Phorbol esters affect skeletal muscle glucose transport in a fiber type-specific manner

Recent evidence has shown that activation of lipid-sensitive protein kinase C (PKC) isoforms leads to skeletal muscle insulin resistance. However, earlier studies demonstrated that phorbol esters increase glucose transport in skeletal muscle. The purpose of the present study was to try to resolve this discrepancy. Treatment with the phorbol ester 12-deoxyphorbol-13-phenylacetate 20-acetate (dPPA) led to an approximately 3.5-fold increase in glucose transport in isolated fast-twitch epitrochlearis and flexor digitorum brevis muscles. Phorbol ester treatment was additive to a maximally effective concentration of insulin in fast-twitch skeletal muscles. Treatment with dPPA did not affect insulin signaling in the epitrochlearis. In contrast, phorbol esters had no effect on basal glucose transport and inhibited maximally insulin-stimulated glucose transport approximately 50% in isolated slow-twitch soleus muscle. Furthermore, dPPA treatment inhibited the insulin-stimulated tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and the threonine and serine phosphorylation of PKB by approximately 50% in the soleus. dPPA treatment also caused serine phosphorylation of IRS-1 in the slow-twitch soleus muscle. In conclusion, our results show that phorbol esters stimulate glucose transport in fast-twitch skeletal muscles and inhibit insulin signaling in slow-twitch soleus muscle of rats. These findings suggest that mechanisms other than PKC activation mediate lipotoxicity-induced whole body insulin resistance.     

Evidence for the involvement of a phospholipase C--protein kinase C signaling pathway in insulin stimulated glucose transport in skeletal muscle

The primary purpose of this investigation was to determine the relationship between phospholipase C (PLC) and diacylglycerol (DAG) sensitive protein kinase C isoforms in insulin signaling in skeletal muscle. Using an in vitro preparation of rat soleus muscle we found that insulin (0.6 nM) stimulated glucose transport was inhibited approximately 20 and 25% by the PKC inhibitor GF109203X and the phospholipase C inhibitor U73122 respectively (p<0.05). The combined effects of these inhibitors were no greater than the inhibitory effects of either compound alone. Western blot analysis revealed that insulin induced a redistribution of PKC beta II from the cytosol to the membrane that was reversed in the presence of GF109203X (1 microM) and U73122 (20 microM). Similarly, U73122 and GF109203X reversed the insulin induced increase in membrane associated phosphorylated (ser 660) PKC beta II. The novel finding of this investigation is that insulin induces an increase in PKC beta II translocation and phosphorylation through a U73122 sensitive pathway in quantatively the most important insulin responsive tissue, skeletal muscle. Furthermore, these results imply that PKC beta II may be one of the DAG sensitive isoforms involved in glucose transport.     

Insulin resistance after a 72-h fast is associated with impaired AS160 phosphorylation and accumulation of lipid and glycogen in human skeletal muscle

During fasting, human skeletal muscle depends on lipid oxidation for its energy substrate metabolism. This is associated with the development of insulin resistance and a subsequent reduction of insulin-stimulated glucose uptake. The underlying mechanisms controlling insulin action on skeletal muscle under these conditions are unresolved. In a randomized design, we investigated eight healthy subjects after a 72-h fast compared with a 10-h overnight fast. Insulin action on skeletal muscle was assessed by a hyperinsulinemic euglycemic clamp and by determining insulin signaling to glucose transport. In addition, substrate oxidation, skeletal muscle lipid content, regulation of glycogen synthesis, and AMPK signaling were assessed. Skeletal muscle insulin sensitivity was reduced profoundly in response to a 72-h fast and substrate oxidation shifted to predominantly lipid oxidation. This was associated with accumulation of both lipid and glycogen in skeletal muscle. Intracellular insulin signaling to glucose transport was impaired by regulation of phosphorylation at specific sites on AS160 but not TBC1D1, both key regulators of glucose uptake. In contrast, fasting did not impact phosphorylation of AMPK or insulin regulation of Akt, both of which are established upstream kinases of AS160. These findings show that insulin resistance in muscles from healthy individuals is associated with suppression of site-specific phosphorylation of AS160, without Akt or AMPK being affected. This impairment of AS160 phosphorylation, in combination with glycogen accumulation and increased intramuscular lipid content, may provide the underlying mechanisms for resistance to insulin in skeletal muscle after a prolonged fast.     

