Caffeine-induced impairment of insulin action but not insulin signaling in human skeletal muscle is reduced by exercise

We investigated the effects of caffeine ingestion on skeletal muscle glucose uptake, glycogen synthase (GS) activity, and insulin signaling intermediates during a 100-min euglycemic-hyperinsulinemic (100 microU/ml) clamp. On two occasions, seven men performed 1-h one-legged knee extensor exercise at 3 h before the clamp. Caffeine (5 mg/kg) or placebo was administered in a randomized, double-blind fashion 1 h before the clamp. During the clamp, whole-body glucose disposal was reduced (P < 0.05) in caffeine (37.5 +/- 3.1 micromol x min(-1) x kg(-1)) vs. placebo (54.1 +/- 2.9 micromol x min(-1) x kg(-1)). In accordance, the total area under the curve over 100 min (AUC(0--100 min)) for insulin-stimulated glucose uptake in caffeine was reduced (P < 0.05) by approximately 50% in rested and exercised muscle. Caffeine also reduced (P < 0.05) GS activity before and during insulin infusion in both legs. Exercise increased insulin sensitivity of leg glucose uptake in both caffeine and placebo. Insulin increased insulin receptor tyrosine kinase (IRTK), insulin receptor substrate 1-associated phosphatidylinositol (PI) 3-kinase activities, and Ser(473) phosphorylation of protein kinase B (PKB)/Akt significantly but similarly in rested and exercised legs. Furthermore, insulin significantly decreased glycogen synthase kinase-3alpha (GSK-3alpha) activity equally in both legs. Caffeine did not alter insulin signaling in either leg. Plasma epinephrine and muscle cAMP concentrations were increased in caffeine. We conclude that 1) caffeine impairs insulin-stimulated glucose uptake and GS activity in rested and exercised human skeletal muscle; 2) caffeine-induced impairment of insulin-stimulated muscle glucose uptake and downregulation of GS activity are not accompanied by alterations in IRTK, PI 3-kinase, PKB/Akt, or GSK-3alpha but may be associated with increases in epinephrine and intramuscular cAMP concentrations; and 3) exercise reduces the detrimental effects of caffeine on insulin action in muscle.     

Dexamethasone-induced insulin resistance in 3T3-L1 adipocytes is due to inhibition of glucose transport rather than insulin signal transduction

Glucocorticoids reportedly induce insulin resistance. In this study, we investigated the mechanism of glucocorticoid-induced insulin resistance using 3T3-L1 adipocytes in which treatment with dexamethasone has been shown to impair the insulin-induced increase in glucose uptake. In 3T3-L1 adipocytes treated with dexamethasone, the GLUT1 protein expression level was decreased by 30%, which possibly caused decreased basal glucose uptake. On the other hand, dexamethasone treatment did not alter the amount of GLUT4 protein in total cell lysates but decreased the insulin-stimulated GLUT4 translocation to the plasma membrane, which possibly caused decreased insulin-stimulated glucose uptake. Dexamethasone did not alter tyrosine phosphorylation of insulin receptors, and it significantly decreased protein expression and tyrosine phosphorylation of insulin receptor substrate (IRS)-1. Interestingly, however, protein expression and tyrosine phosphorylation of IRS-2 were increased. To investigate whether the reduced IRS-1 content is involved in insulin resistance, IRS-1 was overexpressed in dexamethasone-treated 3T3-L1 adipocytes using an adenovirus transfection system. Despite protein expression and phosphorylation levels of IRS-1 being normalized, insulin-induced 2-deoxy-D-[3H]glucose uptake impaired by dexamethasone showed no significant improvement. Subsequently, we examined the effect of dexamethasone on the glucose uptake increase induced by overexpression of GLUT2-tagged p110alpha, constitutively active Akt (myristoylated Akt), oxidative stress (30 mU glucose oxidase for 2 h), 2 mmol/l 5-aminoimidazole-4-carboxamide ribonucleoside for 30 min, and osmotic shock (600 mmol/l sorbitol for 30 min). Dexamethasone treatment clearly inhibited the increases in glucose uptake produced by these agents. Thus, in conclusion, the GLUT1 decrease may be involved in the dexamethasone-induced decrease in basal glucose transport activity, and the mechanism of dexamethasone-induced insulin resistance in glucose transport activity (rather than the inhibition of phosphatidylinositol 3-kinase activation resulting from a decreased IRS-1 content) is likely to underlie impaired glucose transporter regulation.     

