Human triglyceride-rich lipoproteins impair glucose metabolism and insulin signalling in L6 skeletal muscle cells independently of non-esterified fatty acid levels

AIMS/HYPOTHESIS: Elevated fasting and postprandial plasma levels of triglyceride-rich lipoproteins (TGRLs), i.e. VLDL/remnants and chylomicrons/remnants, are a characteristic feature of insulin resistance and are considered a consequence of this state. The aim of this study was to investigate whether intact TGRL particles are capable of inducing insulin resistance. METHODS: We studied the effect of highly purified TGRLs on glycogen synthesis, glycogen synthase activity, glucose uptake, insulin signalling and intramyocellular lipid (IMCL) content using fully differentiated L6 skeletal muscle cells. RESULTS: Incubation with TGRLs diminished insulin-stimulated glycogen synthesis, glycogen synthase activity, glucose uptake and insulin-stimulated phosphorylation of Akt and glycogen synthase kinase 3. Insulin-stimulated tyrosine phosphorylation of IRS-1, and IRS-1- and IRS-2-associated phosphatidylinositol 3-kinase (PI3K) activity were not impaired by TGRLs, suggesting that these steps were not involved in the lipoprotein-induced effects on glucose metabolism. The overall observed effects were time- and dose-dependent and paralleled IMCL accumulation. NEFA concentration in the incubation media did not increase in the presence of TGRLs indicating that the effects observed were solely due to intact lipoprotein particles. Moreover, co-incubation of TGRLs with orlistat, a potent active-site inhibitor of various lipases, did not alter TGRL-induced effects, whereas co-incubation with receptor-associated protein (RAP), which inhibits interaction of TGRL particles with members of the LDL receptor family, reversed the TGRL-induced effects on glycogen synthesis and insulin signalling. CONCLUSIONS/INTERPRETATION: Our data suggest that the accumulation of TGRLs in the blood stream of insulin-resistant patients may not only be a consequence of insulin resistance but could also be a cause for it.     

Cannabinoid type 1 receptors in human skeletal muscle cells participate in the negative crosstalk between fat and muscle

Aims/hypothesis  Cannabinoid type 1 receptor (CB1R) antagonists such as rimonabant (Rim) represent a novel approach to treat obesity and related metabolic disorders. Recent data suggest that endocannabinoids are also produced by human adipocytes. Here we studied the potential involvement of endocannabinoids in the negative crosstalk between fat and muscle. Methods  The protein level of CB1R in human skeletal muscle cells (SkM) during differentiation was analysed using western blotting. SkM were treated with adipocyte-conditioned medium (CM) or anandamide (AEA) in combination with the CB1R antagonists Rim or AM251, and insulin-stimulated Akt phosphorylation and glucose uptake were determined. Furthermore, signalling pathways of CB1R were investigated. Results  We revealed an increase of CB1R protein in SkM during differentiation. Twenty-four hour incubation of SkM with CM or AEA impaired insulin-stimulated Akt(Ser473) phosphorylation by 60% and up to 40%, respectively. Pretreatment of cells with Rim or AM251 reduced the effect of CM by about one-half, while the effect of AEA could be prevented completely. The reduction of insulin-stimulated glucose uptake by CM was completely prevented by Rim. Short-time incubation with AEA activated extracellular regulated kinase 1/2 and p38 mitogen-activated protein kinase, and impaired insulin-stimulated Akt(Ser473) phosphorylation, but had no effect on Akt(Thr308) and glycogen synthase kinase 3α/β phosphorylation. In addition, enhanced IRS-1 (Ser307) phosphorylation was observed. Conclusions/interpretation  Our results show that the CB1R system may play a role in the development of insulin resistance in human SkM. The results obtained with CM support the notion that adipocytes may secrete factors which are able to activate the CB1R. Furthermore, we identified two stress kinases in the signalling pathway of AEA and enhanced IRS-1(Ser307) phosphorylation, potentially underlying the development of insulin resistance. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorised users.     

