L6 Myocytes Research Articles

Isozyme-specific Interaction of Protein Kinase Cδ with Mitochondria Dissected Using Live Cell Fluorescence Imaging

PKCδ signaling to mitochondria has been implicated in both mitochondrial apoptosis and metabolism. However, the mechanism by which PKCδ interacts with mitochondria is not well understood. Using FRET-based imaging, we show that PKCδ interacts with mitochondria by a novel and isozyme-specific mechanism distinct from its canonical recruitment to other membranes such as the plasma membrane or Golgi. Specifically, we show that PKCδ interacts with mitochondria following stimulation with phorbol esters or, in L6 myocytes, with insulin via a mechanism that requires two steps. In the first step, PKCδ translocates acutely to mitochondria by a mechanism that requires its C1A and C1B domains and a Leu-Asn sequence in its turn motif. In the second step, PKCδ is retained at mitochondria by a mechanism that depends on its C2 domain, a unique Glu residue in its activation loop, intrinsic catalytic activity, and the mitochondrial membrane potential. In contrast, of these determinants, only the C1B domain is required for the phorbol ester-stimulated translocation of PKCδ to other membranes. PKCδ also basally localizes to mitochondria and increases mitochondrial respiration via many of the same determinants that promote its agonist-evoked interaction. PKCδ localized to mitochondria has robust activity, as revealed by a FRET reporter of PKCδ-specific activity (δCKAR). These data support a model in which multiple determinants unique to PKCδ drive a specific interaction with mitochondria that promotes mitochondrial respiration.

Abstract 266: Autophagy Reverses Intramuscular Lipid Accumulation in Myocytes

Autophagy, or “self-eating” of the cell, is a tightly regulated “housekeeping” process involved in the degradation and recycling of protein aggregates and damaged organelles. In the failing heart, autophagy has been shown to be an adaptive response. We have previously demonstrated that a common feature of non-ischemic heart failure is lipid accumulation, which in excess can mediate cardiac dysfunction via lipotoxicity. Since autophagy mediates the clearance of lipids through a specific process, known as lipophagy, in hepatocytes, we wondered whether the activation of autophagy in the failing heart protects myocyte from lipotoxicity. To test this hypothesis, L6 myocytes were incubated over a period of 6 days with long chain fatty acids, either 0.5mM or 1.0mM fatty acids (equimolar mixture of oleate and palmitate). On the sixth day of treatment, an autophagic inhibitor (bafilomycin A1, 200nM) or autophagic activator (rapamycin, 1uM) was added to the cell culture medium for 24 hours. Intracellular triglyceride (TG) accumulation was measured in the cells using enzyme quantification assay as well as Oil Red O stain. Immunoblotting was also performed on protein markers to confirm autophagic flux (LC3 and P62) and cell death (Caspase3) in the myocytes. The results indicated that increasing concentrations of fatty acids gradually increased intracellular TG accumulation. Inhibition of autophagy using bafilomycin increased this effect whereas activation of autophagy using rapamycin reduced lipid accumulation in the L6 myocytes. Moreover, fatty acid mediated cell death was increased when autophagy is inhibited. We conclude that autophagy promotes the clearance of lipids from cultured myocytes. These findings suggest that lipophagy may be a protective mechanism in the myocyte through the decrease and reversal of intracellular lipid accumulation.

Piceatannol, a resveratrol derivative, promotes glucose uptake through glucose transporter 4 translocation to plasma membrane in L6 myocytes and suppresses blood glucose levels in type 2 diabetic model db/db mice

The skeletal muscle cells are one of the main sites of glucose uptake through glucose transporter 4 (GLUT4) in response to insulin. In muscle cells, 5′ adenosine monophosphate-activated protein kinase (AMPK) is known as another GLUT4 translocation promoter. Natural compounds that activate AMPK have a possibility to overcome insulin resistance in the diabetic state. Piceatannol is a natural analog and a metabolite of resveratrol, a known AMPK activator. In this study, we investigate the in vitro effect of piceatannol on glucose uptake, AMPK phosphorylation and GLUT4 translocation to plasma membrane in L6 myocytes, and its in vivo effect on blood glucose levels in type 2 diabetic model db/db mice. Piceatannol was found to promote glucose uptake, AMPK phosphorylation and GLUT4 translocation by Western blotting analyses in L6 myotubes under a condition of insulin absence. Promotion by piceatannol of glucose uptake as well as GLUT4 translocation to plasma membrane by immunocytochemistry was also demonstrated in L6 myoblasts transfected with a glut4 cDNA-coding vector. Piceatannol suppressed the rises in blood glucose levels at early stages and improved the impaired glucose tolerance at late stages in db/db mice. These in vitro and in vivo findings suggest that piceatannol may be preventive and remedial for type 2 diabetes and become an antidiabetic phytochemical.