Skeletal muscle insulin signaling defects downstream of phosphatidylinositol 3-kinase at the level of Akt are associated with impaired nonoxidative glucose disposal in HIV lipodystrophy

More than 40% of HIV-infected patients on highly active antiretroviral therapy (HAART) experience fat redistribution (lipodystrophy), a syndrome associated with insulin resistance primarily affecting insulin-stimulated nonoxidative glucose metabolism (NOGM(ins)). Skeletal muscle biopsies, obtained from 18 lipodystrophic nondiabetic patients (LIPO) and 18 nondiabetic patients without lipodystrophy (NONLIPO) before and during hyperinsulinemic (40 mU.m(-2).min(-1))-euglycemic clamps, were analyzed for insulin signaling effectors. All patients were on HAART. Both LIPO and NONLIPO patients were normoglycemic (4.9 +/- 0.1 and 4.8 +/- 0.1 mmol/l, respectively); however, NOGM(ins) was reduced by 49% in LIPO patients (P < 0.001). NOGM(ins) correlated positively with insulin-stimulated glycogen synthase activity (I-form, P < 0.001, n = 36). Glycogen synthase activity (I-form) correlated inversely with phosphorylation of glycogen synthase sites 2+2a (P < 0.001, n = 36) and sites 3a+b (P < 0.001, n = 36) during clamp. Incremental glycogen synthase-kinase-3alpha and -3beta phosphorylation was attenuated in LIPO patients (Ps < 0.05). Insulin-stimulated Akt Ser473 and Akt Thr308 phosphorylation was decreased in LIPO patients (P < 0.05), whereas insulin receptor substrate-1-associated phosphatidylinositol (PI) 3-kinase activity increased significantly (P < 0.001) and similarly (NS) in both groups during clamp. Thus, low glycogen synthase activity explained impaired NOGM(ins) in HIV lipodystrophy, and insulin signaling defects were downstream of PI 3-kinase at the level of Akt. These results suggest mechanisms for the insulin resistance greatly enhancing the risk of type 2 diabetes in HIV lipodystrophy.     

Glucosamine infusion in rats rapidly impairs insulin stimulation of phosphoinositide 3-kinase but does not alter activation of Akt/protein kinase B in skeletal muscle.

Glucosamine, a metabolite of glucose via the hexosamine biosynthetic pathway, potently induces insulin resistance in skeletal muscle by impairing insulin-induced GLUT4 translocation to the plasma membrane. Activation of phosphoinositide (PI) 3-kinase is necessary for insulin-stimulated GLUT4 translocation, and the serine/threonine kinase Akt/protein kinase B (PKB) is a downstream mediator of some actions of PI 3-kinase. To determine whether glucosamine-induced insulin resistance could be due to impaired signaling, we measured insulin receptor substrate (IRS)-1 and insulin receptor tyrosine phosphorylation; PI 3-kinase activity associated with IRS-1, IRS-2, and phosphotyrosine; and Akt activity and phosphorylation in skeletal muscle of rats infused for 2 h with glucosamine (6.0 mg x kg(-1) x min(-1)) or saline. Euglycemic-hyperinsulinemic clamp studies (12 mU x kg(-1) x min(-1) insulin) in awake rats showed that glucosamine infusion resulted in rapid induction of insulin resistance, with a 33% decrease in glucose infusion rate (P < 0.01). Tissues were harvested after saline alone (basal), 1 min after an insulin bolus (10 U/kg), or after 2 h of insulin clamp in saline- and glucosamine-infused rats. After 1 min of insulin stimulation, phosphorylation of IRS-1 and insulin receptor increased 6- to 8-fold in saline-infused rats and 7- to 10-fold in glucosamine-infused rats. In saline-infused rats, 1 min of insulin stimulation increased PI 3-kinase activity associated with IRS-1, IRS-2, or phosphotyrosine 7.6-, 6.4-, and 10-fold, respectively. In glucosamine-infused rats treated for 1 min with insulin, PI 3-kinase activity associated with IRS-1 was reduced 28% (P < 0.01) and that associated with phosphotyrosine was reduced 43% (P < 0.01). Insulin for 1 min stimulated Akt/PKB activity approximately 5-fold in both saline- and glucosamine-infused rats; insulin-induced hyperphosphorylation of Akt/PKB was not different between groups. Glucosamine infusion alone had no effect on tyrosine phosphorylation of the insulin receptor or IRS-1 or on stimulation of PI 3-kinase or Akt/PKB activity. However, 2 h of insulin clamp reduced PI 3-kinase activity associated with IRS-1, IRS-2, or phosphotyrosine to <30% of that seen with 1 min of insulin. No effect of glucosamine was seen on these signaling events when compared with 2 h of insulin clamp without glucosamine. Our data show that 1) glucosamine infusion in rats is associated with an impairment in the early activation of PI 3-kinase by insulin in skeletal muscle, 2) this insulin-resistant state does not involve alterations in the activation of Akt/PKB, and 3) prolonged insulin infusion under clamp conditions results in a blunting of the PI 3-kinase response to insulin.     

Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance of early postreceptor insulin signaling events in skeletal muscle.