Increased beta-oxidation in muscle cells enhances insulin-stimulated glucose metabolism and protects against fatty acid-induced insulin resistance despite intramyocellular lipid accumulation

Skeletal muscle insulin resistance may be aggravated by intramyocellular accumulation of fatty acid-derived metabolites that inhibit insulin signaling. We tested the hypothesis that enhanced fatty acid oxidation in myocytes should protect against fatty acid-induced insulin resistance by limiting lipid accumulation. L6 myotubes were transduced with adenoviruses encoding carnitine palmitoyltransferase I (CPT I) isoforms or beta-galactosidase (control). Two to 3-fold overexpression of L-CPT I, the endogenous isoform in L6 cells, proportionally increased oxidation of the long-chain fatty acids palmitate and oleate and increased insulin stimulation of [(14)C]glucose incorporation into glycogen by 60% while enhancing insulin-stimulated phosphorylation of p38MAPK. Incubation of control cells with 0.2 mm palmitate for 18 h caused accumulation of triacylglycerol, diacylglycerol, and ceramide (but not long-chain acyl-CoA) and decreased insulin-stimulated [(14)C]glucose incorporation into glycogen (60%), [(3)H]deoxyglucose uptake (60%), and protein kinase B phosphorylation (20%). In the context of L-CPT I overexpression, palmitate preincubation produced a relative decrease in insulin-stimulated incorporation of [(14)C]glucose into glycogen (60%) and [(3)H]deoxyglucose uptake (40%) but did not inhibit phosphorylation of protein kinase B. Due to the enhancement of insulin-stimulated glucose metabolism induced by L-CPT I overexpression itself, net insulin-stimulated incorporation of [(14)C]glucose into glycogen and [(3)H]deoxyglucose uptake in L-CPT I-transduced, palmitate-treated cells were significantly greater than in palmitate-treated control cells (71 and 75% greater, respectively). However, L-CPT I overexpression failed to decrease intracellular triacylglycerol, diacylglycerol, ceramide, or long-chain acyl-CoA. We propose that accelerated beta-oxidation in muscle cells exerts an insulin-sensitizing effect independently of changes in intracellular lipid content.     

Invited review: effect of acute exercise on insulin signaling and action in humans.

After a single bout of exercise, insulin action is increased in the muscles that were active during exercise. The increased insulin action has been shown to involve glucose transport, glycogen synthesis, and glycogen synthase (GS) activation as well as amino acid transport. A major mechanism involved in increased insulin stimulation of glucose uptake after exercise seems to be the exercise-associated decrease in muscle glycogen content. Muscle glycogen content also plays a pivotal role for the activity of GS and for the ability of insulin to increase GS activity. Insulin signaling in human skeletal muscle is activated by physiological insulin concentrations, but the increase in insulin action after exercise does not seem to be related to increased insulin signaling [insulin receptor tyrosine kinase activity, insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation (RS1), IRS-1-associated phosphatidylinositol 3-kinase activity, Akt phosphorylation (Ser(473)), glycogen synthase kinase 3 (GSK3) phosphorylation (Ser(21)), and GSK3alpha activity], as measured in muscle lysates. Furthermore, insulin signaling is also largely unaffected by exercise itself. This, however, does not preclude that exercise influences insulin signaling through changes in the spatial arrangement of the signaling compounds or by affecting unidentified signaling intermediates. Finally, 5'-AMP-activated protein kinase has recently entered the stage as a promising player in explaining at least a part of the mechanism by which exercise enhances insulin action.     

Muscle glycogen content affects insulin-stimulated glucose transport and protein kinase B activity

We investigated the possible regulatory role of glycogen in insulin-stimulated glucose transport and insulin signaling in skeletal muscle. Rats were preconditioned to obtain low (LG), normal, or high (HG) muscle glycogen content, and perfused isolated hindlimbs were exposed to 0, 100, or 10,000 microU/ml insulin. In the fast-twitch white gastrocnemius, insulin-stimulated glucose transport was significantly higher in LG compared with HG. This difference was less pronounced in the mixed-fiber red gastrocnemius and was absent in the slow-twitch soleus. In the white gastrocnemius, insulin activation of insulin receptor tyrosine kinase and phosphoinositide 3-kinase was unaffected by glycogen levels, whereas protein kinase B activity was significantly higher in LG compared with HG. In additional incubation experiments on fast-twitch epitrochlearis muscles, insulin-stimulated cell surface GLUT-4 content was significantly higher in LG compared with HG. The data indicate that, in fast-twitch muscle, the effect of insulin on glucose transport and cell surface GLUT-4 content is modulated by glycogen content, which does not involve initial but possibly more downstream signaling events.     

