Development of Glucose-induced Insulin Resistance in Muscle Requires Protein Synthesis*

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.

There are currently three disparate hypotheses regarding the mechanism responsible for glucose-induced insulin resistance of glucose transport. One is that accumulation of large amounts of glycogen, as a result of glucose flooding into the cell, causes insulin resistance (7,11,18), possibly as a consequence of association of GLUT4-containing vesicles with glycogen particles (19,20). A second hypothesis is that glucose toxicity is mediated by activation of protein kinase C (5,21,22) resulting in increased serine and threonine phosphorylation of the insulin receptor, with a decrease in insulin stimulation of insulin receptor tyrosine kinase (21,(23)(24)(25). The third is that glucoseinduced insulin resistance is due to an increased flux of glucose into the hexosamine biosynthetic pathway with accumulation of UDP-N-acetylhexosamines (UDP-HexNAcs) (13, 14, 26 -29). 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 (30) or just exposure to a high concentration of glucose (8,10), 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.

EXPERIMENTAL PROCEDURES
Materials-The antiphospho-protein kinase B (PKB) 1 Ser 473 and Thr 308 polyclonal antibodies were from Upstate Biotechnology. Horseradish peroxidase-conjugated donkey anti-rabbit IgG was obtained from Jackson ImmunoResearch Laboratories. [ 14  Animals-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 ϳ6:00 p.m. * This work was supported National Institutes of Health Grant DK18986 and Diabetes Research and Training Center Grant DK20579. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18  Induction of Insulin Resistance in Vivo-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 (31). 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.
Induction of Insulin Resistance in Vitro-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% O 2 , 5% CO 2 . 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.
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 mM mannitol, 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 (32, 33) using either 2-deoxy-D-glucose (2DG) (33) or 3-O-methyl-D-glucose (3MG) (32).
Plasma Membrane Preparation-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 (34), the homogenate was centrifuged for 20 min at 48,000 ϫ g, and the pellet was used to prepare a plasma membrane fraction as described by Hirshman et al. (35). GLUT4 immunoreactivity in the whole homogenate and the plasma membrane fraction were determined by Western blot analysis as previously described (36) using a rabbit polyclonal antibody directed against the COOH terminus of GLUT4 (F349).
Measurement of Muscle Metabolites-Muscle extracts were prepared as described previously (18) and analyzed for UDP-GlcNAc and UDP-GalNAc by high-performance liquid chromatography using the method of Holstege et al. (37). Glycogen was measured on perchloric acid homogenates of muscle using the amyloglucosidase method (38). Muscle glucose 6-phosphate,2-deoxyglucose-6-phosphate and ATP concentrations were measured fluorometrically (39) on neutralized perchloric acid extracts (33).
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 l 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. (40).
For quantification of phosphorylated PKB, aliquots of the 200,000 ϫ g supernatant were treated with 2ϫ Laemmli sample buffer containing 100 mM dithiothreitol and boiled for 5 min. Samples (80 g 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 Ser 473 antibody or antiphospho-PKB Thr 308 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.
Statistical Analyses-Results are expressed as mean Ϯ 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.

Effect of Glucose Infusion for 24 h-
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 ϳ60% 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 ϳ500 microunits/ml.) The GLUT4 protein content of the plasma membrane fraction was compared in the muscles of 24-h glucose-and salineinfused rats. A maximally effective insulin stimulus was provided by means of an intravenous insulin infusion prior to harvesting of muscles (34). Hypoglycemia was prevented by glucose infusion. Although the plasma membrane fraction prepared by the subcellular fractionation procedure is heavily contaminated with GLUT4 containing vesicles (41), it seems clear that the insulin-induced increase in GLUT4 in the plasma membrane fraction was ϳ50% smaller in the glucose-infused group (Fig. 2).
Induction of Muscle Insulin Resistance in Vitro-To examine the mechanisms by which rapid glucose entry into muscle cells causes a decrease in insulin responsiveness, we developed an in vitro model 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 TABLE I Plasma glucose, insulin, and muscle glycogen levels in glucose-or saline-infused rats Male rats weighing ϳ250 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 mol of glucose/g of muscle wet weight. Values are the means Ϯ S.E. for 8 rats/group.

