Development of Glucose-induced Insulin Resistance in Muscle Requires Protein Synthesis
2001; Elsevier BV; Volume: 276; Issue: 23 Linguagem: Inglês
10.1074/jbc.m010599200
ISSN1083-351X
AutoresKentaro Kawanaka, Dong‐Ho Han, Jiaping Gao, Lorraine A. Nolte, John O. Holloszy,
Tópico(s)Adipose Tissue and Metabolism
ResumoMuscles and fat cells develop insulin resistance when exposed to high concentrations of glucose and insulin. We used an isolated muscle preparation incubated with high levels of glucose and insulin to further evaluate how glucose-induced insulin resistance (GIIR) is mediated. Incubation with 2 milliunits/ml insulin and 36 mm glucose for 5 h resulted in an ∼50% decrease in insulin-stimulated muscle glucose transport. The decrease in insulin responsiveness of glucose transport induced by glucose was not due to impaired insulin signaling, as insulin-stimulated phosphatidylinositol 3-kinase activity and protein kinase B phosphorylation were not reduced. It has been hypothesized that entry of glucose into the hexosamine biosynthetic pathway with accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs) mediates GIIR. However, inhibition of the rate-limiting enzyme GFAT (glutamine:fructose-6-phosphate amidotransferase) did not protect against GIIR despite a marked reduction of UDP-HexNAcs. The mRNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide both completely protected against GIIR despite the massive increases in UDP-HexNAcs and glycogen that resulted from increased glucose entry. Activation of AMP-activated protein kinase also protected against GIIR. These results provide evidence that GIIR can occur in muscle without increased accumulation of hexosamine pathway end products, that neither high glycogen concentration nor impaired insulin signaling is responsible for GIIR, and that synthesis of a protein with a short half-life mediates GIIR. They also suggest that dephosphorylation of a transcription factor may be involved in the induction of GIIR. Muscles and fat cells develop insulin resistance when exposed to high concentrations of glucose and insulin. We used an isolated muscle preparation incubated with high levels of glucose and insulin to further evaluate how glucose-induced insulin resistance (GIIR) is mediated. Incubation with 2 milliunits/ml insulin and 36 mm glucose for 5 h resulted in an ∼50% decrease in insulin-stimulated muscle glucose transport. The decrease in insulin responsiveness of glucose transport induced by glucose was not due to impaired insulin signaling, as insulin-stimulated phosphatidylinositol 3-kinase activity and protein kinase B phosphorylation were not reduced. It has been hypothesized that entry of glucose into the hexosamine biosynthetic pathway with accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs) mediates GIIR. However, inhibition of the rate-limiting enzyme GFAT (glutamine:fructose-6-phosphate amidotransferase) did not protect against GIIR despite a marked reduction of UDP-HexNAcs. The mRNA synthesis inhibitor actinomycin D and the protein synthesis inhibitor cycloheximide both completely protected against GIIR despite the massive increases in UDP-HexNAcs and glycogen that resulted from increased glucose entry. Activation of AMP-activated protein kinase also protected against GIIR. These results provide evidence that GIIR can occur in muscle without increased accumulation of hexosamine pathway end products, that neither high glycogen concentration nor impaired insulin signaling is responsible for GIIR, and that synthesis of a protein with a short half-life mediates GIIR. They also suggest that dephosphorylation of a transcription factor may be involved in the induction of GIIR. protein kinase B UDP-N-acetylhexosamines (UDP-N-acetylglucosamine plus UDP-N-acetylgalactosamine) glutamine:fructose-6-phosphate amidotransferase Krebs-Henseleit bicarbonate buffer 2-deoxy-d-glucose 2-deoxy-d-glucose-6-phosphate 3-O-methyl-d-glucose 6-diazo-5-oxonorleucine 5,6-dichloro-1-β-D ribofuranosylbenzimidazole protein kinase C phosphatidylinositol 3-kinase 5-aminoimidazole-4-carboxamide ribonucleoside AMP-activated protein kinase Tris-buffered saline containing 0.1% Tween 10 Hyperglycemia can lead to the development of insulin resistance, a phenomenon thought to contribute to impaired insulin action in diabetes (1Rossetti L. Giaccari A. DeFronzo R.A. Diabetes Care. 1990; 13: 610-630Crossref PubMed Scopus (895) Google Scholar, 2Yki-Jarvinen H. Endocr. Rev. 1992; 13: 415-431PubMed Google Scholar, 3Rossetti L. Smith D. Shulman G.I. Papachristou D. DeFronzo R.A. J. Clin. 