To explore potential cellular mechanisms by which activation of the hexosamine pathway induces insulin resistance, we have evaluated insulin signaling in conscious fasted rats infused for 2-6 h with saline, insulin (18 mU x kg(-1) x min(-1)), or insulin and glucosamine (30 micromol x kg(-1) x min(-1)) under euglycemic conditions. Glucosamine infusion increased muscle UDP-N-acetylglucosamine concentrations 3.9- and 4.3-fold over saline- or insulin-infused animals, respectively (P < 0.001). Glucosamine induced significant insulin resistance to glucose uptake both at the level of the whole body and in rectus abdominis muscle, and it blunted the insulin-induced increase in muscle glycogen content. At a cellular level, these metabolic effects were paralleled by inhibition of postreceptor insulin signaling critical for glucose transport and glycogen storage, including a 45% reduction in insulin-stimulated insulin receptor substrate (IRS)-1 tyrosine phosphorylation (P = 0.02), a 44% decrease in IRS-1 association with the p85 regulatory subunit of phosphatidylinositol (PI) 3-kinase (P = 0.03), a 34% reduction in IRS-1-associated PI 3-kinase activity (P = 0.03), and a 51% reduction in insulin-stimulated glycogen synthase activity (P = 0.03). These alterations in postreceptor insulin signaling were time-dependent and paralleled closely the progressive inhibition of systemic glucose disposal from 2 to 6 h of glucosamine infusion. We also demonstrated that glucosamine infusion results in O-linked N-acetylglucosamine modification of IRS-1 and IRS-2. These data indicate that activation of the hexosamine pathway may directly modulate early postreceptor insulin signal transduction, perhaps via posttranslation modification of IRS proteins, and thus contribute to the insulin resistance induced by chronic hyperglycemia.     

Prolonged incubation in PUGNAc results in increased protein O-Linked glycosylation and insulin resistance in rat skeletal muscle

Increased flux through the hexosamine biosynthetic pathway and increased O-linked glycosylation (N-acetylglucosamine [O-GlcNAc]) of proteins have been implicated in insulin resistance. Previous research in 3T3-L1 adipocytes indicated that insulin-stimulated glucose uptake and phosphorylation of Akt were reduced after incubation with O-(2-acetamido-2-deoxy-D-glucopyranosylidene)amino-N-phenylcarbamate (PUGNAc; 100 micromol/l), an inhibitor of the O-GlcNAcase that catalyzes removal of O-GlcNAc from proteins. Therefore, in this study, we tested the effects of PUGNAc on skeletal muscle. Incubation of rat epitrochlearis muscles for 19 h with 100 micromol/l PUGNAc resulted in a marked increase in O-GlcNAcylation of multiple proteins. Incubation with PUGNAc reduced glucose transport with a physiologic insulin concentration without affecting glucose transport without insulin or with supraphysiologic insulin. PUGNAc did not significantly alter insulin-stimulated phosphorylation of Akt (serine and threonine) or its substrates glycogen synthase kinase (GSK)3 alpha and GSK3 beta. Insulin stimulated a dose-dependent (12.0 > 0.6 > 0 nmol/l) increase in the phosphorylation of a 160-kDa protein detected using an antibody against an Akt substrate phosphomotif. PUGNAc treatment did not alter phosphorylation of this protein. These results indicate that PUGNAc is an effective inhibitor of O-GlcNAcase in skeletal muscle and suggest that O-GlcNAc modification of proteins can induce insulin resistance in skeletal muscle independent of attenuated phosphorylation of Akt, GSK 3 alpha, GSK3 beta, and a 160-kDa protein with an Akt phosphomotif.     

Ceramide- and oxidant-induced insulin resistance involve loss of insulin-dependent Rac-activation and actin remodeling in muscle cells

In muscle cells, insulin elicits recruitment of the glucose transporter GLUT4 to the plasma membrane. This process engages sequential signaling from insulin receptor substrate (IRS)-1 to phosphatidylinositol (PI) 3-kinase and the serine/threonine kinase Akt. GLUT4 translocation also requires an Akt-independent but PI 3-kinase-and Rac-dependent remodeling of filamentous actin. Although IRS-1 phosphorylation is often reduced in insulin-resistant states in vivo, several conditions eliciting insulin resistance in cell culture spare this early step. Here, we show that insulin-dependent Rac activation and its consequent actin remodeling were abolished upon exposure of L6 myotubes beginning at doses of C2-ceramide or oxidant-producing glucose oxidase as low as 12.5 micromol/l and 12.5 mU/ml, respectively. At 25 micromol/l and 25 mU/ml, glucose oxidase and C2-ceramide markedly reduced GLUT4 translocation and glucose uptake and lowered Akt phosphorylation on Ser473 and Thr308, yet they affected neither IRS-1 tyrosine phosphorylation nor its association with p85 and PI 3-kinase activity. Small interfering RNA-dependent Rac1 knockdown prevented actin remodeling and GLUT4 translocation but spared Akt phosphorylation, suggesting that Rac and actin remodeling do not contribute to overall Akt activation. We propose that ceramide and oxidative stress can each affect two independent arms of insulin signaling to GLUT4 at distinct steps, Rac-GTP loading and Akt phosphorylation.     