Chronic hyperinsulinemia sensitizes myocytes to hyperglycemia‐induced cell death

Recent clinical trials have reported that intensive glucose lowering therapy in type 2 diabetic patients increases death from cardiovascular causes. We therefore investigated whether aggressive insulin therapy sensitizes myocytes to glucose toxicity. Rat L6 myocytes, cultured in low (5.5 mM) glucose conditions, were treated every 24 hours with insulin. After 5 days of treatment, the rates of glucose uptake were measured, and the expression of the glucose transporters GLUT1 and GLUT4 quantified by real‐time PCR and western blotting. Another set of cells was incubated in hyperglycemic (30 mM) conditions, and the activity of the proapoptotic caspase‐3 was measured by spectrophotometry. Chronic insulin treatment increased the rates of glucose uptake compared to non‐treated cells. This increase, which was associated to higher expression levels of both GLUT1 and GLUT4, could not be further potentiated by acute insulin stimulation. In addition, the cells chronically treated with insulin also exhibited an increase in caspase‐3 activity when incubated with high glucose concentrations. In conclusion, simulated chronic hyperinsulinemia upregulates glucose transporters in myocytes, and the ensuing increase in glucose uptake is associated with a sensitization of the cells to hyperglycemia‐induced cell death. This work provides direct evidence for toxic effects of increased glucose metabolism in muscle cells.

Effects of KST48 [(2R,5SR)3-(2-Chloro-benzoyl) -5- (4-chlorophenoxymethyl) -2 (3,4-dichlorophenyl) oxazolidine] on Glucose Transport in L6 Myocytes

KST48 [(2R,5SR)3-(2-chlorobenzoyl)-5-(4-chlorophenoxymethyl)-2-(3,4-dichlorophenyl)oxazolidine] is an oxazolidine derivative that showed significant stimulating effects on glucose transport in L6 myocytes. The effects of KST48 on glucose uptake were assayed by measuring the transport of 2-deoxyglucose in L6 myocytes. The translocation of glucose transport-4 (GLUT-4) was examined by western blot analysis. KST48 enhanced glucose transport in a concentration dependent manner and the maximum effect was observed at 50 micromol/L. The stimulation of glucose transport was characterized as the increase in Vmax without any significant change of Km value. This enhanced glucose transport was found to be decreased by the treatment with phenylarsine oxide and genistein, but not by the treatment with wortmannin and tyrphostin 25. KST48 was also proved to increase the GLUT-4 translocation. These results suggest that KST48 increases glucose transport in L6 myocytes by stimulating the translocation of GLUT4 to plasma membrane and this action might be mediated by protein tyrosine kinase but not by phosphatidyl inositol 3-kinase.

Inhibition of the Protein Tyrosine Phosphatase SHP-1 Increases Glucose Uptake in Skeletal Muscle Cells by Augmenting Insulin Receptor Signaling and GLUT4 Expression

The protein tyrosine phosphatase (PTPase) Src-homology 2-domain-containing phosphatase (SHP)-1 was recently reported to be a novel regulator of insulin's metabolic action. In order to examine the role of this PTPase in skeletal muscle, we used adenovirus (AdV)-mediated gene transfer to express an interfering mutant of SHP-1 [dominant negative (DN)SHP-1; mutation C453S] in L6 myocytes. Expression of DNSHP-1 increased insulin-induced Akt serine-threonine kinase phosphorylation and augmented glucose uptake and glycogen synthesis. Pharmacological inhibition of glucose transporter type 4 (GLUT4) activity using indinavir and GLUT4 translocation assays revealed an important role for this transporter in the increased insulin-induced glucose uptake in DNSHP-1-expressing myocytes. Both GLUT4 mRNA and protein expression were also found to be increased by DNSHP-1 expression. Furthermore, AdV-mediated delivery of DNSHP-1 in skeletal muscle of transgenic mice overexpressing Coxsackie and AdV receptor also enhanced GLUT4 protein expression. Together, these findings confirm that SHP-1 regulates muscle insulin action in a cell-autonomous manner and further suggest that the PTPase negatively modulates insulin action through down-regulation of both insulin signaling to Akt and GLUT4 translocation, as well as GLUT4 expression.