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Maianu L. Garvey W.T. J. Clin. Invest. 1995; 96: 2792-2801Crossref PubMed Scopus (238) Google Scholar, 28Robinson K.A. Weinstein M.L. Lindenmayer G.E. Buse M.G. Diabetes. 1995; 44: 1438-1446Crossref PubMed Scopus (133) Google Scholar, 29Robinson K.A. Sens D.A. Buse M.G. Diabetes. 1993; 42: 1333-1346Crossref PubMed Scopus (155) Google Scholar). In addition to disagreement regarding the mechanism, there is controversy regarding whether rapid entry of glucose into the cell, mediated by high concentrations of both glucose and insulin (30Garvey W.T. Olefsky J.M. Matthaei S. Marshall S. J. Biol. Chem. 1987; 262: 189-197Abstract Full Text PDF PubMed Google Scholar) or just exposure to a high concentration of glucose (8Richter E.A. Hansen B.F. Hansen S.A. Biochem. J. 1988; 252: 733-737Crossref PubMed Scopus (73) Google Scholar, 10Hansen B.F. Hansen S.A. Ploug T. Bak J.F. Richter E.A. Am. J. Physiol. 1992; 262: E440-E446PubMed Google Scholar), is responsible for glucose-induced insulin resistance. In this study we used an in vitro skeletal muscle preparation to evaluate these hypotheses and to further examine the mechanism responsible for glucose-induced insulin resistance. Our results do not support any of the current hypotheses regarding the etiology of glucose toxicity. They show that the decrease in insulin responsiveness of glucose transport results from a rapid influx of glucose and suggest that increased expression of a protein with a short half-life mediates the insulin resistance. The antiphospho-protein kinase B (PKB)1 Ser473 and Thr308 polyclonal antibodies were from Upstate Biotechnology. Horseradish peroxidase-conjugated donkey anti-rabbit IgG was obtained from Jackson ImmunoResearch Laboratories. [14C]Mannitol, 3-O-[3H]methyl-d-glucose, and [γ-32P]ATP were purchased from PerkinElmer Life Sciences. 2-Deoxy-d-[1,2-3H]glucose was purchased from American Radiolabeled Chemicals. Reagents for SDS-polyacrylamide gel electrophoresis were obtained from Bio-Rad. Polyclonal antibody F349 against GLUT4 protein was a gift from Dr. Mike Mueckler of Washington University School of Medicine. All other reagents were purchased from Sigma Aldrich Chemical Co. This research was approved by the Animal Studies Committee of Washington University. Male Wistar rats weighing 100–140 g were obtained from Charles River and fed Purina chow and water until the evening before an experiment, when food was removed at ∼6:00 p.m. Rats were anesthetized with an intraperitoneal injection of sodium pentobarbital, 5 mg/100 g of body weight, and a polyethylene catheter was placed in an external jugular vein. The catheter was tunneled under the skin, exteriorized between the shoulder blades, sutured in place, and filled with saline. The surgical incision was closed, and the externalized catheter was covered with a jacket (Harvard Apparatus). After the rats had recovered from surgery for 3 days, the catheters were threaded through a flexible spring tether attached to a swiveling infusion device (Harvard Apparatus) that allowed the animals to move freely during the infusion. The rats were infused with either 50% glucose at an infusion rate of 50 mg glucose/kg of body weight/min or with 0.9% saline for 24 h. At the end of the infusion period, the rats were anesthetized with sodium pentobarbital, 5 mg/100 g of body weight. Blood samples were obtained for measurement of glucose and insulin (31Morgan D.R. Lazarow A. Diabetes. 