Identification of the glucose transporter in rat skeletal muscle

The glucose transporter in the plasma membrane of rat skeletal muscle has been identified by two approaches. In one, the transporter was detected as the polypeptide that was differentially labeled by photolysis with [³H]cytochalasin B in the presence of l- and d-glucose. [³H]Cytochalasin B is a high-affinity ligand for the transporter that is displaced by d-glucose. In the other, the transporter was detected by means of its reaction with rabbit antibodies against the purified glucose transporter from human erythrocytes. By both procedures, the transporter was found to be a polypeptide with a mobility corresponding to a molecular weight of 45,000–50,000 upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Reconstitution of the glucose transporter from rat skeletal muscle

As a step in the purification and characterization of the glucose transporter from rat skeletal muscle, we have reconstituted glucose transport activity in liposomes. Plasma membranes were prepared from skeletal muscle which display D-glucose reversible binding of cytochalasin B (10 pmol sites/mg protein; KD = 0.3 microM). Older rats gave a slightly lower specific activity and much lower yield of sites per g muscle than young rats. Glucose transport activity was reconstituted into liposomes by the freeze-thaw procedure using either plasma membranes directly or cholate-extracted membrane proteins; the latter gave a 50% higher specific activity. The reconstituted transport activity was stereospecific, saturable, and inhibited by cytochalasin B, phloretin, and mercuric chloride. The optimum cholate concentration for extraction and reconstitution of transport activity was about 1.5%, and the highest specific activity of reconstituted transport was seen only at low ratios of protein to lipid in the reconstitution. Chromatography on agarose lentil lectin and agarose ethanethiol doubled both the specific activity of reconstituted transport and the fraction of glucose uptake which was stereospecific. In all of these respects the results were similar to our results with the bovine heart transporter (T. J. Wheeler and M. A. Hauck, Biochim. Biophys. Acta 818, 171-182 (1985)). Our findings suggest that further purification procedures developed for the heart transporter may be applicable to the skeletal muscle transporter as well.     

Developmental changes in intestinal glucose transporter mRNA levels.

Developmental changes in glucose transporter mRNA levels in the jejunum of rats of different ages were examined by using slot blot RNA analysis. The level of SGLT1 mRNA did not change significantly through life. The GLUT5 mRNA level was highest in 10-day-old rats and then decreased reaching the adult level by day 20 after birth. The GLUT2 mRNA level was low in rats of 5 and 10 days old, but then increased progressively reaching the adult value by day 25 after birth. These results indicate that the expressions of intestinal facilitative glucose transporter genes change markedly in the third week after birth.     

Increase in liver glucose transporter mRNA levels during rat liver regeneration

Gene expression of liver facilitated glucose transporter was rapidly induced during the liver regenerating process in rats. It reached maximum of 2.7 times at 8 hr of the regenerating course and returned to normal by 48 hr. The protein synthesis inhibitor, cycloheximide, did not interfere with the increased gene expression of liver facilitated glucose transporter. By contrast, erythrocyte/brain-type glucose transporter mRNA could not be detected in the livers of partially hepatectomized rats and sham-operated rats. The plasma glucose levels were transiently increased within 2 hr of the regenerative course and then decreased to a nadir at 4 hr. These results suggest that the increased gene expression of liver facilitated glucose transporter contributes to the decrease in plasma glucose levels.     

Regulation of glucose transporter messenger RNA levels in rat adipose tissue by insulin

Analysis of glucose transporter mRNA levels in adipose tissue from streptozotocin (STZ)-induced diabetic rats demonstrated a specific decrease (10-fold) in adipose tissue GLUT-4 mRNA with no significant effect on GLUT-1 mRNA levels. Treatment of STZ-diabetic rats with twice daily injections of insulin for 1-3 days resulted in a 16-fold increase in the relative amount of GLUT-4 mRNA to levels approximately 2-fold greater than those in control animals. However, after 7 days of insulin therapy the amount of GLUT-4 mRNA decreased approximately 2-fold back to the levels in the control animals. Normalization of the STZ-induced serum hyperglycemia by phlorizin treatment, which inhibits renal tubular reabsorption of glucose, had no effect on GLUT-4 mRNA in the absence of insulin. Similar to STZ-diabetes, fasting for 48 h also reduced adipose GLUT-4 mRNA levels. Parenteral administration of insulin with glucose over 7.5 h, but not glucose alone, increased the levels of the GLUT-4 mRNA 3- to 4-fold. These studies demonstrate that the relative glycemic state does not influence GLUT-4 glucose transporter mRNA expression in vivo and strongly suggests that insulin is a major factor regulating the levels of GLUT-4 mRNA in adipose tissue.     