High glucose and glucosamine induce insulin resistance via different mechanisms in 3T3-L1 adipocytes

Sustained hyperglycemia induces insulin resistance, but the mechanism is still incompletely understood. Glucosamine (GlcN) has been extensively used to model the role of the hexosamine synthesis pathway (HSP) in glucose-induced insulin resistance. 3T3-L1 adipocytes were preincubated for 18 h in media +/- 0.6 nmol/l insulin containing either low glucose (5 mmol/l), low glucose plus GlcN (0.1-2.5 mmol/l), or high glucose (25 mmol/l). Basal and acute insulin-stimulated (100 nmol/l) glucose transport was measured after re-equilibration in serum and insulin-free media. Preincubation with high glucose or GlcN (1-2.5 mmol/l) inhibited basal and acute insulin-stimulated glucose transport only if insulin was present during preincubation. However, only preincubation with GlcN plus insulin inhibited insulin-stimulated GLUT4 translocation. GLUT4 and GLUT1 protein expression were not affected. GlcN (2.5 mmol/l) increased cellular UDP-N-acetylhexosamines (UDP-HexNAc) by 400 and 900% without or with insulin, respectively. High glucose plus insulin increased UDP-HexNAc by 30%. GlcN depleted UDP-hexoses, whereas high glucose plus insulin increased them. Preincubation with 0.5 mmol/l GlcN plus insulin maximally increased UDP-HexNAc without affecting insulin-stimulated or basal glucose transport. GlcN plus insulin (but not high glucose plus insulin) caused marked GlcN dose-dependent accumulation of GlcN-6-phosphate, which correlated with insulin resistance of glucose transport (r = 0.935). GlcN plus insulin (but not high glucose plus insulin) decreased ATP (10-30%) and UTP (>50%). GTP was not measured, but GDP increased. Neither high glucose plus insulin nor GlcN plus insulin prevented acute insulin stimulation (approximately 20-fold) of insulin receptor substrate 1-associated phosphatidylinositol (PI)-3 kinase. We have come to the following conclusions. 1) Chronic exposure to high glucose or GlcN in the presence of low insulin caused insulin resistance of glucose transport by different mechanisms. 2) GlcN inhibited GLUT4 translocation, whereas high glucose impaired GLUT4 "intrinsic activity" or membrane intercalation. 3) Both agents may act distally to PI-3 kinase. 4) GlcN has metabolic effects not shared by high glucose. GlcN may not model HSP appropriately, at least in 3T3-L1 adipocytes.     

Postexercise Improvement in Insulin-Stimulated Glucose Uptake Occurs Concomitant With Greater AS160 Phosphorylation in Muscle From Normal and Insulin-Resistant Rats

Earlier research on rats with normal insulin sensitivity demonstrated that acute exercise increased insulin-stimulated glucose uptake (GU) concomitant with greater phosphorylation of Akt substrate of 160 kDa (pAS160). Because mechanisms for exercise effects on GU in insulin-resistant muscle are unknown, our primary objective was to assess insulin-stimulated GU, proximal insulin signaling (insulin receptor [IR] tyrosine phosphorylation, IR substrate 1–phosphatidylinositol-3-kinase, and Akt phosphorylation and activity), and pAS160 in muscles from acutely exercised (one session) and sedentary rats fed either chow (low-fat diet [LFD]; normal insulin sensitivity) or a high-fat diet (HFD; for 2 weeks, insulin-resistant). At 3 h postexercise (3hPEX), isolated epitrochlearis muscles were used for insulin-stimulated GU and insulin signaling measurements. Although exercise did not enhance proximal signaling in either group, insulin-stimulated GU at 3hPEX exceeded respective sedentary control subjects (Sedentary) in both diet groups. Furthermore, insulin-stimulated GU for LFD-3hPEX was greater than HFD-3hPEX values. For HFD-3hPEX muscles, pAS160 exceeded HFD-Sedentary, but in muscle from LFD-3hPEX rats, pAS160 was greater still than HFD-3hPEX values. These results implicated pAS160 as a potential determinant of the exercise-induced elevation in insulin-stimulated GU for each diet group and also revealed pAS160 as a possible mediator of greater postexercise GU of insulin-stimulated muscles from the insulin-sensitive versus insulin-resistant group.     

Effects of endurance exercise training on insulin signaling in human skeletal muscle: interactions at the level of phosphatidylinositol 3-kinase, Akt, and AS160

The purpose of this study was to investigate the mechanisms explaining improved insulin-stimulated glucose uptake after exercise training in human skeletal muscle. Eight healthy men performed 3 weeks of one-legged knee extensor endurance exercise training. Fifteen hours after the last exercise bout, insulin-stimulated glucose uptake was approximately 60% higher (P < 0.01) in the trained compared with the untrained leg during a hyperinsulinemic-euglycemic clamp. Muscle biopsies were obtained before and after training as well as after 10 and 120 min of insulin stimulation in both legs. Protein content of Akt1/2 (55 +/- 17%, P < 0.05), AS160 (25 +/- 8%, P = 0.08), GLUT4 (52 +/- 19%, P < 0.001), hexokinase 2 (HK2) (197 +/- 40%, P < 0.001), and insulin-responsive aminopeptidase (65 +/- 15%, P < 0.001) increased in muscle in response to training. During hyperinsulinemia, activities of insulin receptor substrate-1 (IRS-1)-associated phosphatidylinositol 3-kinase (PI3-K) (P < 0.005), Akt1 (P < 0.05), Akt2 (P < 0.005), and glycogen synthase (GS) (percent I-form, P < 0.05) increased similarly in both trained and untrained muscle, consistent with increased phosphorylation of Akt Thr(308), Akt Ser(473), AS160, glycogen synthase kinase (GSK)-3alpha Ser(21), and GSK-3beta Ser(9) and decreased phosphorylation of GS site 3a+b (all P < 0.005). Interestingly, training improved insulin action on thigh blood flow, and, furthermore, in both basal and insulin-stimulated muscle tissue, activities of Akt1 and GS and phosphorylation of AS160 increased with training (all P < 0.05). In contrast, training reduced IRS-1-associated PI3-K activity (P < 0.05) in both basal and insulin-stimulated muscle tissue. Our findings do not support generally improved insulin signaling after endurance training; rather it seems that improved insulin-stimulated glucose uptake may result from hemodynamic adaptations as well as increased cellular protein content of individual insulin signaling components and molecules involved in glucose transport and metabolism.     