1963; 12: 115-126Crossref Google Scholar). The epitrochlearis muscles were dissected out and incubated for 60 min with 60 microunits/ml or 2 milliunits/ml insulin prior to measurement of glucose transport activity. After removal of the epitrochlearis muscles, insulin was infused at a rate of 7.2 milliunits/min/100 g of body weight for 30 min to induce a maximal insulin stimulus; glucose was also infused to prevent hypoglycemia. After 30 min of insulin infusion, the gastrocnemius/plantaris muscle group was excised for determination of GLUT4 in the sarcolemma. Epitrochlearis muscles were placed in 2 ml of oxygenated Krebs-Henseleit bicarbonate buffer (KHB) with or without 2 milliunits/ml insulin, 0.1% radioimmunoassay grade bovine serum albumin, various concentrations of glucose, and sufficient mannitol so that the concentration of glucose plus mannitol was 40 mm. Routinely, 36 mm glucose was used to induce muscle insulin resistance, and 5 mm glucose was used as the control. The other additions to the incubation medium are described for each experiment under "Results" and/or the figure legends. The muscles were incubated with shaking at 35 °C, and the flasks were gassed continuously with 95% O2, 5% CO2. During 5-h long incubations, the muscles were placed in fresh incubation medium after 2.5 h. After the incubations some muscles were blotted and used for measurement of metabolites, while others were used for measurement of glucose transport activity. After the initial incubation period, the muscles were quickly rinsed twice in KHB and transferred to flasks containing 2 ml of KHB with 40 mmmannitol, with the same concentration of insulin as in the initial incubation, and incubated with shaking at 30 °C to remove glucose. This procedure was repeated once. Glucose transport activity was then measured as described previously (32Young D.A. Uhl J.J. Cartee G.D. Holloszy J.O. J. Biol. Chem. 1986; 261: 16049-16053Abstract Full Text PDF PubMed Google Scholar, 33Ren J.-M. Marshall B.A. Gulve E.A. Gao J. Johnson D.W. Holloszy J.O. Mueckler M. J. Biol. Chem. 1993; 268: 16113-16115Abstract Full Text PDF PubMed Google Scholar) using either 2-deoxy-d-glucose (2DG) (33Ren J.-M. Marshall B.A. Gulve E.A. Gao J. Johnson D.W. Holloszy J.O. Mueckler M. J. Biol. Chem. 1993; 268: 16113-16115Abstract Full Text PDF PubMed Google Scholar) or 3-O-methyl-d-glucose (3MG) (32Young D.A. Uhl J.J. Cartee G.D. Holloszy J.O. J. Biol. Chem. 1986; 261: 16049-16053Abstract Full Text PDF PubMed Google Scholar). The increase in GLUT4 protein in the plasma membrane in response to a maximally effective insulin stimulus was compared in muscles of the rats infused for 24 h with 50% glucose or 0.9% saline. The muscles were processed and homogenized as described previously (34Ren J.-M. Semenkovich C.F. Gulve E.A. Gao J. Holloszy J.O. J. Biol. Chem. 1994; 269: 14396-14401Abstract Full Text PDF PubMed Google Scholar), the homogenate was centrifuged for 20 min at 48,000 × g, and the pellet was used to prepare a plasma membrane fraction as described by Hirshman et al. (35Hirshman M.F. Goodyear L.J. Wardzala L.J. Horton E.D. Horton E.S. J. Biol. Chem. 1990; 265: 987-991Abstract Full Text PDF PubMed Google Scholar). GLUT4 immunoreactivity in the whole homogenate and the plasma membrane fraction were determined by Western blot analysis as previously described (36Hansen P.A. Han D.-H. Nolte L.A. Chen M. Holloszy J.O. Am. J. Physiol. 1997; 273: R1704-R1708Crossref PubMed Google Scholar) using a rabbit polyclonal antibody directed against the COOH terminus of GLUT4 (F349). Muscle extracts were prepared as described previously (18Kawanaka K. Han D.-H. Nolte L.A. Hansen P.A. Nakatani A. Holloszy J.O. Am. J. Physiol. 1999; 276: E907-E912Crossref PubMed Google Scholar) and analyzed for UDP-GlcNAc and UDP-GalNAc by high-performance liquid chromatography using the method of Holstege et al. (37Holstege A. Schulz-Holstege C. Henninger H. Reiffen K.A. Scheinder F. Keppler D.O. Eur. J. Biochem. 1982; 121: 469-474Crossref PubMed Scopus (36) Google Scholar). Glycogen was measured on perchloric acid homogenates of muscle using the amyloglucosidase method (38Passoneau J.V. Lauderdale V.R. Anal. Biochem. 1974; 60: 405-412Crossref PubMed Scopus (628) Google Scholar). Muscle glucose 6-phosphate,2-deoxyglucose-6-phosphate and ATP concentrations were measured fluorometrically (39Lowry O.H. Passoneau J.V. A Flexible System of Enzymatic Analysis. Academic Press, New York1972: 123-124Google Scholar) on neutralized perchloric acid extracts (33Ren J.