Focal adhesion kinase regulates insulin resistance in skeletal muscle

Aims/hypothesis On the basis of our previous studies, we investigated the possible role of focal adhesion kinase (FAK) in the development of insulin resistance in skeletal muscle, a major organ responsible for insulin-stimulated glucose uptake. Materials and methods Insulin-resistant C2C12 skeletal muscle cells were transfected with FAK wild-type or FAK mutant plasmids, knocked down using small interfering RNA (siRNA), and their effects on the levels and activities of insulin-signalling molecules and on glucose uptake were determined. Results A significant decrease in tyrosine phosphorylation of FAK in insulin-resistant C2C12 cells was observed. A similar decrease was observed in skeletal muscle obtained from insulin-resistant Sprague–Dawley rats fed a high-fat diet. Increased levels of FAK in insulin-resistant C2C12 skeletal muscle cells increased insulin sensitivity and glucose uptake. These effects were reversed by an increase in the level of kinase activity mutant FAK or suppression of endogenous FAK by siRNA. FAK was also found to interact downstream with insulin receptor substrate-1, phosphatidylinositol 3-kinase and protein kinase C and glycogen synthase kinase 3β, leading to translocation of glucose transporter 4 and resulting in the regulation of glucose uptake. Conclusions/interpretation The present study provides strong evidence that the modulation of FAK level regulates the insulin sensitivity of skeletal muscle cells. The results demonstrate a direct role of FAK in insulin-resistant skeletal muscle cells for the first time. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorised users. This contains details of the authors’ contributions to the study. Niper communication no. 389     

Acute elevation of circulating fatty acids impairs downstream insulin signalling in rat skeletal muscle in vivo independent of effects on stress signalling

The aim of this study was to examine the effect of an acute, physiological increase in plasma free fatty acid (FFA) on initial signalling events in rat red quadriceps muscle (RQ). Male Wistar rats received a 7% glycerol (GLYC) or 7% Intralipid/heparin (LIP) infusion for 3 h, after which they were either killed or infused with insulin at a rate of 0.5 U/kg per h for 5 min, before RQ collection. Plasma FFAs were elevated to approximately 2 mM in the LIP rats only. Insulin-stimulated insulin receptor (IR) Tyr1162/Tyr1163 phosphorylation and IR substrate (IRS)-1 Tyr612 phosphorylation were increased at least twofold over basal in GLYC rats with insulin and this increase was not significantly impaired in the LIP rats. However, there was no insulin-stimulated protein kinase B (PKB) Ser473 or glycogen synthase kinase (GSK)-3beta Ser9 phosphorylation in the LIP rats, compared with at least a twofold increase over basal in GLYC rats for both proteins. c-Jun N-terminal kinase, inhibitor of kappa kinase beta and inhibitor of nuclear factor-kappaB phosphorylation and total protein expression, as well as Ser307-IRS-1 phosphorylation, were not altered by lipid infusion compared with GLYC infusion. These data indicate that acute, physiological elevation in FFA has a greater impact on insulin signalling downstream of IR and IRS-1, at the level of PKB and GSK-3beta, and that under these conditions stress signalling pathways are not significantly stimulated. Decreased PKB and GSK-3beta phosphorylation in RQ may therefore be primary determinants of the reduced insulin action observed in situations of acute FFA oversupply.     

Fat storage in pancreas and in insulin-sensitive tissues in pathogenesis of type 2 diabetes

Obesity is associated with increased storage of lipids in nonadipose tissues like skeletal muscle, liver, and pancreatic beta cells. These lipids constitute a continuous source of long-chain fatty acyl CoA (LC-CoA) and derived metabolites like diacylglycerol and ceramide, acting as signalling molecules on protein kinases activities (in particular, the family of PKCs), ion channel, gene expression, and protein acylation. In skeletal muscle, the increase in LC-CoA and diacylglycerol translocates and activates specific protein kinase C (PKC) isoforms, which will phosphorylate IRS-1 on serine, preventing its phosphorylation on tyrosine and association with PI3 kinase. This interrupts the insulin signalling pathway leading to the stimulation of glucose transport. In pancreatic beta cells, short-term excess of fatty acids or LC-CoA activates PKC and also directly stimulates insulin exocytosis. Long-term exposure to free fatty acids (FFA) leads to an increased basal and blunted glucose-stimulated insulin secretion by affecting gene expression, increase in K(ATP) channel activity, and uncoupling of the mitochondria. In addition, the saturated FFA palmitate increases cell death by apoptosis via increase in ceramide synthesis.     