Resveratrol inhibition of inducible nitric oxide synthase in skeletal muscle involves AMPK but not SIRT1

The plant-derived polyphenol resveratrol (RSV) modulates life span and metabolism, and it is thought that these effects are largely mediated by activating the deacetylase enzyme SIRT1. However, RSV also activates the cell energy sensor AMP-activated protein kinase (AMPK). We have previously reported that AMPK activators inhibit inducible nitric oxide synthase (iNOS), a key proinflammatory mediator of insulin resistance in endotoxemia and obesity. The aim of this study was to evaluate whether RSV inhibits iNOS induction in insulin target tissues and to determine the role of SIRT1 and AMPK activation in this effect. We found that RSV (40 mg/kg ip) treatment decreased iNOS induction and NO production in skeletal muscle and white adipose tissue, but not in liver, of endotoxin (LPS)-challenged mice. This effect of the polyphenol was recapitulated in vitro, where RSV (10-80 μM) robustly inhibited iNOS protein induction and NO production in cytokine/LPS-treated L6 myocytes and 3T3-L1 adipocytes. However, no effect of RSV was observed on iNOS induction in FAO hepatocytes. Further studies using inhibitors of SIRT1 revealed that the deacetylase enzyme is not involved in RSV action on iNOS. In marked contrast, RSV activates AMPK in L6 myocytes, and blunting its activation using Compound C or RNA interference partly blocked the inhibitory effect of RSV on NO production. These results show that RSV specifically inhibits iNOS induction in muscle through a mechanism involving AMPK but not SIRT1 activation. This anti-inflammatory action of RSV likely contributes to the therapeutic effect of this plant polyphenol.

Piperidine alkaloids from Piperretrofractum Vahl. protect against high-fat diet-induced obesity by regulating lipid metabolism and activating AMP-activated protein kinase

The fruits of Piperretrofractum Vahl. have been used for their anti-flatulent, expectorant, antitussive, antifungal, and appetizing properties in traditional medicine, and they are reported to possess gastroprotective and cholesterol-lowering properties. However, their anti-obesity activity remains unexplored. The present study was conducted to isolate the anti-obesity constituents from P. retrofractum Vahl. and evaluate their effects in high-fat diet (HFD)-induced obese mice. Piperidine alkaloids from P. retrofractum Vahl. (PRPAs), including piperine, pipernonaline, and dehydropipernonaline, were isolated as the anti-obesity constituents through a peroxisome proliferator-activated receptor δ (PPARδ) transactivation assay. The molecular mechanism was investigated in 3T3-L1 adipocytes and L6 myocytes. PRPA treatment activated AMP-activated protein kinase (AMPK) signaling and PPARδ protein and also regulated the expression of lipid metabolism-related proteins. In the animal model, oral PRPA administration (50, 100, or 300mg/kg/day for 8weeks) significantly reduced HFD-induced body weight gain without altering the amount of food intake. Fat pad mass was reduced in the PRPA treatment groups, as evidenced by reduced adipocyte size. In addition, elevated serum levels of total cholesterol, low-density lipoprotein cholesterol, total lipid, leptin, and lipase were suppressed by PRPA treatment. PRPA also protected against the development of nonalcoholic fatty liver by decreasing hepatic triglyceride accumulation. Consistent with the in vitro results, PRPA activated AMPK signaling and altered the expression of lipid metabolism-related proteins in liver and skeletal muscle. Taken together, these findings demonstrate that PRPAs attenuate HFD-induced obesity by activating AMPK and PPARδ, and regulate lipid metabolism, suggesting their potential anti-obesity effects.

