen synthase and glycogen phosphorylase. These changes were still evident on day 3. This initial accumulation of glycogen was likely due to its decreased degradation in response to muscle disuse. Thereafter, the increased glycogen concentration apparently diminished the activity ratio of glycogen synthase, leading to a subsequent fall in the total glycogen content after day 1. After 24 hours of unloading, when no significant atrophy was detectable, there was no differential response to insulin for in vitro glucose metabolism. As reported for day 6 (reference 6), on day 3 the soleus atrophied significantly and displayed a greater sensitivity to insulin for most of these parameters compared to the weight-bearing control muscle. However, insulin sensitivity for glycogen synthesis was unchanged. These results showed that the increased sensitivity to insulin of the unloaded soleus is associated with the degree of muscle atrophy, likely due to an increased insulin binding capacity relative to muscle mass. This study also showed that insulin regulation of glucose uptake and of glycogen synthesis is affected differentially in the unloaded soleus muscle.

Hind leg muscles of female rats (85-99 g) were unloaded by tail cast suspension for 6 days. In the fresh-frozen unloaded soleus, the significantly greater concentration of glycogen correlated with a lower activity ratio of glycogen phosphorylase (p less than 0.02). The activity ratio of glycogen synthase also was lower (p less than 0.001), possibly due to the higher concentration of glycogen. In isolated unloaded soleus, insulin (0.1 milliunit/ml) increased the oxidation of D-[U-14C]glucose, release of lactate and pyruvate, incorporation of D-[U-14C]glucose into glycogen, and the concentration of glucose 6-phosphate more (p less than 0.05) than in the weight-bearing soleus. At physiological doses of insulin, the percent of maximal uptake of 2-deoxy-D-[1,2-3H]glucose/muscle also was greater in the unloaded soleus. Unloading of the soleus increased by 50% the concentration of insulin receptors, due to no decrease in total receptor number during muscle atrophy. This increase may account for the greater response of glucose metabolism to insulin in this muscle. The extensor digitorum longus, which generally shows little response to unloading, displayed no differential response of glucose metabolism to insulin.

Glucose transport can be stimulated via two separate pathways in muscle. One is activated by insulin, the other by contractile activity and hypoxia. Polymyxin B, a cationic antibiotic that displaces Ca2+ from anionic phospholipids, is reported to selectively inhibit the stimulation of glucose transport by insulin in muscle. A purpose of the present study was to determine whether the inhibition by polymyxin B is actually restricted to insulin. We found that polymyxin B (250 micrograms/ml) significantly inhibited the stimulation of glucose transport in rat skeletal muscles not only by insulin and vanadate but also by hypoxia, electrical stimulation, and K+. Polymyxin B also decreased the tension developed in response to electrical stimulation or K+. Although polymyxin B inhibited the increase in sugar transport activity induced by insulin and hypoxia, it had no inhibitory effect on sugar transport after it had been stimulated by these agents. These results show that the inhibitory effect of polymyxin B on the stimulation of glucose transport is not specific for insulin action. They suggest that polymyxin B inhibits a step that is common to the two pathways for stimulating glucose transport in skeletal muscle.

It has been hypothesized on the basis of studies on BC3H-1 myocytes that diacylglycerol generation with activation of protein kinase C (PKC) is involved in the stimulation of glucose transport in muscle by insulin (Standaert, M. L., Farese, R. V., Cooper, R. D., and Pollet, R. J. (1988) J. Biol. Chem. 263, 8696-8705). In the present study, we used the rat epitrochlearis muscle to evaluate the possibility that PKC activity mediates the stimulation of glucose transport by insulin in mammalian skeletal muscle. Phospholipase C from Clostridium perfringens (PLC-Cp), which generates diacylglycerol from membrane phospholipids, and 4 beta-phorbol 12 beta-myristate 13 alpha-acetate (PMA) induced increases in glucose transport activity (assessed using 3-O-methylglucose transport) that were approximately 80 and approximately 20% as great, respectively, as that induced by a maximal insulin stimulus. PLC-Cp and PMA both caused a approximately 2-fold increase in membrane-associated PKC activity. In contrast, insulin did not affect PKC activity. These findings argue against a role of diacylglycerol-mediated PKC activation in the stimulation of skeletal muscle glucose transport by insulin. They also show that the BC3H-1 myocyte is not a good model for studying regulation of glucose transport in skeletal muscle. Neither the submaximal nor maximal effects of PLC-Cp and insulin on glucose transport were additive, suggesting that PLC-Cp interferes with insulin action. The maximal effects of PLC-Cp and hypoxia or muscle contractions were also not additive. However, the submaximal effects of hypoxia and PLC-Cp were completely additive. These findings raise the possibility that PLC-Cp stimulates glucose transport by the exercise/hypoxia-activated, not the insulin-activated, pathway in skeletal muscle. Exposure to PLC-Cp activated glycogen phosphorylase and potentiated twitch tension in response to electrical stimulation, providing evidence that PLC-Cp increases cytoplasmic Ca2+ concentration. Dantrolene, an inhibitor of Ca2+ release from the sarcoplasmic reticulum, completely blocked both the activation of phosphorylase and the stimulation of glucose transport by PLC-Cp. These findings provide evidence that an increase in cytoplasmic Ca2+ concentration is involved in the activation of glucose transport in skeletal muscle by PLC-Cp.

The influence of glucocorticoids on muscle glutamine production in starvation was studied by using cortisol-treated or non-cortisol-treated, starved, adrenalectomized rats. Administration of cortisol at physiological doses in vivo (1 mg/100 g body weight) to fasted, adrenalectomized rats increased the muscle ratio of glutamine/glutamate and the activity of glutamine synthetase after only 6 hours. Prior treatment of fasted, adrenalectomized animals with actinomycin D or proflavine abolished these increases by cortisol. Therefore, cortisol induces muscle glutamine synthetase, and this induction can be detected by changes in the fresh-muscle ratio of glutamine/glutamate. Using this ratio as a qualitative indicator of muscle glutamine synthesis, the role of glucocorticoids in modifying muscle glutamine production in starvation was studied. In fresh-frozen soleus, extensor digitorum longus, and diaphragm muscle, starvation led to greater ratios of glutamine/glutamate and higher levels of tyrosine, which are indicative of enhanced muscle protein turnover. These effects were not apparent in starved, adrenalectomized animals but were restored, at least partially, by administering a physiological dose of cortisol. Therefore, glucocorticoids seem essential for promoting muscle glutamine production in starvation probably by inducing the activity of glutamine synthetase.