Saline infusion
Glucose infusion a Insulin, microunits/ml 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 vitro incubation 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 Ϯ 0.2 mol/g) or 36 mM glucose (4.6 Ϯ 0.2 mol/g) compared with 4.6 Ϯ 0.3 mol/g for muscles clamp-frozen in situ (values are means Ϯ 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 (Table II).
Exposure of Muscles to High Glucose in the Absence of Insulin-It has been reported that exposure of muscles to a high concentration of glucose in the absence of insulin causes insulin resistance (8). 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.
Evaluation of the Role of Hexosamine Pathway End Products-Studies by Marshall and co-workers (13,14) 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 biosyn- FIG. 1. Effect of 24 h of glucose infusion on insulin-stimulated muscle 2DG transport. Rats were given glucose by intravenous infusion to raise plasma glucose and insulin (black bars). Controls were infused with saline (white bars). After 24 h of glucose or saline infusion, rats were anesthetized and epitrochlearis muscles were excised. The muscles were incubated for 60 min in the absence (Basal) or presence of insulin, either 60 microunits/ml or 2 milliunits/ml. Muscles were then washed in glucose-free medium for 10 min followed by measurement of glucose transport activity using 2DG as described under "Experimental Procedures." Values were the means Ϯ S.E. for 6 -8 muscles/group. Black bars, saline-infused; white bars, glucose-infused. *, p Ͻ 0.05; **, p Ͻ 0.01; glucose-infused versus saline-infused.
FIG. 2. Increase in GLUT4 in plasma membrane. Rats infused for 24 h with glucose or saline were anesthetized and given an intravenous infusion of insulin for 30 min to raise plasma insulin concentration above 2.5 milliunits/ml. The gastrocnemius/plantaris muscle group was then excised and used for preparation of a plasma membrane fraction, which was used to measure GLUT4 content (see "Experimental Procedures"). A representative Western blot of GLUT4 in the plasma membrane fraction is shown at the top of the figure. Filled-in portions of bars represent the increase in GLUT4 in response to insulin. Each bar represents the mean Ϯ S.E. for muscles from 6 rats. *, p Ͻ 0.05, glucose-infused versus saline-infused.
FIG. 3. Insulin responsiveness of muscles exposed to different concentrations of glucose for 5 h. Epitrochlearis muscles were incubated for 5 h in the absence (Basal) or presence of insulin (2 milliunits/ml) for 5 h with different concentrations of glucose. Muscles were then washed for two 10-min periods in glucose-free medium followed by measurement of 3MG transport as described under "Experimental Procedures." When insulin was present during the initial incubation it was also present during the washes and 3MG transport measurement. Values are the means Ϯ S.E. for 12-25 muscles/group.

TABLE II
Comparison of 3MG transport rates and glycogen concentrations in muscles incubated for 1 or 5 h 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 Ϯ S.E. The number of muscles per group is given in parentheses. thetic pathway, with the glutamine analog 6-diazo-5-oxonorleucine (DON), protected fat cells against glucose-induced insulin resistance (13). As shown in Fig. 5A 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-Hex-NAcs, DON had no protective effect against the development of glucose-induced insulin resistance (Fig. 5B).

Inhibition of mRNA and Protein Synthesis Protects against Glucose-induced Insulin
Resistance-In their experiments on primary cultures of adipocytes, Marshall et al. (14) found that the inhibitors of mRNA synthesis, actinomycin D and 5,6dichloro-1-␤-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. 5B) 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 (32), has a similar protective effect (Fig. 5B). None of these inhibitors had any effect on insulin-stimulated glucose transport in control muscles incubated with 5 mM glucose (Fig. 5B). They also had no effect on basal transport, and in the case of cycloheximide, basal 3MG transport averaged 0.203 Ϯ 0.05 mol⅐ml Ϫ1 ⅐10 min Ϫ1 in muscles incubated for 5 h with 5 mM glucose and 0.217 Ϯ 0.02 mol⅐ml Ϫ1 ⅐10 min Ϫ1 for muscles incubated for 5 h with glucose and 75 M cycloheximide (means Ϯ S.E. for 6 muscles/group). The prevention of glucoseinduced 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. 5A). This greater increase in UDP-HexNAcs is likely due to more glucose being transported into the muscle cells and entering the hexosamine synthetic pathway because of the prevention of insulin resistance. Similarly, glycogen accumulation was increased in muscles in which insulin resistance was prevented by actinomycin D or cycloheximide (Fig. 6). This effect on glycogen was greater with actinomycin D, possibly because of an inhibitory effect of cycloheximide on the activation of glycogen synthase by insulin (42).