Glucosamine and glucose induce insulin resistance by different mechanisms in rat skeletal muscle

It has been hypothesized that glucose-induced insulin resistance is mediated by accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs). In a previous study on rat epitrochlearis muscles incubated with high concentrations of glucose and insulin (Kawanaka K, D-H Han, J Gao, LA Nolte, and JO Holloszy. J Biol Chem 276: 20101-20107, 2001), we found that insulin resistance developed even when the increase in UDP-Hex-NAcs was prevented. Furthermore, actinomycin D completely prevented glucose-induced insulin resistance despite a greater accumulation of UDP-HexNAcs. In the present study, we used the same epitrochlearis muscle preparation, as well as the rat hemidiaphragm, to determine whether, like glucose, glucosamine causes insulin resistance by an actinomycin D-inhibitable process. Incubation of diaphragm muscles with 10 mM glucosamine for 3 h resulted in an approximately fivefold increase in UDP-HexNAcs, an approximately 50% reduction in insulin responsiveness of glucose transport, and a 58% reduction in ATP concentration. These effects of glucosamine were not prevented by actinomycin D. Incubation of epitrochlearis muscles with 20 mM glucosamine for 3 h or with 10 mM glucosamine for 5 h also caused large decreases in insulin responsiveness of glucose transport but with no reduction in ATP concentration. Actinomycin D did not prevent the glucosamine-induced insulin resistance. The insulin-induced increases in tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the binding of PI 3-kinase to IRS-1 were decreased approximately 60% in epitrochlearis muscles exposed to glucosamine. This is in contrast to glucose-induced insulin resistance, which was not associated with impaired insulin signaling. These results provide evidence that glucosamine and glucose induce insulin resistance by different mechanisms.     

Characterization of skeletal muscle insulin receptor

The insulin receptor from rat skeletal muscle was characterized. Treatment of muscle membranes with the photoactive insulin analog, 125I[N-epsilonB29-monoazidobenzoyl]-insulin revealed a single protein band of 135,000 Da, the alpha subunit. Iodination of total membrane protein followed by Triton X-100 solubilization and immunoprecipitation demonstrated the presence of a protein band of 90,000 Da, the beta subunit, together with a protein band of 190,000 Da, which may be the receptor precursor. In partially purified receptor preparations, the beta subunit exhibited dose-dependent, insulin-stimulated phosphorylation with incorporation of phosphate solely into tyrosine residues, which was also observed in the 190,000-Da receptor precursor. Purified plasma membranes contained a large amount of insulin-degrading activity which had to be inactivated prior to performing insulin-binding studies. If degradation of insulin was not prevented, apparent enhanced binding in the presence of unlabeled insulin was observed.     

Recruitment of GLUT-4 glucose transporters by insulin in diabetic rat skeletal muscle

The cause of reduced insulin-stimulated glucose transport in skeletal muscle of diabetic rats was investigated. Basal and insulin-stimulated glucose uptake into hindquarter muscles of 7-day diabetic rats were 70% and 50% lower, respectively, than in nondiabetic controls. Subcellular fractionation of hindquarter muscles yielded total crude membranes, plasma membranes and intracellular membranes. The number of GLUT-4 glucose transporters was lower in crude membranes, plasma membranes and intracellular membranes, relative to non-diabetic rat muscles. These results were paralleled by reductions in D-glucose-protectable binding of cytochalasin B. Insulin caused a redistribution of GLUT-4 transporters from intracellular membranes to plasma membranes, in both control and diabetic rat muscles. This redistribution was also recorded using binding of cytochalasin B. The insulin-dependent decrement in glucose transporters in intracellular membranes was similar for both animal groups, but the gain and final amount of transporters in the plasma membrane were 50% lower in the diabetic group. The results suggest that insulin signalling and recruitment of GLUT-4 glucose transporters occur in diabetic rat muscle, and that the diminished insulin response may be due to fewer glucose transporters operating in the muscle plasma membrane.     

Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.     