Insulin receptor substrate-4 is expressed in muscle tissue without acting as a substrate for the insulin receptor

Insulin receptor substrate (IRS) proteins represent key elements of the insulin-signaling cascade. IRS-4 is the most recently characterized member of the IRS family with an undefined in vivo function. In contrast to IRS-1 and IRS-2, IRS-4 exhibits a limited tissue expression, and IRS-4 protein has not been detected in any mouse or primary human tissue so far. The purpose of the present study was to analyze the expression of IRS-4 in rat muscle and human skeletal muscle cells and assess involvement of IRS-4 in initial insulin signaling. Using immunoblotting and immunoprecipitation, the specific expression of IRS-4 protein could be demonstrated in rat soleus and cardiac muscle and human skeletal muscle cells, but it was not significantly detectable in quadriceps and gastrocnemius. A prominent down-regulation of IRS-4 was observed in heart and soleus muscle of WOKW rats, an animal model of the metabolic syndrome. In human skeletal muscle cells, both IRS-1 and IRS-2 are rapidly phosphorylated on tyrosine in response to insulin, whereas essentially no tyrosine phosphorylation of IRS-4 was observed in response to both insulin and IGF-I. Instead, a 2-fold increase in IRS-4 tyrosine phosphorylation was observed in myocytes subjected to osmotic stress. In conclusion, IRS-4 protein is expressed in heart and skeletal muscle in a fiber type specific fashion. Our data suggest that IRS-4 does not function as a substrate of the insulin and the IGF-I receptor in primary muscle cells but may be involved in nonreceptor tyrosine kinase signaling.     

A sustained increase in plasma NEFA upregulates the Toll-like receptor network in human muscle

Aims/hypothesis

Insulin-sensitive tissues (muscle, liver) of individuals with obesity and type 2 diabetes mellitus are in a state of low-grade inflammation, characterised by increased Toll-like receptor (TLR) expression and TLR-driven signalling. However, the cause of this mild inflammatory state is unclear. We tested the hypothesis that a prolonged mild increase in plasma NEFA will increase TLR expression and TLR-driven signalling (nuclear factor κB [NFκB] and mitogen-activated kinase [MAPK]) and impair insulin action in muscle of lean healthy individuals.

Methods

Twelve lean, normal-glucose-tolerant participants were randomised to receive a 48 h infusion (30 ml/h) of saline or Intralipid followed by a euglycaemic–hyperinsulinaemic clamp. Vastus lateralis muscle biopsies were performed before and during the clamp.

Results

Lipid infusion impaired insulin-stimulated IRS-1 tyrosine phosphorylation and reduced peripheral insulin sensitivity (p?<?0.01). The elevation in circulating NEFA increased expression of TLR3, TLR4 and TLR5, and several MAPK (MAPK8, MAP4K4, MAP2K3) and inhibitor of κB kinase-NFκB (CHUK [IKKA], c-REL [REL] and p65 [RELA, NFKB3, p65]) signalling genes (p?<?0.05). The lipid infusion also increased extracellular signal-regulated kinase (ERK) phosphorylation (p?<?0.05) and tended to reduce the content of inhibitor of kappa Bα (p?=?0.09). The muscle content of most diacylglycerol, ceramide and acylcarnitine species was unaffected. In summary, insulin resistance induced by prolonged low-dose lipid infusion occurs together with increased TLR-driven inflammatory signalling and impaired insulin-stimulated IRS-1 tyrosine phosphorylation.

Conclusions/interpretation

A sustained, mild elevation in plasma NEFA is sufficient to increase TLR expression and TLR-driven signalling (NFκB and MAPK) in lean individuals. The activation of this pathway by NEFA may be involved in the pathogenesis of insulin resistance in humans. Trial registration ClinicalTrials.gov NCT01740817     

Lipid second messenger regulation: the role of diacylglycerol kinases and their relevance to hypertension