Effects of 3T3 adipocytes on interleukin-6 expression and insulin signaling in L6 skeletal muscle cells

Objective Central adiposity and inflammation play key roles in the development of insulin resistance through the effects of pro-inflammatory adipokines such as IL-6, but the effect of infiltrating adipocytes in skeletal muscle tissues is not known. Communications between muscle cells and fat cells may contribute to the inflammatory response associated with insulin resistance. Methods In this study we used a co-culture system of skeletal muscle (L6) and adipocyte (3T3-L1) cell lines to study expression of the inflammatory cytokine IL-6 and changes in insulin signaling. This model could mimic the adipocytes infiltrating myocytes that is commonly seen in obese patients. Results When plated alone the L6 cells express IL-6 mRNA and secrete IL-6 protein, both of which are increased when the cells are challenged with the bacterial lipopolysaccharide (LPS). In contrast, the 3T3-L1 cells had very little expression of IL-6 mRNA or protein. Co-culture of 3T3-L1 pre-adipocytes with L6 cells, at a density ratio of 1:10, respectively, increased IL-6 expression significantly and decreased insulin-stimulated Akt phosphorylation. To examine the role of IL-6 in insulin sensitivity we incubated the L6 cells with IL-6. A brief challenge of L6 cells with IL-6 enhanced insulin-stimulated Akt phosphorylation. In contrast, incubation of the L6 cells with IL-6 for 96 h markedly decreased insulin-stimulated Akt phosphorylation. Conclusion The enhanced IL-6 mRNA expression and IL-6 release in L6 myocytes co-cultured with 3T3-L1 cells indicate an important interaction between adipocytes and myocytes. This observation may shed some light on the long-standing enigma of obesity-induced insulin resistance where infiltration of the skeletal muscle by preadipocytes/adipocytes is evident.

Role Of ARC And Caspase-3 During Skeletal Muscle Cell Differentiation And Apoptosis

Apoptosis is a highly regulated form of cell death that is essential during development and to maintain tissue homeostasis. Healthy skeletal muscle has a relatively apoptotic resistant phenotype which is critical for the preservation of this terminally differentiated tissue. Despite previous studies showing dramatic increases in caspase-3 activity during differentiation, skeletal muscle can retain its resistance to apoptosis. Apoptosis repressor with caspase recruitment domain (ARC) is a potent inhibitor of apoptosis by acting on both the death receptor and mitochondrial pathway. In addition, ARC is mainly expressed in post-mitotic tissues such as skeletal muscle, heart and neurons. PURPOSE: To examine ARC levels during myocyte differentiation in relation to caspase-3 activity, apoptosis and myosin expression. METHODS: L6 myocytes were plated on collagen coated dishes or glass cover slips and grown to approximately 70% confluence. Following this, cells were switched to a differentiation medium containing 2% horse serum in DMEM. Cells were isolated after 0, 3, 5 and 7 days of differentiation, and analyzed for ARC and myosin protein expression, as well as caspase-3 activity. Immunofluorescent microscopy was performed to evaluate myotube formation in conjunction with ARC and myosin expression. RESULTS: After 3, 5 and 7 days of differentiation, ARC levels increased 64%, 314% and 1814% respectively, while caspase-3 activity increased 42%, 84% and 285%, respectively. At day 0, myosin expression was undetectable, but increased exponentially between then and day 7. Immunofluorescent detection of myosin was only observed in myotubes which expressed ARC, whereas some myotubes that expressed ARC did not necessarily contain myosin. CONCLUSION: During myotube formation, ARC levels increase dramatically. This increase in ARC mirrors that of caspase-3 activity and myosin expression. Interestingly, no cells that contained myosin were without ARC protein. Taken together, these results suggest that ARC may be responsible for attenuating apoptosis in differentiating myocytes despite increases in caspase-3 activity. Furthermore, it appears that ARC expression preceded that of myosin, possibly establishing an anti-apoptotic environment within the cell which may be essential for muscle cell differentiation.