Protein kinase Cdelta mediates insulin-induced glucose transport in primary cultures of rat skeletal muscle

Insulin activates certain protein kinase C (PKC) isoforms that are involved in insulin-induced glucose transport. In this study, we investigated the possibility that activation of PKCdelta by insulin participates in the mediation of insulin effects on glucose transport in skeletal muscle. Studies were performed on primary cultures of rat skeletal myotubes. The role of PKCdelta in insulin-induced glucose uptake was evaluated both by selective pharmacological blockade and by over-expression of wild-type and point-mutated inactive PKCdelta isoforms in skeletal myotubes. We found that insulin induces tyrosine phosphorylation and translocation of PKCdelta to the plasma membrane and increases the activity of this isoform. Insulin-induced effects on translocation and phosphorylation of PKCdelta were blocked by a low concentration of rottlerin, whereas the effects of insulin on other PKC isoforms were not. This selective blockade of PKCdelta by rottlerin also inhibited insulin-induced translocation of glucose transporter 4 (GLUT4), but not glucose transporter 3 (GLUT3), and significantly reduced the stimulation of glucose uptake by insulin. When overexpressed in skeletal muscle, PKCdelta and PKCdelta were both active. Overexpression of PKCdelta induced the translocation of GLUT4 to the plasma membrane and increased basal glucose uptake to levels attained by insulin. Moreover, insulin did not increase glucose uptake further in cells overexpressing PKCdelta. Overexpression of PKCdelta did not affect basal glucose uptake or GLUT4 location. Stimulation of glucose uptake by insulin in cells overexpressing PKCdelta was similar to that in untransfected cells. Transfection of skeletal myotubes with dominant negative mutant PKCdelta did not alter basal glucose uptake but blocked insulin-induced GLUT4 translocation and glucose transport. These results demonstrate that insulin activates PKCdelta and that activated PKCdelta is a major signaling molecule in insulin-induced glucose transport.     

Palmitate oxidation rate and action on glycogen synthase in myoblasts from insulin-resistant subjects

Elevated plasma lipid and nonesterified fatty acid concentrations reduce insulin-mediated glucose disposal in skeletal muscle. Cultured myoblasts from 21 subjects were studied for rates of palmitate oxidation and the effect of palmitate on glycogen synthase activity at the end of an 18-h incubation in serum- and glucose-free media. Oxidation rates of 40 microM palmitate in cultured myoblasts correlated with the fasting glucose (r = 0.71, P = 0.001), log fasting insulin (r = 0.52, P = 0.03), and insulin-mediated glucose storage rate (r = -0.50, P = 0.04) of the muscle donors. Myoblast glycogen synthase activity can be regulated by 240 microM palmitate, but the changes are associated with the basal respiratory quotient and not with the insulin resistance of the muscle donor. These results indicate that myoblasts producing elevated palmitate oxidation rates in vitro can be used to identify skeletal muscle abnormalities which are primary contributors to insulin resistance in vivo. Effects of 240 microM palmitate on myoblast glycogen synthase activity appear to be mechanistically different from the relationship between myoblast palmitate oxidation rates and insulin resistance of the muscle donor.     

Protein kinase Cδ but not PKCα is involved in insulin-induced glucose metabolism in hepatocytes

The liver is a major insulin‐responsive tissue responsible for glucose regulation. One important mechanism in this phenomenon is insulin‐induced glycogen synthesis. Studies in our laboratory have shown that protein kinase Cs delta (PKCδ) and alpha (α) have important roles in insulin‐induced glucose transport in skeletal muscle, and that their expression and activity are regulated by insulin. Their importance in glucose regulation in liver cells is unclear. In this study we investigated the possibility that these isoforms are involved in the mediation of insulin‐induced glycogen synthesis in hepatocytes. Studies were done on rat hepatocytes in primary culture and on the AML‐12 (alpha mouse liver) cell line. Insulin increased activity and tyrosine phosphorylation of PKCδ within 5 min. In contrast, activity and tyrosine phosphorylation of PKCα were not increased by insulin. PKCδ was constitutively associated with IR, and this was increased by insulin stimulation. Suppression of PKCδ expression by transfection with RNAi, or overexpression of kinase dead (dominant negative) PKCδ reduced both the insulin‐induced activation of PKB/Akt and the phosphorylation of glycogen synthase kinase 3 (GSK3) and reduced significantly insulin‐induced glucose uptake. In addition, treatment of primary rat hepatocytes with rottlerin abrogated insulin‐induced increase in glycogen synthesis. Neither overexpression nor inhibition of PKCα appeared to alter activation of PKB, phosphorylation of GSK3 or glucose uptake in response to insulin. We conclude that PKCδ, but not PKCα, plays an essential role in insulin‐induced glucose uptake and glycogenesis in hepatocytes. J. Cell. Biochem. 113: 2064–2076, 2012. © 2012 Wiley Periodicals, Inc.     