Akt2 is essential for the full effect of calorie restriction on insulin-stimulated glucose uptake in skeletal muscle

Brief calorie restriction (CR; 20 days of 60% of ad libitum [AL] intake) improves insulin-stimulated glucose transport, concomitant with enhanced phosphorylation of Akt2. The purpose of this study was to determine whether Akt2 is essential for the calorie restriction-induced enhancement in skeletal muscle insulin sensitivity. We measured insulin-stimulated 2-deoxyglucose (2DG) uptake in isolated extensor digitorum longus (EDL) and soleus muscles from male and female wild-type (WT) and Akt2-null (knockout [KO]) mice after ad libitum or calorie-restricted (20 days at 60% of AL) feeding. In WT mice, calorie restriction significantly enhanced insulin-stimulated 2DG uptake in both muscles regardless of sex. However, in KO mice, calorie restriction did not enhance insulin-stimulated 2DG in male or female EDL or in female soleus. Only in male KO soleus did calorie restriction significantly increase insulin-stimulated 2DG through an Akt2-independent mechanism, although 2DG uptake of the KO-CR group was reduced compared with the WT-CR soleus group. Akt2 serine phosphorylation was enhanced approximately two- to threefold in insulin-stimulated WT-CR versus WT-AL muscles. Calorie restriction induced an approximately 1.5- to 2-fold elevation in Akt1 phosphorylation of insulin-treated muscles, regardless of genotype, but this increase was insufficient to replace Akt2 for insulin-stimulated 2DG in Akt2-deficient muscles. These results indicate that Akt2 is essential for the full effect of brief calorie restriction on insulin-stimulated glucose uptake in skeletal muscle with physiologic insulin.     

Insulin signaling and insulin sensitivity after exercise in human skeletal muscle

Muscle glucose uptake, glycogen synthase activity, and insulin signaling were investigated in response to a physiological hyperinsulinemic (600 pmol/l)-euglycemic clamp in young healthy subjects. Four hours before the clamp, the subjects performed one-legged exercise for 1 h. In the exercised leg, insulin more rapidly activated glucose uptake (half activation time [t1/2] = 11 vs. 34 min) and glycogen synthase activity (t1/2 = 8 vs. 17 min), and the magnitude of increase was two- to fourfold higher compared with the rested leg. However, prior exercise did not result in a greater or more rapid increase in insulin-induced receptor tyrosine kinase (IRTK) activity (t1/2 = 50 min), serine phosphorylation of Akt (t1/2 = 1-2 min), or serine phosphorylation of glycogen synthase kinase-3 (GSK-3) (t1/2 = 1-2 min) or in a larger or more rapid decrease in GSK-3 activity (t1/2 = 3-8 min). Thirty minutes after cessation of insulin infusion, glucose uptake, glycogen synthase activity, and signaling events were partially reversed in both the rested and the exercised leg. We conclude the following: 1) physiological hyperinsulinemia induces sustained activation of insulin-signaling molecules in human skeletal muscle; 2) the more distal insulin-signaling components (Akt, GSK-3) are activated much more rapidly than the proximal signaling molecules (IRTK as well as insulin receptor substrate 1 and phosphatidylinositol 3-kinase [Wojtaszewski et al., Diabetes 46:1775-1781, 1997]); and 3) prior exercise increases insulin stimulation of both glucose uptake and glycogen synthase activity in the absence of an upregulation of signaling events in human skeletal muscle.     

The antihyperglycemic drug alpha-lipoic acid stimulates glucose uptake via both GLUT4 translocation and GLUT4 activation: potential role of p38 mitogen-activated protein kinase in GLUT4 activation