UCP4 overexpression improves fatty acid oxidation and insulin sensitivity in L6 myocytes

Obesity, which is caused by energy uptake being greater than energy expenditure, is widely prevalent today. Currently, only a limited number of efficient interventional strategies are available for the prevention of obesity. Previous studies have shown that UCP4 transcription occurs at a considerable level in mouse skeletal muscle; however, the exact functions of UCP4 remain unclear. In this study, we investigated the effect of UCP4 on mitochondrial function and insulin sensitivity in mature L6 myocytes. UCP4 overexpression in L6 myocytes induced increased mitochondrial carnitine palmitoyltransferase 1A (CPT1A) and decreased citrate synthase (CS) mRNA in the basal condition (i.e., in the absence of insulin). UCP4 overexpression significantly improved insulin sensitivity, increased tyrosine phosphorylation of IRS-1 in the presence of insulin, and significantly reduced intracellular triglyceride (TG). Additionally, intracellular ATP content and mitochondrial membrane potential were downregulated. We also observed that intracellular ROS, mitochondrial morphology, and mitochondrial mtDNA copy number were maintained upon UCP4 expression, with no change in mitochondrial fusion and fission. In summary, our findings provide evidence to show that UCP4 overexpression reduced the insulin sensitivity and mitochondrial fatty acid oxidation of L6 myocytes. These findings support the notion that UCPs are ideal targets for treatment of insulin resistance.

Endotoxin Mediated-iNOS Induction Causes Insulin Resistance via ONOO− Induced Tyrosine Nitration of IRS-1 in Skeletal Muscle

BackgroundIt is believed that the endotoxin lipopolysaccharide (LPS) is implicated in the metabolic perturbations associated with both sepsis and obesity (metabolic endotoxemia). Here we examined the role of inducible nitric oxide synthase (iNOS) in skeletal muscle insulin resistance using LPS challenge in rats and mice as in vivo models of endotoxemia.Methodology/Principal FindingsPharmacological (aminoguanidine) and genetic strategies (iNOS−/− mice) were used to counter iNOS induction in vivo. In vitro studies using peroxynitrite (ONOO−) or inhibitors of the iNOS pathway, 1400 W and EGCG were conducted in L6 myocytes to determine the mechanism by which iNOS mediates LPS-dependent insulin resistance. In vivo, both pharmacological and genetic invalidation of iNOS prevented LPS-induced muscle insulin resistance. Inhibition of iNOS also prevented insulin resistance in myocytes exposed to cytokine/LPS while exposure of myocytes to ONOO− fully reproduced the inhibitory effect of cytokine/LPS on both insulin-stimulated glucose uptake and PI3K activity. Importantly, LPS treatment in vivo and iNOS induction and ONOO− treatment in vitro promoted tyrosine nitration of IRS-1 and reduced insulin-dependent tyrosine phosphorylation.Conclusions/SignificanceOur work demonstrates that iNOS-mediated tyrosine nitration of IRS-1 is a key mechanism of skeletal muscle insulin resistance in endotoxemia, and presents nitrosative modification of insulin signaling proteins as a novel therapeutic target for combating muscle insulin resistance in inflammatory settings.

Berberine inhibits PTP1B activity and mimics insulin action

Type 2 diabetes patients show defects in insulin signal transduction that include lack of insulin receptor, decrease in insulin stimulated receptor tyrosine kinase activity and receptor-mediated phosphorylation of insulin receptor substrates (IRSs). A small molecule that could target insulin signaling would be of significant advantage in the treatment of diabetes. Berberine (BBR) has recently been shown to lower blood glucose levels and to improve insulin resistance in db/db mice partly through the activation of AMP-activated protein kinase (AMPK) signaling and induction of phosphorylation of insulin receptor (IR). However, the underlying mechanism remains largely unknown. Here we report that BBR mimics insulin action by increasing glucose uptake ability by 3T3-L1 adipocytes and L6 myocytes in an insulin-independent manner, inhibiting phosphatase activity of protein tyrosine phosphatase 1B (PTP1B), and increasing phosphorylation of IR, IRS1 and Akt in 3T3-L1 adipocytes. In diabetic mice, BBR lowers hyperglycemia and improves impaired glucose tolerance, but does not increase insulin release and synthesis. The results suggest that BBR represents a different class of anti-hyperglycemic agents.