Effect of Protein Kinase C Inhibitor Calphostin C-Activation of protein kinase C (PKC) has been reported to cause insulin resistance by means of serine phosphorylation of the insulin receptor (21,24). Furthermore, the insulin resistance induced by a high glucose concentration has been attributed to PKC activation (5, 21, 22). Therefore, to evaluate the role of PKC in glucose-induced insulin resistance in our muscle preparation, we examined the effect of the PKC inhibitor calphostin FIG. 4. Muscles exposed to 36 mM glucose without insulin do not become insulin-resistant. Muscles were incubated with either 5 mM (Control) or 36 mM (High Glucose) glucose for 5 h with insulin, 2 milliunits/ml (black bars), or without insulin (white bars). All muscles were then washed for two 10-min periods in glucose-free medium containing 2 milliunits/ml insulin followed by measurement of 3MG transport in medium containing 2 milliunits/ml insulin as described under "Experimental Procedures." Values are the means Ϯ S.E. for 8 -10 muscles/group. *, p Ͻ 0.001 versus other groups.

FIG. 5. Effects of inhibition of GFAT and of mRNA and protein synthesis on UDP-HexNAc accumulation and insulin resistance
in muscles exposed to high glucose. After a 60-min preincubation with or without 200 M DON, 75 M cycloheximide (CYH), or 2 M actinomycin D (AD), muscles were incubated for 5 h with 5 mM (Control) or 36 mM (High Glucose) glucose, 2 milliunits/ml insulin and, when present in the prior incubation, DON, cycloheximide, or actimomycin D. A, some muscles were then frozen and used for measurement of UDP-GlcNAc and UDP-GalNAc. Each bar represents the mean Ϯ S.E. for the sum of UDP-GlcNAc and UDP-GalNAc (UDP-HexNAcs) for 5 or 6 sets of pooled muscles. *, p Ͻ 0.001 for high glucose with no inhibitor versus control and high glucose plus DON; ϩ , p Ͻ 0.001 versus high glucose with no inhibitor. B, other muscles were then washed in glucose-free medium to remove glucose from the extracellular space followed by measurement of 3MG transport. Each bar represents the mean Ϯ S.E. for 7-14 muscles. *, p Ͻ 0.001 versus control groups and cycloheximide and actinomycin, high glucose groups.

FIG. 6. Glycogen accumulation is increased in muscles incubated with actinomycin D or cycloheximide.
Muscles were incubated with or without 2 M actinomycin D (AD) or 75 M cycloheximide (CYH), 2 milliunits/ml insulin, and 5 (Control) or 36 mM glucose using the same protocol as described in Fig. 5 except that instead of measurement of 3MG transport, muscles were clamp-frozen and used for measurement of glycogen. Values are the means Ϯ S.E. for 12-18 muscles. *, p Ͻ 0.05; **, p Ͻ 0.001, versus high glucose with no inhibitor. C, which acts at the diacylglycerol and phorbol ester binding site (43). Calphostin C, at a concentration (0.5 M) that has an inhibitory effect on phorbol ester-stimulated PKC activity in our muscle preparation (44), did not protect against the decrease in insulin responsiveness induced by high glucose and insulin (Fig. 7).
Insulin Signaling-Activation of PI 3-kinase by insulin is usually measured after brief exposure of muscle to a high insulin concentration. This approach was not possible in the present study as we were studying the effects of prolonged exposure of muscle to high concentrations of glucose and insulin. We therefore measured PI 3-kinase activity in muscles subjected to the same treatment as in the experiments in which 3MG transport was measured, i.e. after incubation with 2 milliunits/ml insulin and either 5 or 36 mM glucose for 5 h. As shown in Fig. 8, phosphotyrosine-associated PI 3-kinase activity was increased ϳ3-fold in muscles treated with insulin. There was no significant difference in insulin-stimulated PI 3-kinase activity between muscles incubated with 5 or 36 mM glucose.
Phosphorylation of PKB on Ser 473 and Thr 308 was also determined in muscles that were incubated with glucose and insulin for 5 h. Phosphorylated PKB, which was too low to quantify in muscles incubated without insulin, increased to the same extent in response to insulin in the muscles incubated with 5 or 36 mM glucose (Fig. 9).