Effects of vanadate and insulin on glucose 1,6-P2 and fructose 2,6-P2 levels in rat skeletal muscle

Levels of glucose 1,6-P2 but not fructose 2,6-P2 were found decreased in skeletal muscle of alloxan-diabetic ketotic rats. Administration of both insulin and vanadate restored the altered values without affecting fructose 2,6-P2 concentrations. In normal rats, insulin increased muscle levels of both sugars, and vanadate decreased glucose 1,6-P2 without changing fructose 2,6-P2 levels. Enzymatic activities involved in glucose 1,6-P2 and fructose 2,6-P2 metabolism were not affected under any experimental condition.     

1,2-Diacylglycerol and ceramide levels in rat skeletal muscle and liver in vivo. Studies with insulin, exercise, muscle denervation, and vasopressin

Studies on BC3H-1 myocytes suggest that the insulin-induced increase in cellular diacylglycerol level mediates the insulin-stimulated glucose transport in these cells (Standaert, M. L., Farese, R. V., Cooper, D. R., and Pollet, R. J. (1988) J. Biol. Chem. 263, 8696-8705). The present study tested whether diacylglycerol could mediate the insulin-induced and exercise-induced increases in glucose uptake by rat skeletal muscle in vivo. Glucose uptake by calf muscles of the rat was assessed by measuring cellular 2-deoxyglucose uptake in vivo. Diacylglycerol and ceramides in muscles frozen in situ were assayed with diacylglycerol kinase. Intravenous injection of 0.1 unit of insulin/rat resulted in a 6-fold increase in muscle 2-deoxyglucose uptake during the subsequent 25-min period. In contrast, no statistically significant changes in muscle diacylglycerol or ceramide levels were observed at 2, 5, 10, and 25 min after insulin injection. When calf muscles of the hindlimb were exercised in vivo for 25 min by electrical stimulation inducing one contraction/s, 2-deoxyglucose uptake by muscles was increased 15-fold. However, no statistically significant changes in muscle diacylglycerol or ceramide content were observed at 5, 10, 15, and 25 min of exercise. Although the findings do not exclude the possibility of a compartmentalized increase in diacylglycerol level, the present data suggest that diacylglycerol is not a mediator of the insulin-induced or exercise-induced augmentation of glucose uptake by skeletal muscle in vivo. Since interruption of nerve supply to the muscles makes the muscles insulin resistant (Turinsky, J., (1987) Am. J. Physiol. 252, R531-R537), the effect of denervation on diacylglycerol and ceramide levels in calf muscles of the rat was also examined. The denervation resulted in 21, 51, and 117% increases in muscle diacylglycerol levels at 3, 16, and 32 days after denervation, respectively. No statistically significant changes in muscle ceramide levels were observed at any postdenervation interval. Finally, the measured lipids were studied in muscles and livers of rats infused with supraphysiological doses of vasopressin (86 pmol/min). In controls, diacylglycerol concentrations of the muscles and liver did not significantly differ, but the liver exhibited a 5-fold higher level of ceramides than the muscles. Infusion of vasopressin for 5 min did not have a statistically significant effect on diacylglycerol concentration of the liver but continuation of the same infusion for 10 min resulted in a 63% increase in liver diacylglycerol. The 10-min infusion had no effect on muscle diacylglycerol concentration or ceramide levels in any of the tissues studied.(ABSTRACT TRUNCATED AT 400 WORDS)     

Co-localization of a glucose transporter and the insulin receptor in microsomes of insulin-treated rat adipocytes

Microsomal vesicles prepared from rat adipocytes were immuno-adsorbed to formaldehyde-fixed Staphylococcus aureus cells (Pansorbin) coated with anti-human-erythrocyte-glucose-transporter IgG. More than 75% of the glucose transporter detected was precipitated. The glucose transporter was about 10-fold enriched by the adsorption procedure. On insulin treatment, the insulin receptor in plasma membranes was internalized and the receptor in the microsome fraction increased 5-fold. Thirty-five % of the insulin receptor in the microsome fraction was recovered with the glucose-transporter-containing vesicles. These observations indicate that on insulin treatment a considerable portion of the microsome vesicles containing the insulin receptor fuses or becomes tightly associated with ones containing the glucose transporter.     