SPARC interacts with AMPK and regulates GLUT4 expression

AMP-activated protein kinase (AMPK) is a critical regulator of glucose metabolism. To elucidate the biochemical mechanisms by which AMPK regulates glucose and fat metabolism, we conducted a yeast two-hybrid screen to identify its interacting partners. A yeast two-hybrid system was used to screen a mouse embryo cDNA library for proteins able to bind mouse AMPKα1. We also demonstrated an endogenous interaction between AMPKα1 and its interacting partner by co-immunoprecipitation of the endogenous proteins using specific antibodies in HepG2 cells, and in rat kidney, liver, skeletal muscle, and fat tissue. We show that secreted protein acidic and rich in cysteine (SPARC) is an AMPK-interacting protein, and the two proteins enhance each other. AMPK activation increases SPARC expression, and knockdown of AMPK to inhibit endogenous AMPK expression reduces SPARC protein levels. On the other hand, SPARC siRNA reduces AICAR-stimulated AMPK phosphorylation. SPARC affects AMPK-mediated glucose metabolism through regulation of Glut4 expression in L6 myocytes. Our findings suggest that SPARC may be involved in regulating glucose metabolism via AMPK activation. These results provide a starting point for efforts to clarify the relationship between AMPK and SPARC, and deepen our understanding of their roles in fat and glucose metabolism.

Central role of nitric oxide synthase in AICAR and caffeine-induced mitochondrial biogenesis in L6 myocytes

5-Aminoimidazole-4-carboxamide-ribonucleoside (AICAR) and caffeine, which activate AMP-activated protein kinase (AMPK) and cause sarcoplasmic reticulum calcium release, respectively, have been shown to increase mitochondrial biogenesis in L6 myotubes. Nitric oxide (NO) donors also increase mitochondrial biogenesis. Since neuronal and endothelial NO synthase (NOS) are calcium dependent and are also phosphorylated by AMPK, we hypothesized that NOS inhibition would attenuate the activation of mitochondrial biogenesis in response to AICAR and caffeine. L6 myotubes either were not treated (control) or were exposed acutely or for 5 h/day over 5 days to 100 microM of N(G)-nitro-L-arginine methyl ester (L-NAME, NOS inhibitor), 100 microM S-nitroso-N-acetyl-penicillamine (SNAP) (NO donor) +/- 100 microM L-NAME, 2 mM AICAR +/- 100 microM L-NAME, or 5 mM caffeine +/- 100 microM L-NAME (n = 12/treatment). Acute AICAR administration increased (P < 0.05) phospho- (P-)AMPK, but also increased P-CaMK, with resultant chronic increases in peroxisome proliferator-activated receptor-gamma coactivator-1 alpha (PGC-1 alpha), cytochrome-c oxidase (COX)-1, and COX-4 protein expression compared with control cells. NOS inhibition, which had no effect on AICAR-stimulated P-AMPK, surprisingly increased P-CaMK and attenuated the AICAR-induced increases in COX-1 and COX-4 protein. Caffeine administration, which increased P-CaMK without affecting P-AMPK, increased COX-1, COX-4, PGC-1 alpha, and citrate synthase activity. NOS inhibition, surprisingly, greatly attenuated the effect of caffeine on P-CaMK and attenuated the increases in COX-1 and COX-4 protein. SNAP increased all markers of mitochondrial biogenesis, and it also increased P-AMPK and P-CaMK. In conclusion, AICAR and caffeine increase mitochondrial biogenesis in L6 myotubes, at least in part, via interactions with NOS.

Functional involvement of protein kinase C-βII and its substrate, myristoylated alanine-rich C-kinase substrate (MARCKS), in insulin-stimulated glucose transport in L6 rat skeletal muscle cells