Does Glucose-6-Phosphate Mediate the Effect of High Glucose?-In liver and fat cells, rapid influx of glucose induces an increase in the expression of a number of enzymes involved in glucose and fat metabolism (45). It has been proposed that glucose-6-phosphate provides the signal for the increase in enzyme transcription; this hypothesis is based on the finding that 2-deoxy-D-glucose-6-phosphate (2DG-6-P) mimics the effect of high glucose on fatty acid synthase expression in adipocytes (46). In this context we determined whether 2DG-6-P accumulation decreases insulin responsiveness of glucose transport in muscle. Muscles were incubated for 5 h with 4 mM pyruvate as substrate, 2 milliunits/ml insulin, and 8 mM 2DG for the first 30 min and with 0.5 mM 2DG for the last 4.5 h. This procedure resulted in the accumulation of 5.4 Ϯ 0.78 mol/g 2DG-6-P after 30 min and 5.74 Ϯ 1.07 mol/g 2DG-6-P after 5 h (means Ϯ S.E. for 8 muscles/muscle group) but had no effect on maximally insulin-stimulated 3MG transport (1.21 Ϯ 0.13 mol⅐ml Ϫ1 ⅐10 min Ϫ1 for muscles incubated with 2DG and 1.26 Ϯ 0.11 mol⅐ml Ϫ1 ⅐10 min Ϫ1 for control muscles (n ϭ 8/group)). The concentration of 2DG-6-P attained in the muscles incubated with 2DG (5.74 Ϯ 1.07 mol/g) was much higher than that of glucose-6-phosphate (0.14 Ϯ 0.03 mol/g; n ϭ 4) in muscles incubated with 36 mM glucose and 2 milliunits/ml insulin. These findings do not, of course, rule out the possibility that glucose-6-phosphate plays a role in inducing glucose toxicity by a mechanism that is not mimicked by 2DG-6-P.
Protection against Glucose-induced Insulin Resistance by AICAR-In liver and fat cells, the rapid influx of glucose induces a number of enzymes involved in glucose and fat metab- 7. The protein kinase C inhibitor calphostin C does not protect against muscle insulin resistance. Muscles were incubated for 5 h with 36 mM glucose and 2 milliunits/ml insulin with or without 0.5 M calphostin C. Control muscles were incubated for 5 h with 5 mM glucose and 2 milliunits of insulin. Muscles were then washed with glucose-free medium followed by measurement of 3MG transport. Each bar represents the mean Ϯ S.E. for 7 muscles. *, p Ͻ 0.001 versus control group.
FIG. 8. Phosphotyrosine-associated PI 3-kinase activity is similar in muscles exposed to 5 or 36 mM glucose. Muscles were incubated in the absence (white bar) or presence (black bars) of insulin (2 milliunits/ml) and 5 mM (Control) or 36 mM glucose for 5 h and then clamp-frozen. Muscles extracts were used to prepare phosphotyrosine immunoprecipitates, which were assayed for PI 3-kinase activity as described under "Experimental Procedures." A representative autoradiogram showing 32 P incorporation into phosphatidylinositol is shown at the top of the figure. The average of the optical density values for the basal samples was set at 100. Values for the insulin-stimulated samples were then calculated relative to basal. Values are means Ϯ S.E. for 8 -9 muscles.