A novel role of neuregulin in skeletal muscle. Neuregulin stimulates glucose uptake, glucose transporter translocation, and transporter expression in muscle cells

Neuregulins regulate the expression of acetylcholine receptor genes and induce development of the neuromuscular junction in muscle. In studying whether neuregulins regulate glucose uptake in muscle, we analyzed the effect of a recombinant neuregulin, (r)heregulin-beta1-(177-244) (HRG), on L6E9 muscle cells, which express the neuregulin receptors ErbB2 and ErbB3. L6E9 responded acutely to HRG by a time- and concentration-dependent stimulation of 2-deoxyglucose uptake. HRG-induced stimulation of glucose transport was additive to the effect of insulin. The acute stimulation of the glucose transport induced by HRG was a consequence of the translocation of GLUT4, GLUT1, and GLUT3 glucose carriers to the cell surface. The effect of HRG on glucose transport was dependent on phosphatidylinositol 3-kinase activity. HRG also stimulated glucose transport in the incubated soleus muscle and was additive to the effect of insulin. Chronic exposure of L6E9 cells to HRG potentiated myogenic differentiation, and under these conditions, glucose transport was also stimulated. The activation of glucose transport after chronic HRG exposure was due to enhanced cell content of GLUT1 and GLUT3 and to increased abundance of these carriers at the plasma membrane. However, under these conditions, GLUT4 expression was markedly down-regulated. Muscle denervation is associated with GLUT1 induction and GLUT4 repression. In this connection, muscle denervation caused a marked increase in the content of ErbB2 and ErbB3 receptors, which occurred in the absence of alterations in neuregulin mRNA levels. This fact suggests that neuregulins regulate glucose transporter expression in denervated muscle. We conclude that neuregulins regulate glucose uptake in L6E9 muscle cells by mechanisms involving the recruitment of glucose transporters to the cell surface and modulation of their expression. Neuregulins may also participate in the adaptations in glucose transport that take place in the muscle fiber after denervation.     

Increased IGFR activity and glucose transport in cultured skeletal muscle from insulin receptor null mice

We have studied the role of the insulin receptor (IR) in metabolic and growth-promoting effects of insulin on primary cultures of skeletal muscle derived from the limb muscle of IR null mice. Cultures of IR null skeletal muscle displayed normal morphology and spontaneous contractile activity. Expression of muscle-differentiating proteins was slightly reduced in myoblasts and myotubes of the IR null skeletal muscle cells, whereas that of the Na+/K+ pump appeared to be unchanged. Insulin-like growth factor receptor (IGFR) expression was higher in myoblasts from IR knockout (IRKO) than from IR wild-type (IRWT) mice but was essentially unchanged in myotubes. Expression of the GLUT-1 and GLUT-4 transporters appeared to be higher in IRKO than in IRWT myoblasts and was significantly greater in myotubes from IRKO than from IRWT cultures. Consistent with GLUT expression, both basal and insulin or insulin-like growth factor I (IGF-I)-stimulated glucose uptakes were higher in IR null skeletal myotubes than in wild-type skeletal myotubes. Interestingly, autophosphorylation of IGFR induced by insulin and IGF-I was markedly increased in IR null skeletal myotubes. These results indicate that, in the absence of IR, there is a compensatory increase in basal as well as in insulin- and IGF-I-induced glucose transport, the former being mediated via increased activation of the IGF-I receptor.     

mRNA levels for alpha-subunit of prolyl 4-hydroxylase and fibrillar collagens in immobilized rat skeletal muscle.

There is evidence that immobilization causes a decrease in total collagen synthesis in skeletal muscle within a few days. In this study, early immobilization effects on the expression of prolyl 4-hydroxylase (PH) and the main fibrillar collagens at mRNA and protein levels were investigated in rat skeletal muscle. The right hindlimb was immobilized in full plantar flexion for 1, 3, and 7 days. Steady-state mRNAs for alpha- and beta-subunits of PH and type I and III procollagen, PH activity, and collagen content were measured in gastrocnemius and plantaris muscles. Type I and III procollagen mRNAs were also measured in soleus and tibialis anterior muscles. The mRNA level for the PH alpha-subunit decreased by 49 and 55% (P < 0.01) in gastrocnemius muscle and by 41 and 39% (P < 0.05) in plantaris muscle after immobilization for 1 and 3 days, respectively. PH activity was decreased (P < 0.05-0.01) in both muscles at days 3 and 7. The mRNA levels for type I and III procollagen were decreased by 26-56% (P < 0.05-0.001) in soleus, tibialis anterior, and plantaris muscles at day 3. The present results thus suggest that pretranslational downregulation plays a key role in fibrillar collagen synthesis in the early phase of immobilization-induced muscle atrophy.     