Alterations of nPKC distribution,but normal Akt/PKB activation in denervated rat soleus muscle

Denervation has been shown to impair the ability of insulin to stimulate glycogen synthesis and, to a lesser extent, glucose transport in rat skeletal muscle. Insulin binding to its receptor, activation of the receptor tyrosine kinase and phosphatidylinositol 3'-kinase do not appear to be involved. On the other hand, it has been shown that denervation causes an increase in the total diacylglycerol (DAG) content and membrane-associated protein kinase C (PKC) activity. In this study, we further characterize these changes in PKC and assess other possible signaling abnormalities that might be related to the decrease of glycogen synthesis. The results reveal that PKC-epsilon and -theta;, but not -alpha or -zeta, are increased in the membrane fraction 24 h after denervation and that the timing of these changes parallels the impaired ability of insulin to stimulate glycogen synthesis. At 24 h, these changes were associated with a 65% decrease in glycogen synthase (GS) activity ratio and decreased electrophoretic mobility, indicative of phosphorylation in GS in muscles incubated in the absence of insulin. Incubation of the denervated soleus with insulin for 30 min minimally increased glucose incorporation into glycogen; however, it increased GS activity threefold, to a value still less than that of control muscle, and it eliminated the gel shift. In addition, insulin increased the apparent abundance of GS kinase (GSK)-3 and protein phosphatase (PP)1 alpha in the supernatant fraction of muscle homogenate to control values, and it caused the same increases in GSK-3 and Akt/protein kinase B (PKB) phosphorylation and Akt/PKB activity that it did in nondenervated muscle. No alterations in hexokinase I or II activity were observed after denervation; however, in agreement with a previous report, glucose 6-phosphate levels were diminished in 24-h-denervated soleus, and they did not increase after insulin stimulation. These results indicate that alterations in the distribution of PKC-epsilon and -theta; accompany the impairment of glycogen synthesis in the 24-h-denervated soleus. They also indicate that the basal rate of glycogen synthesis and its stimulation by insulin in these muscles are diminished despite a normal activation of Akt/PKB and phosphorylation of GSK-3. The significance of the observed alterations to GSK-3 and PP1 alpha distribution remain to be determined.     

Functional studies of Akt isoform specificity in skeletal muscle in vivo; maintained insulin sensitivity despite reduced insulin receptor substrate-1 expression

The phosphoinositide 3-kinase/Akt pathway is thought to be essential for normal insulin action and glucose metabolism in skeletal muscle and has been shown to be dysregulated in insulin resistance. However, the specific roles of and signaling pathways triggered by Akt isoforms have not been fully assessed in muscle in vivo. We overexpressed constitutively active (ca-) Akt-1 or Akt-2 constructs in muscle using in vivo electrotransfer and, after 1 wk, assessed the roles of each isoform on glucose metabolism and fiber growth. We achieved greater than 2.5-fold increases in total Ser473 phosphorylation in muscles expressing ca-Akt-1 and ca-Akt-2, respectively. Both isoforms caused hypertrophy of muscle fibers, consistent with increases in p70S6kinase phosphorylation, and a 60% increase in glycogen accumulation, although only Akt-1 increased glycogen synthase kinase-3beta phosphorylation. Akt-2, but not Akt-1, increased basal glucose uptake (by 33%, P = 0.004) and incorporation into glycogen and lipids, suggesting a specific effect on glucose transport. Consistent with this, short hairpin RNA-mediated silencing of Akt-2 caused reductions in glycogen storage and glucose uptake. Consistent with Akt-mediated insulin receptor substrate 1 (IRS-1) degradation, we observed approximately 30% reductions in IRS-1 protein in muscle overexpressing ca-Akt-1 or ca-Akt-2. Despite this, we observed no decrease in insulin-stimulated glucose uptake. Furthermore, a 68% reduction in IRS-1 levels induced using short hairpin RNAs targeting IRS-1 also did not affect glucose disposal after a glucose load. These data indicate distinct roles for Akt-1 and Akt-2 in muscle glucose metabolism and that moderate reductions in IRS-1 expression do not result in the development of insulin resistance in skeletal muscle in vivo.     