-M. Marshall B.A. Gulve E.A. Gao J. Johnson D.W. Holloszy J.O. Mueckler M. J. Biol. Chem. 1993; 268: 16113-16115Abstract Full Text PDF PubMed Google Scholar). Muscle samples were homogenized in 50 mm HEPES (pH 7.4), 150 mmNaCl, 10% glycerol, 1% Triton X-100, 1.5 mmMgCl2, 1.0 mm aprotinin (10 μg/ml), leupeptin (10 μg/ml), pepstatin (0.5 μg/ml), and phenylmethylsulfonyl fluoride (2 mm). Homogenates were incubated with end-over-end rotation at 4 °C for 60 min and then centrifuged at 200,000 × g for 50 min at 4 °C. For analysis of PI 3-kinase activity associated with phosphorylated tyrosine, aliquots of supernatant containing 1 mg of protein were immunoprecipitated overnight with end-over-end rotation at 4 °C in the presence of 40 μl of monoclonal antiphosphotyrosine antibody coupled to protein A-Sepharose. Immunocomplexes were collected by centrifugation and washed, suspended in assay medium, and analyzed for PI 3-kinase activity as described by Goodyear et al.(40Goodyear L.J. Giorgino F. Balon T.W. Condorelli G. Smith R.J. Am. J. Physiol. 1995; 268: E987-E995Crossref PubMed Google Scholar). For quantification of phosphorylated PKB, aliquots of the 200,000 × g supernatant were treated with 2× Laemmli sample buffer containing 100 mm dithiothreitol and boiled for 5 min. Samples (80 μg of protein) were subjected to SDS-polyacrylamide gel electrophoresis (10% resolving gel) and were then transferred to nitrocellulose membranes. The membranes were blocked in 5% nonfat dry milk in Tris-buffered saline containing 0.1% Tween 10 (TBST), pH 7.5, overnight. The membranes were rinsed in TBST and incubated with either antiphospho-PKB Ser473antibody or antiphospho-PKB Thr308 antibody for 4 h. The membranes were rinsed in TBST and incubated with horseradish peroxidase-conjugated donkey anti-rabbit IgG for 60 min. Antibody-bound protein was visualized by ECL. The intensity of the bands corresponding to phosphorylated PKB was assessed by densitometry. Results are expressed as mean ± S.E. The significance of differences between two groups was assessed using Student's unpaired t test. For multiple comparisons, the significance was evaluated by analysis of variance. A Newman-Keul's post hoc test was used to locate significant mean differences. In our initial experiment, we gave rats glucose by intravenous infusion for 24 h. The glucose infusion resulted in large increases in plasma glucose and insulin, to levels in the range sometimes found in untreated patients with type 2 diabetes (Table I). Muscle glycogen was also increased. Fig. 1 shows insulin-stimulated 2DG transport rates in epitrochlearis muscles studied in vitro after the 24-h infusion period. The increase in 2DG transport above basal induced by either 60 microunits/ml or 2 milliunits/ml of insulin was ∼60% smaller in muscles from the glucose-infused as compared with the saline-infused animals. (In our epitrochlearis muscle preparation, glucose transport is stimulated maximally by an insulin concentration of ∼500 microunits/ml.)Table IPlasma glucose, insulin, and muscle glycogen levels in glucose- or saline-infused ratsSaline infusionGlucose infusion 1-ap < 0.01.Insulin, microunits/ml15.4 ± 2.8459 ± 52Glucose, mm7.1 ± 0.213.0 ± 0.5Glycogen, μmol/g21 ± 147 ± 2Male rats weighing ∼250 g were given glucose intravenously at a rate of 50 mg/min/kg body weight for 24 h via a catheter in the jugular vein. Control rats were given saline. The catheter was attached to a swivel that enabled rats to move freely in their cages. Rat chow and water were available ad libitum. Muscle glycogen is expressed as μmol of glucose/g of muscle wet weight. Values are the means ± S.E. for 8 rats/group.1-a p < 0.01. Open table in a new tab Male rats weighing ∼250 g were given glucose intravenously at a rate of 50 mg/min/kg body weight for 24 h via a catheter in the jugular vein. Control rats were given saline. The catheter was attached to a swivel that enabled rats to move freely in their cages. Rat chow and water were available ad libitum. Muscle glycogen is expressed as μmol of glucose/g of muscle wet weight. Values are the means ± S.E. for 8 rats/group. The GLUT4 protein content of the plasma membrane fraction was compared in the muscles of 24-h glucose- and saline-infused rats. A maximally effective insulin stimulus was provided by means of an intravenous insulin infusion prior to harvesting of muscles (34Ren J.