The cofactor of mitochondrial dehydrogenase complexes and potent antioxidant alpha-lipoic acid has been shown to lower blood glucose in diabetic animals. alpha-Lipoic acid enhances glucose uptake and GLUT1 and GLUT4 translocation in 3T3-L1 adipocytes and L6 myotubes, mimicking insulin action. In both cell types, insulin-stimulated glucose uptake is reduced by inhibitors of p38 mitogen-activated protein kinase (MAPK). Here we explore the effect of alpha-lipoic acid on p38 MAPK, phosphatidylinositol (PI) 3-kinase, and Akt1 in L6 myotubes. alpha-Lipoic acid (2.5 mmol/l) increased PI 3-kinase activity (31-fold) and Akt1 (4.9-fold). Both activities were inhibited by 100 nmol/l wortmannin. alpha-Lipoic acid also stimulated p38 MAPK phosphorylation by twofold within 10 min. The phosphorylation persisted for at least 30 min. Like insulin, alpha-lipoic acid increased the kinase activity of the alpha (2.8-fold) and beta (2.1-fold) isoforms of p38 MAPK, measured by an in vitro kinase assay. Treating cells with 10 micromol/l of the p38 MAPK inhibitors SB202190 or SB203580 reduced the alpha-lipoic acid-induced stimulation of glucose uptake by 66 and 55%, respectively. In contrast, SB202474, a structural analog that does not inhibit p38 MAPK, was without effect on glucose uptake. In contrast to 2-deoxyglucose uptake, translocation of GLUT4myc to the cell surface by either alpha-lipoic acid or insulin was unaffected by 20 micromol/l of SB202190 or SB203580. The results suggest that inhibition of 2-deoxyglucose uptake in response to alpha-lipoic acid by inhibitors of p38 MAPK is independent of an effect on GLUT4 translocation. Instead, it is likely that regulation of transporter activity is sensitive to these inhibitors.     

Mechanisms of hyperglycemia-induced insulin resistance in whole body and skeletal muscle of type I diabetic patients.

To examine the mechanisms of hyperglycemia-induced insulin resistance, eight insulin-dependent (type I) diabetic men were studied twice, after 24 h of hyperglycemia (mean blood glucose 20.0 +/- 0.3 mM, i.v. glucose) and after 24 h of normoglycemia (7.1 +/- 0.4 mM, saline) while receiving identical diets and insulin doses. Whole-body and forearm glucose uptake were determined during a 300-min insulin infusion (serum free insulin 359 +/- 22 and 373 +/- 29 pM, after hyper- and normoglycemia, respectively). Muscle biopsies were taken before and at the end of the 300-min insulin infusion. Plasma glucose levels were maintained constant during the 300-min period by keeping glucose for 150 min at 16.7 +/- 0.1 mM after 24-h hyperglycemia and increasing it to 16.5 +/- 0.1 mM after normoglycemia and by allowing it thereafter to decrease in both studies to normoglycemia. During the normoglycemic period (240-300 min), total glucose uptake (25.0 +/- 2.8 vs. 33.8 +/- 3.9 mumol.kg-1 body wt.min-1, P less than 0.05) was 26% lower, forearm glucose uptake (11 +/- 4 vs. 18 +/- 3 mumol.kg-1 forearm.min-1, P less than 0.05) was 35% lower, and nonoxidative glucose disposal (8.9 +/- 2.2 vs. 19.4 +/- 3.3 mumol.kg-1 body wt-1min-1, P less than 0.01) was 54% lower after 24 h of hyper- and normoglycemia, respectively. Glucose oxidation rates were similar. Basal muscle glycogen content was similar after 24 h of hyperglycemia (234 +/- 23 mmol/kg dry muscle) and normoglycemia (238 +/- 22 mmol/kg dry muscle). Insulin increased muscle glycogen to 273 +/- 22 mmol/kg dry muscle after 24 h of hyperglycemia and to 296 +/- 33 mmol/kg dry muscle after normoglycemia (P less than 0.05 vs. 0 min for both). Muscle ATP, free glucose, glucose-6-phosphate, and fructose-6-phosphate concentrations were similar after both 24-h treatment periods and did not change in response to insulin. We conclude that a marked decrease in whole-body, muscle, and nonoxidative glucose disposal can be induced by hyperglycemia alone.     

Troglitazone induces GLUT4 translocation in L6 myotubes

A number of studies have demonstrated that insulin resistance in the skeletal muscle plays a pivotal role in the insulin resistance associated with obesity and type 2 diabetes. A decrease in GLUT4 translocation from the intracellular pool to the plasma membranes in skeletal muscles has been implicated as a possible cause of insulin resistance. Herein, we examined the effects of an insulin-sensitizing drug, troglitazone (TGZ), on glucose uptake and the translocation of GLUT4 in L6 myotubes. The prolonged exposure (24 h) of L6 myotubes to TGZ (10(-5) mol/l) caused a substantial increase in the 2-deoxy-[3H]D-glucose (2-DG) uptake without changing the total amount of the glucose transporters GLUT4, GLUT1, and GLUT3. The TGZ-induced 2-DG uptake was completely abolished by cytochalasin-B (10 micromol/l). The ability of TGZ to translocate GLUT4 from light microsomes to the crude plasma membranes was greater than that of insulin. Both cycloheximide treatment (3.5 x 10(-6) mol/l) and the removal of TGZ by washing reversed the 2-DG uptake to the basal level. Moreover, insulin did not enhance the TGZ-induced 2-DG uptake additively. The TGZ-induced 2-DG uptake was only partially reversed by wortmannin to 80%, and TGZ did not change the expression and the phosphorylation of protein kinase B; the expression of protein kinase C (PKC)-lambda, PKC-beta2, and PKC-zeta; or 5'AMP-activated protein kinase activity. a-Tocopherol, which has a molecular structure similar to that of TGZ, did not increase 2-DG uptake. We conclude that the glucose transport in L6 myotubes exposed to TGZ for 24 h is the result of an increased translocation of GLUT4. The present results imply that the effects of troglitazone on GLUT4 translocation may include a new mechanism for improving glucose transport in skeletal muscle.     