Insulin stimulates phosphorylation cascades, including phosphatidylinositol-3-kinase (PI3K), phosphatidylinositol-dependent kinase (PDK1), Akt, and protein kinase C (PKC). Myristoylated alanine-rich C-kinase substrate (MARCKS), a PKCbetaII substrate, could link the effects of insulin to insulin-stimulated glucose transport (ISGT) via phosphorylation of its effector domain since MARCKS has a role in cytoskeletal rearrangements. We examined phosphoPKCbetaII after insulin treatment of L6 myocytes, and cytosolic and membrane phosphoMARCKS, MARCKS and phospholipase D1 in cells pretreated with LY294002 (PI3K inhibitor), CG53353 (PKCbetaII inhibitor) or W13 (calmodulin inhibitor), PI3K, PKCbetaII and calmodulin inhibitors, respectively, before insulin treatment, using western blots. ISGT was examined after cells had been treated with inhibitors, small inhibitory RNA (siRNA) for MARCKS, or transfection with MARCKS mutated at a PKC site. MARCKS, PKCbetaII, GLUT4 and insulin receptor were immunoblotted in subcellular fractions with F-actin antibody immunoprecipitates to demonstrate changes following insulin treatment. GLUT4 membrane insertion was followed after insulin with or without CG53353. Insulin increased phosphoPKCbetaII(Ser660 and Thr641); LY294002 blocked this, indicating its activation by PI3K. Insulin treatment increased cytosolic phosphoMARCKS, decreased membrane MARCKS and increased membrane phospholipase D1 (PLD1), a protein regulating glucose transporter vesicle fusion resulted. PhosphoMARCKS was attenuated by CG53353 or MARCKS siRNA. MARCKS siRNA blocked ISGT. Association of PKCbetaII and GLUT4 with membrane F-actin was enhanced by insulin, as was that of cytosolic and membrane MARCKS. ISGT was attenuated in myocytes transfected with mutated MARCKS (Ser152Ala), whereas overproduction of wild-type MARCKS enhanced ISGT. CG53353 blocked insertion of GLUT4 into membranes of insulin treated cells. The results suggest that PKCbetaII is involved in mediating downstream steps of ISGT through MARCKS phosphorylation and cytoskeletal remodelling.

Characteristics of signalling properties mediated by long-acting insulin analogue glargine and detemir in target cells of insulin

Glargine and detemir are long-acting human insulin analogues with a smooth peakless profile of action. Although their binding affinities to the insulin receptor have been studied, little is known about the subsequent signalling properties activated after the binding. We directly compared intracellular signalling properties of them in various cultured cells. Regarding the metabolic signalling, glargine and insulin-induced comparable dose-dependent phosphorylation of insulin receptor, IRS-1, Akt, and GSK3, whereas detemir-induced kinetics were markedly lower in 3T3-L1 adipocytes and L6 myocytes. A similar pattern of phosphorylation induction was observed in primary hepatocytes and vascular smooth muscle cells (VSMCs). Because of the binding of detemir to albumin with high affinity, the phosphorylation kinetics and glucose uptake of detemir, but not glargine, decreased with increasing concentrations of BSA. Concerning the mitogenic properties, glargine and insulin-induced comparable dose-dependent phosphorylation of MAP kinase (MAPK) and 5-bromo-2’-deoxyuridine (BrdU) incorporation. Detemir-induced phosphorylation of MAPK was apparently reduced, whereas it stimulated BrdU incorporation with relatively similar dose-dependent manner in VSMCs. These results indicate that glargine has comparable properties to human insulin in metabolic and mitogenic signalling and action. In contrast, detemir-induced metabolic signaling is less potent in all cell types studied, and is reduced further by increasing concentrations of albumin.