-M. Semenkovich C.F. Gulve E.A. Gao J. Holloszy J.O. J. Biol. Chem. 1994; 269: 14396-14401Abstract Full Text PDF PubMed Google Scholar). Hypoglycemia was prevented by glucose infusion. Although the plasma membrane fraction prepared by the subcellular fractionation procedure is heavily contaminated with GLUT4 containing vesicles (41Lund S. Holman G.D. Schmitz O. Pedersen O. FEBS Lett. 1993; 330: 312-318Crossref PubMed Scopus (96) Google Scholar), it seems clear that the insulin-induced increase in GLUT4 in the plasma membrane fraction was ∼50% smaller in the glucose-infused group (Fig.2). To examine the mechanisms by which rapid glucose entry into muscle cells causes a decrease in insulin responsiveness, we developed an in vitromodel in which this phenomenon can be studied under controlled conditions in a reasonable time period. Fig.3 shows the effect of incubating rat epitrochlearis muscles for 5 h with 2 milliunits/ml insulin and various concentrations of glucose. Insulin responsiveness decreased with increasing glucose concentration. Incubation of epitrochlearis muscles with 36 mm glucose and insulin for 5 h resulted in as great a decrease in insulin responsiveness as that induced by the 24-h glucose infusion used in our initial experiment. This experimental protocol, i.e. incubation of muscles for 5 h with 36 mm glucose and 2 milliunits of insulin, was therefore routinely used to induce insulin resistance in subsequent experiments. Epitrochlearis muscles tolerated the 5 h of in vitroincubation well. This was evidenced by maintenance of a normal ATP concentration in muscles, rapid glycogen synthesis (see below), and normal extracellular space. ATP concentration was the same in muscles incubated for 5 h with 5 mm (4.4 ± 0.2 μmol/g) or 36 mm glucose (4.6 ± 0.2 μmol/g) compared with 4.6 ± 0.3 μmol/g for muscles clamp-frozen in situ(values are means ± S.E. for 6 muscles/group). Insulin-stimulated glucose transport activity was also normal after 5 h, as evidenced by the finding that 3MG transport rate was not significantly different after 5 h, as compared with 1 h of incubation with 5 mm glucose plus 2 milliunits of insulin (TableII).Table IIComparison of 3MG transport rates and glycogen concentrations in muscles incubated for 1 or 5 hGlucoseInsulin1-h incubation5-h incubation3-Methylglucose transportmm2 milliunits/mlμmol/ml/10 min5−0.18 ± 0.02 (8)0.20 ± 0.05 (6)36−0.26 ± 0.06 (6)0.28 ± 0.02 (8)5+1.00 ± 0.09 (15)1.19 ± 0.06 (13)36+0.98 ± 0.05 (5)0.60 ± 0.04 (20) 2-ap < 0.01, 36versus 5 mm glucose.Glycogenμmol/g muscle5+11.2 ± 1.9 (6)22.2 ± 1.1 (10)36+20.3 ± 1.6 (6)42.1 ± 3.1 (9)2-ap < 0.01, 36versus 5 mm glucose.Rat epitrochlearis muscles were incubated for 1 or 5 h in oxygenated KHB containing either 5 or 36 mm glucose. Muscles were then washed in glucose-free medium for two 10-min periods to remove glucose from the extracellular space followed by measurement of glucose transport activity using 3MG, as described under "Experimental Procedures." Muscles were incubated with 2 milliunits/ml insulin throughout the experiment when insulin-stimulated glucose transport was measured. Results are expressed as micromoles of 3MG taken up per milliliter of intracellular water in 10 min. Values are the means ± S.E. The number of muscles per group is given in parentheses.2-a p < 0.01, 36versus 5 mm glucose. Open table in a new tab Rat epitrochlearis muscles were incubated for 1 or 5 h in oxygenated KHB containing either 5 or 36 mm glucose. Muscles were then washed in glucose-free medium for two 10-min periods to remove glucose from the extracellular space followed by measurement of glucose transport activity using 3MG, as described under "Experimental Procedures." Muscles were incubated with 2 milliunits/ml insulin throughout the experiment when insulin-stimulated glucose transport was measured. Results are expressed as micromoles of 3MG taken up per milliliter of intracellular water in 10 min. Values are the means ± S.E. The number of