Knockout of PKC alpha enhances insulin signaling through PI3K

Insulin stimulates glucose transport and certain other metabolic processes by activating atypical PKC isoforms (lambda, zeta, iota) and protein kinase B (PKB) through increases in D3-polyphosphoinositides derived from the action of PI3K. The role of diacylglycerol-sensitive PKC isoforms is less clear as they have been suggested to be both activated by insulin and yet inhibit insulin signaling to PI3K. Presently, we found that insulin signaling to insulin receptor substrate 1-dependent PI3K, PKB, and PKC lambda, and downstream processes, glucose transport and activation of ERK, were enhanced in skeletal muscles and adipocytes of mice in which the ubiquitous conventional diacylglycerol-sensitive PKC isoform, PKC alpha, was knocked out by homologous recombination. On the other hand, insulin provoked wortmannin-insensitive increases in immunoprecipitable PKC alpha activity in adipocytes and skeletal muscles of wild-type mice and rats. We conclude that 1) PKC alpha is not required for insulin-stimulated glucose transport, and 2) PKC alpha is activated by insulin at least partly independently of PI3K, and largely serves as a physiological feedback inhibitor of insulin signaling to the insulin receptor substrate 1/PI3K/PKB/PKC lambda/zeta/iota complex and dependent metabolic processes.     

Inhibition of glycogen synthesis by fatty acid in C(2)C(12) muscle cells is independent of PKC-alpha, -epsilon,and -theta

We have previously shown that glycogen synthesis is reduced in lipid-treated C(2)C(12) skeletal muscle myotubes and that this is independent of changes in glucose uptake. Here, we tested whether mitochondrial metabolism of these lipids is necessary for this inhibition and whether the activation of specific protein kinase C (PKC) isoforms is involved. C(2)C(12) myotubes were pretreated with fatty acids and subsequently stimulated with insulin for the determination of glycogen synthesis. The carnitine palmitoyltransferase-1 inhibitor etomoxir, an inhibitor of beta-oxidation of acyl-CoA, did not protect against the inhibition of glycogen synthesis caused by the unsaturated fatty acid oleate. In addition, although oleate caused translocation, indicating activation, of individual PKC isoforms, inhibition of PKC by pharmacological agents or adenovirus-mediated overexpression of dominant negative PKC-alpha, -epsilon, or -theta mutants was unable to prevent the inhibitory effects of oleate on glycogen synthesis. We conclude that neither mitochondrial lipid metabolism nor activation of PKC-alpha, -epsilon, or -theta plays a role in the direct inhibition of glycogen synthesis by unsaturated fatty acids.     

Essential role of p38 MAPK for activation of skeletal muscle glucose transport by lithium

Lithium increases glucose transport and glycogen synthesis in insulin-sensitive cell lines and rat skeletal muscle, and has been used as a non-selective inhibitor of glycogen synthase kinase-3 (GSK-3). However, the molecular mechanisms underlying lithium action on glucose transport in mammalian skeletal muscle are unknown. Therefore, we examined the effects of lithium on glucose transport activity, glycogen synthesis, insulin signaling elements (insulin receptor (IR), Akt, and GSK-3beta), and the stress-activated p38 mitogen-activated protein kinase (p38 MAPK) in the absence or presence of insulin in isolated soleus muscle from lean Zucker rats. Lithium (10 mM LiCl) enhanced basal glucose transport by 62% (p < 0.05) and augmented net glycogen synthesis by 112% (p < 0.05). Whereas lithium did not affect basal IR tyrosine phosphorylation or Akt ser(473) phosphorylation, it did enhance (41%, p < 0.05) basal GSK-3beta ser(9) phosphorylation. Lithium further enhanced (p < 0.05) the stimulatory effects of insulin on glucose transport (43%), glycogen synthesis (44%), and GSK-3beta ser(9) phosphorylation (13%). Lithium increased (p < 0.05) p38 MAPK phosphorylation both in the absence (37%) and presence (41%) of insulin. Importantly, selective inhibition of p38 MAPK (using 10 microM A304000) completely prevented the basal activation of glucose transport by lithium, and also significantly reduced (52%, p < 0.05) the lithium-induced enhancement of insulin-stimulated glucose transport. Theses results demonstrate that lithium enhances basal and insulin-stimulated glucose transport activity and glycogen synthesis in insulin-sensitive rat skeletal muscle, and that these effects are associated with a significant enhancement of GSK-3beta phosphorylation. Importantly, we have documented an essential role of p38 MAPK phosphorylation in the action lithium on the glucose transport system in isolated mammalian skeletal muscle.     