Improvement of insulin sensitivity by antagonism of the renin-angiotensin system

The reduced capacity of insulin to stimulate glucose transport into skeletal muscle, termed insulin resistance, is a primary defect leading to the development of prediabetes and overt type 2 diabetes. Although the etiology of this skeletal muscle insulin resistance is multifactorial, there is accumulating evidence that one contributor is overactivity of the renin-angiotensin system (RAS). Angiotensin II (ANG II) produced from this system can act on ANG II type 1 receptors both in the vascular endothelium and in myocytes, with an enhancement of the intracellular production of reactive oxygen species (ROS). Evidence from animal model and cultured skeletal muscle cell line studies indicates ANG II can induce insulin resistance. Chronic ANG II infusion into an insulin-sensitive rat produces a markedly insulin-resistant state that is associated with a negative impact of ROS on the skeletal muscle glucose transport system. ANG II treatment of L6 myocytes causes impaired insulin receptor substrate (IRS)-1-dependent insulin signaling that is accompanied by augmentation of NADPH oxidase-mediated ROS production. Further critical evidence has been obtained from the TG(mREN2)27 rat, a model of RAS overactivity and insulin resistance. The TG(mREN2)27 rat displays whole body and skeletal muscle insulin resistance that is associated with local oxidative stress and a significant reduction in the functionality of the insulin receptor (IR)/IRS-1-dependent insulin signaling. Treatment with a selective ANG II type 1 receptor antagonist leads to improvements in whole body insulin sensitivity, enhanced insulin-stimulated glucose transport in muscle, and reduced local oxidative stress. In addition, exercise training of TG(mREN2)27 rats enhances whole body and skeletal muscle insulin action. However, these metabolic improvements elicited by antagonism of ANG II action or exercise training are independent of upregulation of IR/IRS-1-dependent signaling. Collectively, these findings support targeting the RAS in the design of interventions to improve metabolic and cardiovascular function in conditions of insulin resistance associated with prediabetes and type 2 diabetes.

TNF-α and glucocorticoid receptor interaction in L6 muscle cells: A cooperative downregulation of myosin heavy chain

Sepsis is associated with increased expression of TNF-α with subsequent activation of nuclear factor-kappa B (NF-κB). The glucocorticoid receptor (GR) and NF-κB function as mutual antagonists and induction of the latter is believed to play a major role in the acquired glucocorticoid resistance that occurs in some septic patients. GR expression and function has been reported to be elevated in septic muscle suggesting a limited effect of the activated NF-κB on GR function in this context. In this study, the L6 myocyte cell line was used as an in vitro model for a sepsis-like condition in skeletal muscle. While short or long term treatment with TNF-α had no effect on GR expression, glucocorticoid-dependent downregulation of GR occurred with a kinetic profile that is accelerated relative to that observed in most cells. This downregulation was not affected by co-treatment or prior priming of L6 cells with TNF-α. The synthetic glucocorticoid, dexamethasone (DEX) blunted TNF-α-stimulated NF-κB activation in L6 cells. However, although effective at activating an NF-κB transcriptional response, TNF-α treatment exerted a minimal effect in myoblasts and no effect in myotubes on GR transcriptional activity. This limited impact of TNF-α on GR activity was not universal as TNF-α and DEX exerted an additive effect on the reduction in myosin heavy chain (MHC) protein expression caused by either agent alone. Thus, the selective perseverance of GR function in the presence of increased levels of glucocorticoids and TNF-α during sepsis or other inflammatory states may exacerbate muscle protein breakdown.

Lactate as a Metabolic Signal in Gene Expression

PURPOSE: Lactate plays an important role in adaptive response of muscle to exercise. The possible contribution gene transcription in causing lactate-induced adaptive responses remains to be discovered. The goal of this study was to assess genome-wide responses of L6 myocytes to lactate. METHODS: L6 myocytes were cultured in Dulbecco's modified Eagle's Medium (high glucose). Eight days following seeding of the cells they were incubated with media containing 0 mM or 20 mM of sodium lactate. The cells were then harvested 1 hr or 6 hr after incubation with lactate. GeneChip analysis (Rat genome 230A2.0, Affymetrix, Santa Clara, CA) was performed as described by manufacturer. Quantitative real-time RT-PCR was performed to validate the data of GeneChip analysis. RESULTS: Twenty mM lactate significantly increased the expression of 33 genes and decreased the expression of 76 genes at 1 hr, and increased the expression of 673 genes and decreased the expression of only 3 genes at 6 hr. Interestingly, 79 genes involved in metabolism and mitochondria were up-regulated at 6 hr and among them, large numbers of genes are related to mitochondria biogenesis. Furthermore, large numbers of genes which are related to trans crip tional activation, transporter, oxidative stress, apoptosis, cell growth, or Ca2+ and muscle were up-regulated at 6 hr. The inductions of monocarboxylate transporter MCT1 and cytochrome c oxidase genes detected by the GeneChip assay were confirmed by real-time RT-PCR. CONCLUSIONS: The GeneChip analysis suggests that lactate accumulation increases mitochondrial biogenesis in L6 myocytes.