Caffeine modulates phosphorylation of insulin receptor substrate-1 and impairs insulin signal transduction in rat skeletal muscle

Caffeine decreases insulin sensitivity and insulin-stimulated glucose transport in skeletal muscle; however, the precise mechanism responsible for this deleterious effect is not understood fully. We investigated the effects of incubation with caffeine on insulin signaling in rat epitrochlearis muscle. Caffeine (≥1 mM, ≥15 min) suppressed insulin-stimulated insulin receptor substrate (IRS)-1 Tyr(612) phosphorylation in a dose- and time-dependent manner. These responses were associated with inhibition of the insulin-stimulated phosphorylation of phosphatidylinositol 3-kinase (PI3K) Tyr(458), Akt Ser(473), and glycogen synthase kinase-3β Ser(9) and with inhibition of insulin-stimulated 3-O-methyl-d-glucose (3MG) transport but not with inhibition of the phosphorylation of insulin receptor-β Tyr(1158/62/63). Furthermore, caffeine enhanced phosphorylation of IRS-1 Ser(307) and an IRS-1 Ser(307) kinase, inhibitor-κB kinase (IKK)-α/β Ser(176/180). Blockade of IKK/IRS-1 Ser(307) by caffeic acid ameliorated the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation and 3MG transport. Caffeine also increased the phosphorylation of IRS-1 Ser(789) and an IRS-1 Ser(789) kinase, 5'-AMP-activated protein kinase (AMPK). However, inhibition of IRS-1 Ser(789) and AMPK phosphorylation by dantrolene did not rescue the caffeine-induced downregulation of IRS-1 Tyr(612) phosphorylation or 3MG transport. In addition, caffeine suppressed the phosphorylation of insulin-stimulated IRS-1 Ser(636/639) and upstream kinases, including the mammalian target of rapamycin and p70S6 kinase. Intravenous injection of caffeine at a physiological dose (5 mg/kg) in rats inhibited the phosphorylation of insulin-stimulated IRS-1 Tyr(612) and Akt Ser(473) in epitrochlearis muscle. Our results indicate that caffeine inhibits insulin signaling partly through the IKK/IRS-1 Ser(307) pathway, via a Ca(2+)- and AMPK-independent mechanism in skeletal muscle.     

Differential regulation of glucose transporter activity and expression in red and white skeletal muscle.

Insulin-stimulated glucose transport activity and GLUT4 glucose transporter protein expression in rat soleus, red-enriched, and white-enriched skeletal muscle were examined in streptozotocin (STZ)-induced insulin-deficient diabetes. Six days of STZ-diabetes resulted in a nearly complete inhibition of insulin-stimulated glucose transport activity in perfused soleus, red, and white muscle which recovered following insulin therapy. A specific decrease in the GLUT4 glucose transporter protein was observed in soleus (3-fold) and red (2-fold) muscle which also recovered to control values with insulin therapy. Similarly, cardiac muscle displayed a marked STZ-induced decrease in GLUT4 protein that was normalized by insulin therapy. White muscle displayed a small but statistically significant decrease in GLUT4 protein (23%), but this could not account for the marked inhibition of insulin-stimulated glucose transport activity observed in this tissue. In addition, GLUT4 mRNA was found to decrease in red muscle (2-fold) with no significant alteration in white muscle. The effect of STZ-induced diabetes was time-dependent with maximal inhibition of insulin-stimulated glucose transport activity at 24 h in both red and white skeletal muscle and half-maximal inhibition at approximately 8 h. In contrast, GLUT4 protein in red and white muscle remained unchanged until 4 and 7 days following STZ treatment, respectively. These data demonstrate that red skeletal muscle displays a more rapid hormonal/metabolic-dependent regulation of GLUT4 glucose transporter protein and mRNA expression than white skeletal muscle. In addition, the inhibition of insulin-stimulated glucose transport activity in both red and white muscle precedes the decrease in GLUT4 protein and mRNA levels. Thus, STZ treatment initially results in a rapid uncoupling of the insulin-mediated signaling of glucose transport activity which is independent of GLUT4 protein and mRNA levels.