muscles per group is given in parentheses. It has been reported that exposure of muscles to a high concentration of glucose in the absence of insulin causes insulin resistance (8Richter E.A. Hansen B.F. Hansen S.A. Biochem. J. 1988; 252: 733-737Crossref PubMed Scopus (73) Google Scholar). We therefore examined the effect of exposure of muscles to 36 mm glucose in the absence of insulin. Muscles were incubated for 5 h without insulin and then incubated with 2 milliunits/ml insulin for the 20-min period during which glucose was washed out of the extracellular space and during the measurement of 3MG transport. As shown in Fig. 4, muscles incubated with 36 mm glucose for 5 h without insulin showed no decrease in insulin responsiveness, whereas muscles that underwent the same procedure in the presence of insulin became markedly insulin-resistant. Thus, the insulin resistance is due to rapid entry of large amounts of glucose into muscle rather than to exposure to either a high glucose or a high insulin concentration. That a high insulin concentration per se is not responsible for the insulin resistance is evidenced by the finding that muscles exposed to 5 mm glucose and 2 milliunits/ml insulin for 5 h did not become insulin-resistant. Studies by Marshall and co-workers (13Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar, 14Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 10155-10161Abstract Full Text PDF PubMed Google Scholar) on fat cells have led to the concept that glucose toxicity-induced insulin resistance is due to the accumulation of hexosamine pathway end products. One finding that led to this conclusion was that inhibition of glutamine:fructose-6-phosphate amidotransferase (GFAT), the rate-limiting enzyme in the hexosamine biosynthetic pathway, with the glutamine analog 6-diazo-5-oxonorleucine (DON), protected fat cells against glucose-induced insulin resistance (13Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 4706-4712Abstract Full Text PDF PubMed Google Scholar). As shown in Fig.5 A treatment of muscles with DON was effective in inhibiting GFAT, as evidenced by a large decrease in the concentration of UDP-N-acetylhexosamines. However, despite the marked decrease in UDP-HexNAcs, DON had no protective effect against the development of glucose-induced insulin resistance (Fig. 5 B). In their experiments on primary cultures of adipocytes, Marshall et al. (14Marshall S. Bacote V. Traxinger R.R. J. Biol. Chem. 1991; 266: 10155-10161Abstract Full Text PDF PubMed Google Scholar) found that the inhibitors of mRNA synthesis, actinomycin D and 5,6-dichloro-1-β-D ribofuranosylbenzimidazole (DRB), prevented glucose-induced insulin resistance. They attributed this protective effect to inhibition of the synthesis of a short-lived protein, which they thought was probably GFAT. We found that both actinomycin D (Fig.5 B) and DRB (data not shown) also completely prevent the insulin resistance induced by high glucose and insulin in skeletal muscle. Cycloheximide, at a concentration that inhibits protein synthesis in our muscle preparation (32Young D.A. Uhl J.J. Cartee G.D. Holloszy J.O. J. Biol. Chem. 1986; 261: 16049-16053Abstract Full Text PDF PubMed Google Scholar), has a similar protective effect (Fig. 5 B). None of these inhibitors had any effect on insulin-stimulated glucose transport in control muscles incubated with 5 mm glucose (Fig. 5 B). They also had no effect on basal transport, and in the case of cycloheximide, basal 3MG transport averaged 0.203 ± 0.05 μmol·ml−1·10 min−1 in muscles incubated for 5 h with 5 mm glucose and 0.217 ± 0.02 μmol·ml−1·10 min−1 for muscles incubated for 5 h with glucose and 75 μm cycloheximide (means ± S.E. for 6 muscles/group). The prevention of glucose-induced insulin resistance by inhibition of protein synthesis is clearly not mediated by a decrease in GFAT in muscle; this is evident from the finding that UDP-HexNAc concentrations were markedly higher in the muscles treated with cycloheximide or actinomycin D (Fig. 5 A). This greater increase in UDP-HexNAcs is likely due to more glucose being transported into the muscle cells and ent
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