FIG. 9. Exposure of muscles to 36 mM glucose has no effect on insulin stimulation of PKB phosphorylation. Muscles were incubated in the absence or presence of insulin (2 milliunits/ml) and 5 mM (Control) or 36 mM (High Glucose) glucose for 5 h. Muscle extracts were resolved by SDS-polyacrylamide gel electrophoresis (10% gels), transferred to nitrocellulose membranes, and immunoblotted with antibodies against phospho-PKB Ser 473 and Thr 308 as described under "Experimental Procedures." A representative blot is shown at the top of the figure. The phospho-Ser 473 and phospho-Thr 308 bands were quantified by densitometry. PKB phosphorylation in the absence of insulin was too low to detect. The average value for the 5 mM glucose group was set at 100, and the values for the 36 mM group were expressed as percentages of the average for the muscles exposed to 5 mM glucose. There were 12 muscles/group. olism within ϳ4 h (45)(46)(47)(48). There is evidence suggesting that the dephosphorylation of transcription factor(s) by protein phosphatases 1 and/or 2A is involved in this effect, which is prevented by okadaic acid or calyculin (46,47). We could not use okadaic acid to evaluate whether a similar mechanism mediates glucose-induced insulin resistance in muscle, because it causes a marked inhibition of insulin-stimulated glucose transport, which does not reverse in the time frame of our experiments. It has also been shown that various kinases (45)(46)(47)(48), including AMP-activated protein kinase (AMPK) (48), prevent activation of gene transcription by glucose in hepatocytes, presumably by keeping the transcription factor(s) in the phosphorylated, inactive state. 5-Aminoimidazole-4-carboxamide ribonucleoside (AICAR) enters cells and is converted to the AMP analog ZMP (5-aminoimidaazole-4-carboxamide-1-␤-D-ribofuranosyl-5Ј-monophosphate) resulting in the activation of AMPK (49). In this context, we evaluated the possibility that AICAR might prevent the decrease in insulin-stimulated glucose transport. A complication with the use of this approach is that activation of AMPK by AICAR results in stimulation of glucose transport, and this effect is additive to that of a maximal insulin stimulus (50,51). We therefore included AICAR in the incubation medium for only the first 3 h of the 5-h incubation to allow its effect on glucose transport to wear off. As shown in Fig. 10, this strategy was successful, as there was no significant difference in 3MG transport between the 5 mM glucose-exposed muscles incubated with or without AICAR. Treatment with AICAR for the first 3 h of a 5-h incubation with 36 mM glucose and insulin protected muscles against the glucose-induced decrease in insulin responsiveness of glucose transport (Fig. 10). DISCUSSION Previous studies have shown that high muscle glycogen concentrations are associated with decreased responsiveness of glucose transport to insulin (7,9,11,18) and that low glycogen levels are associated with enhanced insulin action (52,53). The smaller increase in glucose transport in response to insulin in muscles that have a high glycogen content is due to translocation of fewer GLUT4 to the cell surface (18). It has been hypothesized that the insulin resistance of glucose transport associated with a high glycogen concentration is mediated by the association of GLUT4-containing vesicles with the glycogenprotein complex, making them unavailable for translocation to the cell surface (19,20). Despite considerable investigation, experimental support for this attractive hypothesis has remained elusive, and our finding that actinomycin D completely protects against glucose-induced insulin resistance despite a massive increase in glycogen argues strongly against it. We therefore conclude that a high muscle glycogen level serves as a marker for rapid influx of glucose into muscle cells but is not the cause of the decrease in insulin action.
There is evidence that a high glucose concentration inhibits insulin action by inducing a protein kinase C-mediated serine phosphorylation of the insulin receptor in fibroblasts transfected with the human insulin receptor (21), and it has been hypothesized that this is the mechanism that mediates glucose toxicity-induced insulin resistance (5,21,22). However, decreased insulin receptor activation, resulting in impaired insulin signaling, is not the mechanism responsible for the glucoseinduced insulin resistance in our muscle preparation. This is evidenced by the finding that the stimulation of PI 3-kinase activity and the phosphorylation of PKB by insulin were not significantly reduced. Furthermore, the protein kinase C inhibitor calphostin C, which acts at the diacylglycerol binding site (43), did not protect against glucose-induced insulin resistance.
Much evidence has accumulated suggesting that hexosamine pathway end products are responsible for glucose-induced insulin resistance. The concept that the hexosamine synthetic pathway mediates glucose toxicity originated from an elegant series of studies by Marshall and co-workers (13,14,30,54). Their initial studies showed that rapid influx of glucose, mediated by exposure of cells to high concentrations of both glucose and insulin, resulted in the development of insulin resistance of glucose transport (30) but only when glutamine was included in the incubation medium (54). The finding that glutamine was necessary suggested the possible involvement of the hexosamine biosynthetic pathway. Support for this possibility was provided by experiments in which inhibition of GFAT, the initial and rate-limiting step in the hexosamine pathway, prevented the development of glucose induced-insulin resistance (13). Further evidence for involvement of the hexosamine pathway was provided by the finding that glucosamine, which enters the pathway beyond GFAT, is an ϳ40-fold more potent inducer of insulin resistance than glucose (13). Subsequent studies in which insulin resistance of glucose transport developed in rats infused with glucosamine (26,27) and in muscles incubated with glucosamine (28,29) showed that the phenomenon also occurs in skeletal muscle, supporting the concept that accumulation of UDP-HexNAcs inhibits insulin stimulation of glucose transport. Further evidence for involvement of the hexosamine pathway was provided by the finding that glucose disposal was reduced in transgenic mice overexpressing GFAT in muscle and adipocytes (55).