Nitric oxide increases glucose uptake through a mechanism that is distinct from the insulin and contraction pathways in rat skeletal muscle

Insulin, contraction, and the nitric oxide (NO) donor, sodium nitroprusside (SNP), all increase glucose transport in skeletal muscle. Some reports suggest that NO is a critical mediator of insulin- and/or contraction-stimulated transport. To determine if the mechanism leading to NO-stimulated glucose uptake is similar to the insulin- or contraction-dependent signaling pathways, isolated soleus and extensor digitorum longus (EDL) muscles from rats were treated with various combinations of SNP (maximum 10 mmol/l), insulin (maximum 50 mU/ml), electrical stimulation to produce contractions (maximum 10 min), wortmannin (100 nmol/l), and/or the NO synthase (NOS) inhibitor NG-monomethyl-L-arginine (L-NMMA) (0.1 mmol/l). The combinations of SNP plus insulin and SNP plus contraction both had fully additive effects on 2-deoxyglucose uptake. Wortmannin completely inhibited insulin-stimulated glucose transport and only slightly inhibited SNP-stimulated 2-deoxyglucose uptake, whereas L-NMMA did not inhibit contraction-stimulated 2-deoxyglucose uptake. SNP significantly increased the activity of the alpha1 catalytic subunit of 5'AMP-activated protein kinase (AMPK), a signaling molecule that has been implicated in mediating glucose transport in fuel-depleted cells. Addition of the NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME) (1 mg/ml) to the drinking water of rats for 2 days failed to affect the increase in muscle 2-deoxyglucose uptake in response to treadmill exercise. These data suggest that NO stimulates glucose uptake through a mechanism that is distinct from both the insulin and contraction signaling pathways.     

Inhibition of glycogen synthase kinase 3 improves insulin action and glucose metabolism in human skeletal muscle

Glycogen synthase kinase (GSK)-3 has been implicated in the regulation of multiple cellular physiological processes in skeletal muscle. Selective cell-permeable reversible inhibitors (INHs) of GSK-3 (CT98014 and CHIR98023 [Chiron, Emeryville, CA] and LiCl) were used to evaluate the role of GSK-3 in controlling glucose metabolism. Acute treatment (30 min) of cultured human skeletal muscle cells with either INH resulted in a dose-dependent activation of glycogen synthase (GS) with a maximally effective concentration of approximately 2 micromol/l. The maximal acute effect of either INH on GS (103 +/- 25% stimulation over basal) was greater than the maximal insulin response (48 +/- 9%, P < 0.05 vs. INH); LiCl was as effective as insulin. The GSK-3 inhibitor effect, like that of insulin, was on the activation state (fractional velocity [FV]) of GS. Cotreatment of muscle cells with submaximal doses of INH and insulin resulted in an additive effect on GS FV (103 +/- 10% stimulation, P < 0.05 vs. either agent alone). Glucose incorporation into glycogen was also acutely stimulated by INH. While prolonged (6-24 h) insulin exposure led to desensitization of GS, INH continued to activate GS FV for at least 24 h. Insulin and LiCl acutely activated glucose uptake, whereas INH stimulation of glucose uptake required more prolonged exposure, starting at 6 h and continuing to 24 h. Chronic (4-day) treatment with INH increased both basal (154 +/- 32% of control) and insulin-stimulated (219 +/- 74%) glucose uptake. Upregulation of uptake activity occurred without any change in total cellular GLUT1 or GLUT4 protein content. Yet the same chronic treatment resulted in a 65 +/- 6% decrease in GSK-3 protein and a parallel decrease (61 +/- 11%) in GSK-3 total activity. Together with the INH-induced increase in insulin-stimulated glucose uptake, there was an approximately 3.5-fold increase (P < 0.05) in insulin receptor substrate (IRS)-1 protein abundance. Despite upregulation of IRS-1, maximal insulin stimulation of Akt phosphorylation was unaltered by INH treatment. The results suggest that selective inhibition of GSK-3 has an impact on both GS and glucose uptake, including effects on insulin action, using mechanisms that differ from and are additive to those of insulin.     

The diabetic phenotype is conserved in myotubes established from diabetic subjects: evidence for primary defects in glucose transport and glycogen synthase activity

The most well-described defect in the pathophysiology of type 2 diabetes is reduced insulin-mediated glycogen synthesis in skeletal muscles. It is unclear whether this defect is primary or acquired secondary to dyslipidemia, hyperinsulinemia, or hyperglycemia. We determined the glycogen synthase (GS) activity; the content of glucose-6-phosphate, glucose, and glycogen; and the glucose transport in satellite cell cultures established from diabetic and control subjects. Myotubes were precultured in increasing insulin concentrations for 4 days and subsequently stimulated acutely by insulin. The present study shows that the basal glucose uptake as well as insulin-stimulated GS activity is reduced in satellite cell cultures established from patients with type 2 diabetes. Moreover, increasing insulin concentrations could compensate for the reduced GS activity to a certain extent, whereas chronic supraphysiological insulin concentrations induced insulin resistance in GS and glucose transport activity. Our data suggest that insulin resistance in patients with type 2 diabetes comprises at least two important defects under physiological insulin concentrations: a reduced glucose transport under basal conditions and a reduced GS activity under acute insulin stimulation, implicating a reduced glucose uptake in the fasting state and a diminished insulin-mediated storage of glucose as glycogen after a meal.     