Human glycated albumin affects glucose metabolism in L6 skeletal muscle cells by impairing insulin-induced insulin receptor substrate (IRS) signaling through a protein kinase C alpha-mediated mechanism

Nonenzymatic glycation is increased in diabetes and leads to increased levels of glycated proteins. Most studies have focused on the role of glycation products in vascular complications. Here, we have investigated the action of human glycated albumin (HGA) on insulin signaling in L6 skeletal muscle cells. Exposure of these cells to HGA inhibited insulin-stimulated glucose uptake and glycogen synthase activity by 95 and 80%, respectively. These effects were time- and dose-dependent, reaching a maximum after 12 h incubation with 0.1 mg/ml HGA. In contrast, exposure of the cells to HGA had no effect on thymidine incorporation. Further, HGA reduced insulin-stimulated serine phosphorylation of PKB and GSK3, but did not alter ERK1/2 activation. HGA did not affect either insulin receptor kinase activity or insulin-induced Shc phosphorylation on tyrosine. In contrast, insulin-dependent IRS-1 and IRS-2 tyrosine phosphorylation was severely reduced in cells preincubated with HGA for 24 h. Insulin-stimulated association of PI3K with IRS-1 and IRS-2, and PI3K activity were reduced by HGA in parallel with the changes in IRS tyrosine phosphorylation, while Grb2-IRS association was unchanged. In L6 myotubes, exposure to HGA increased PKC activity by 2-fold resulting in a similar increase in Ser/Thr phosphorylation of IRS-1 and IRS-2. These phosphorylations were blocked by the PKC inhibitor bisindolylmaleimide (BDM). BDM also blocked the action of HGA on insulin-stimulated PKB and GSK3 alpha. Simultaneously, BDM rescued insulin-stimulation of glucose uptake and glycogen synthase activity in cells exposed to HGA. The use of antibodies specific to PKC isoforms shows that this effect appears to be mediated by activated PKC alpha, independent of reactive oxygen species production. In summary, in L6 skeletal muscle cells, exposure to HGA leads to insulin resistance selectively in glucose metabolism with no effect on growth-related pathways regulated by the hormone.     

Activation of glucose transport in skeletal muscle by phospholipase C and phorbol ester. Evaluation of the regulatory roles of protein kinase C and calcium

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

Oxidative stress-induced insulin resistance in rat skeletal muscle: role of glycogen synthase kinase-3

Oxidative stress can contribute to the multifactorial etiology of whole body and skeletal muscle insulin resistance. No investigation has directly assessed the effect of an in vitro oxidant stress on insulin action in intact mammalian skeletal muscle. Therefore, the purpose of the present study was to characterize the molecular actions of a low-grade oxidant stress (H(2)O(2)) on insulin signaling and glucose transport in isolated skeletal muscle of lean Zucker rats. Soleus strips were incubated in 8 mM glucose for 2 h in the absence or presence of 100 mU/ml glucose oxidase, which produces H(2)O(2) at approximately 90 microM. By itself, H(2)O(2) significantly (P < 0.05) activated basal glucose transport activity, net glycogen synthesis, and glycogen synthase activity and increased phosphorylation of insulin receptor (Tyr), Akt (Ser(473)), and GSK-3beta (Ser(9)). In contrast, this oxidant stress significantly inhibited the expected insulin-mediated enhancements in glucose transport, glycogen synthesis, and these signaling factors and allowed GSK-3beta to retain a more active form. In the presence of CT-98014, a selective GSK-3 inhibitor, the ability of insulin to stimulate glucose transport and glycogen synthesis during exposure to this oxidant stress was enhanced by 20% and 39% (P < 0.05), respectively, and insulin stimulation of the phosphorylation of insulin receptor, Akt, and GSK-3 was significantly increased by 36-58% (P < 0.05). These results indicate that an oxidant stress can directly and rapidly induce substantial insulin resistance of skeletal muscle insulin signaling, glucose transport, and glycogen synthesis. Moreover, a small, but significant, portion of this oxidative stress-induced insulin resistance is associated with a reduced insulin-mediated suppression of the active form of GSK-3beta.