In our experiments on isolated muscles, we found that, as in the studies on fat cells (30), rapid influx of glucose induced by incubation with high concentrations of glucose and insulin, rather that just exposure to high glucose, is necessary for the development of insulin resistance of glucose transport. However, in contrast to the finding that inclusion of glutamine in the medium was essential for development of glucose toxicity in fat cells (54), glucose-induced insulin resistance developed in skeletal muscle despite the absence of glutamine in the incubation medium. Also, unlike the findings on fat cells (13), the GFAT inhibitor DON did not protect muscle against development of glucose toxicity despite prevention of an increase in UPD-HexNAcs concentration. Marshall et al. (14) found that inhibition of mRNA synthesis with actinomycin D or DRB completely protected against glucose-induced insulin resistance in fat cells. They attributed this protective effect to the inhibition of synthesis of a protein with a short half-life and hypothesized that the protein might be GFAT (14). This interpretation was clouded by the puzzling finding that the protein synthesis inhibitor cycloheximide did not protect against glu-FIG. 10. The activator of AMP kinase, AICAR, protects against glucose-induced insulin resistance. Muscles were incubated for 3 h with 2 milliunits/ml insulin and 5 mM glucose (Control) or 36 mM high glucose with (black bars) or without (white bars) 500 M AICAR. The muscles were then transferred to fresh medium with the same insulin and glucose concentrations but without AICAR and incubated for another 2 h. Muscles were then washed in glucose-free medium followed by measurement of 3MG transport. Each bar represents the mean Ϯ S.E. for 13 muscles. *, p Ͻ 0.01 versus all other groups. cose-induced insulin resistance (15). As in the studies on fat cells, we found that actinomycin D and DRB completely protected against development of insulin resistance in muscles incubated with high glucose and insulin. However, in contrast to the finding in fat cells (15), cycloheximide also prevented the decrease in insulin-stimulated glucose transport in muscle.
These findings are in keeping with the conclusion of Marshall et al. (14) that inhibition of synthesis of a protein with a short half-life protects against development of glucose toxicity. However, it is clear from our results that this protein is not GFAT, as we found that treatment with cycloheximide or actinomycin D resulted in large increases in UDP-HexNAcs in muscles incubated with high glucose and insulin. This finding is probably explained by increased glucose influx due to prevention of insulin resistance. The findings that cycloheximide prevents development of insulin resistance despite markedly increasing UDP-HexNAcs concentration, and that DON does not protect against glucose toxicity despite lowering UDP-Hex-NAcs, provides strong evidence that accumulation of hexosamine pathway end products is not responsible for glucoseinduced insulin resistance in muscle.
How then can the evidence that glucosamine causes insulin resistance (26,27,29) be explained? One hypothesis, proposed by Hresko et al. (56), on the basis of their findings in fat cells, is that the insulin resistance induced by glucosamine is caused by depletion of ATP. Although it is clear from the study by Hresko et al. (56) and from similar findings obtained by us in isolated muscle 2 that this mechanism can account for glucosamine-induced insulin resistance under some experimental conditions, glucosamine can also cause insulin resistance under conditions that do not result in a decrease in ϳP (57). However, it appears from the findings of Nelson et al. (58) that glucose and glucosamine induce insulin resistance by different mechanisms. In any case, our findings provide evidence that conversion of glucose to glucosamine is not necessary for development of glucose-induced insulin resistance in muscle, because inhibition of GFAT does not have a protective effect.
In liver and fat cells, a rapid influx of glucose induces increased expression of several enzymes involved in glucose and fat metabolism within ϳ4 h (45)(46)(47)(48). The activity of many transcription factors is regulated by their phosphorylation state. There is evidence that protein dephosphorylation is involved in glucose-induced gene expression (46 -48) and that activation of either of two closely related protein kinases, AMPK or Ca 2ϩ /calmodulin-dependent protein kinase, prevents activation of gene transcription by glucose in hepatocytes (47,48). Our finding that AICAR, which activates AMPK (49), protects against glucose toxicity suggests that dephosphorylation of a transcription factor may be involved in induction by glucose of a protein that causes insulin resistance and that activation of AMPK prevents insulin resistance by keeping the transcription factor in the phosphorylated state.