Local effect of burn on skeletal muscle insulin responsiveness

Basal and insulin-stimulated glucose metabolism by skeletal muscle were studied 3 days after a 3-sec scald of one hindlimb of the rat. Soleus muscles from the burned and unburned limb of the burned rats, as well as from timed controls, were incubated with [U-¹⁴C]glucose and 0, 0.1, 1.0, or 10.0 mU insulin/ml. Basal glucose uptake by soleus muscle from the burned limb was 144% (P < 0.001) greater than that of the controls. The glucose uptake by muscle from the unburned limb did not differ from that of controls. Insulin increased glucose uptake in control and unburned muscles but had no effect in burned muscles. Basal lactic acid release by soleus muscle from the burned limb and the contralateral unburned limb of burned rats was 123% (P < 0.001) and 24% (P < 0.01) higher, respectively, than that of soleus muscle from control rats. Of the lactic acid released by muscles from control rats or the unburned limb of burned rats, 35% was derived from exogenous glucose. In contrast, 50–55% of the lactic acid released by muscles from the burned limb was derived from exogenous glucose reflecting a predominant conversion of glucose to lactic acid. Insulin had no effect on the rate of lactic acid release and did not change the proportion of glucose converted to lactic acid by any of the three muscle groups. The basal rate of glucose conversion to CO₂ by muscle from the burned limb was elevated 133% (P < 0.01) and that by muscle from the unburned limb was not altered as compared to controls. Insulin did not stimulate the conversion of glucose to CO₂ by any of the three muscle groups, and in the presence of insulin the release of CO₂ by burned, unburned, and control muscle did not differ. The basal rate of glucose incorporation into glycogen was the same in all three muscle groups. Insulin stimulated glucose incorporation into glycogen to a similar degree in muscles from control rats and the unburned limb of burned rats. However, the stimulatory effect of insulin was completely absent in soleus muscle from the burned limb. It is concluded that thermal injury suppresses the insulin-induced augmentation of glucose uptake and glucose incorporation into glycogen in skeletal muscles in the region of the burn but does not alter insulin sensitivity of skeletal muscles in the unburned region.     

Dose-response effect of elevated plasma free fatty acid on insulin signaling

The dose-response relationship between elevated plasma free fatty acid (FFA) levels and impaired insulin-mediated glucose disposal and insulin signaling was examined in 21 lean, healthy, normal glucose-tolerant subjects. Following a 4-h saline or Liposyn infusion at 30 (n = 9), 60 (n = 6), and 90 (n = 6) ml/h, subjects received a 2-h euglycemic insulin (40 mU . m(-2) . min(-1)) clamp. Basal plasma FFA concentration ( approximately 440 micromol/l) was increased to 695, 1,251, and 1,688 micromol/l after 4 h of Liposyn infusion and resulted in a dose-dependent reduction in insulin-stimulated glucose disposal (R(d)) by 22, 30, and 34%, respectively (all P < 0.05 vs. saline control). At the lowest lipid infusion rate (30 ml/h), insulin receptor and insulin receptor substrate (IRS)-1 tyrosine phosphorylation, phosphatidylinositol (PI) 3-kinase activity associated with IRS-1, and Akt serine phosphorylation were all significantly impaired (P < 0.05-0.01). The highest lipid infusion rate (90 ml/h) caused a further significant reduction in all insulin signaling events compared with the low-dose lipid infusion (P < 0.05-0.01) whereas the 60-ml/h lipid infusion caused an intermediate reduction in insulin signaling. However, about two-thirds of the maximal inhibition of insulin-stimulated glucose disposal already occurred at the rather modest increase in plasma FFA induced by the low-dose (30-ml/h) lipid infusion. Insulin-stimulated glucose disposal was inversely correlated with both the plasma FFA concentration after 4 h of lipid infusion (r = -0.50, P = 0.001) and the plasma FFA level during the last 30 min of the insulin clamp (r = -0.54, P < 0.001). PI 3-kinase activity associated with IRS-1 correlated with insulin-stimulated glucose disposal (r = 0.45, P < 0.01) and inversely with both the plasma FFA concentration after 4 h of lipid infusion (r = -0.39, P = 0.01) and during the last 30 min of the insulin clamp (r = -0.43, P < 0.01). In summary, in skeletal muscle of lean, healthy subjects, a progressive increase in plasma FFA causes a dose-dependent inhibition of insulin-stimulated glucose disposal and insulin signaling. The inhibitory effect of plasma FFA was already significant following a rather modest increase in plasma FFA and develops at concentrations that are well within the physiological range (i.e., at plasma FFA levels observed in obesity and type 2 diabetes).