Extracellular stimuli elicit cellular responses through generation of intracellular second messengers. The lipid second messenger diacylglycerol is produced following activation of the phosphoinositide signalling system. Diacylglycerol is the physiological activator of protein kinase C but also interacts indirectly with other signalling molecules such as small G proteins. Diacylglycerol kinases convert diacylglycerol to phosphatidic acid so terminating signalling through diacylglycerol. However, phosphatidic acid itself has a lipid second messenger role, with targets distinct from those of its precursor diacylglycerol. Therefore, diacylglycerol kinases occupy a central position in signal transduction and regulation of their activity is crucial to cellular function. A family of nine mammalian diacylglycerol kinases have been identified. Their structural diversity and complex pattern of tissue expression suggests that they function in distinct cellular processes. In addition to the plasma membrane, diacylglycerol kinases are found at the nucleus and cytoskeleton and translocation between subcellular compartments occurs with agonist stimulation. In small arteries diacylglycerol kinase activity is increased by adrenergic stimulation implying a role in vascular smooth muscle responses. Due to their role as key regulators of protein kinase C activity diacylglycerol kinases may play a role in the cardiovascular changes that occur in hypertension and as such could represent novel therapeutic targets.     

Paradoxical effects of increased expression of PGC-1α on muscle mitochondrial function and insulin-stimulated muscle glucose metabolism

Peroxisome proliferator-activated receptor-γ coactivator (PGC)-1α has been shown to play critical roles in regulating mitochondria biogenesis, respiration, and muscle oxidative phenotype. Furthermore, reductions in the expression of PGC-1α in muscle have been implicated in the pathogenesis of type 2 diabetes. To determine the effect of increased muscle-specific PGC-1α expression on muscle mitochondrial function and glucose and lipid metabolism in vivo, we examined body composition, energy balance, and liver and muscle insulin sensitivity by hyperinsulinemic-euglycemic clamp studies and muscle energetics by using ³¹P magnetic resonance spectroscopy in transgenic mice. Increased expression of PGC-1α in muscle resulted in a 2.4-fold increase in mitochondrial density, which was associated with an ≈60% increase in the unidirectional rate of ATP synthesis. Surprisingly, there was no effect of increased muscle PGC-1α expression on whole-body energy expenditure, and PGC-1α transgenic mice were more prone to fat-induced insulin resistance because of decreased insulin-stimulated muscle glucose uptake. The reduced insulin-stimulated muscle glucose uptake could most likely be attributed to a relative increase in fatty acid delivery/triglyceride reesterfication, as reflected by increased expression of CD36, acyl-CoA:diacylglycerol acyltransferase1, and mitochondrial acyl-CoA:glycerol-sn-3-phosphate acyltransferase, that may have exceeded mitochondrial fatty acid oxidation, resulting in increased intracellular lipid accumulation and an increase in the membrane to cytosol diacylglycerol content. This, in turn, caused activation of PKCθ, decreased insulin signaling at the level of insulin receptor substrate-1 (IRS-1) tyrosine phosphorylation, and skeletal muscle insulin resistance.     

Regulation of insulin receptor substrate-2 tyrosine phosphorylation in animal models of insulin resistance

Insulin induces a wide variety of growth and metabolic responses in many cell types. These actions are initiated by insulin binding to its receptor and involve a series of alternative and complementary pathways created by the multiple substrates of the insulin receptor (insulin receptor substrates [IRSs]). We investigated IRS-1 and IRS-2 tyrosine phosphorylation; their association with phosphatidylinositol-3-OH kinase (PI3-K); and the phosphorylation of Akt, a serine-threonine kinase situated downstream of PI3-K, in liver and muscle of two animal models of insulin resistance: epinephrine- or dexamethasone-treated rats. We used in vivo insulin infusion followed by tissue extraction, immunoprecipitation, and immunoblotting. IRS-1 and IRS-2 protein expression did not change in liver and muscle of the epinephrine-treated rats, but in dexamethasone-treated rats IRS-1 presented an increase in liver and a decrease in muscle tissue. PI3-K and Akt protein expression did not change in liver or muscle of the two animal models of insulin resistance. There was a downregulation in insulin-induced IRS-1 and IRS-2 tyrosine phosphorylation and association with PI3-K in both models of insulin resistance. In parallel, insulin-induced Akt phosphorylation was reduced in both tissues of epinephrine-treated rats, and in liver but not in muscle of dexamethasonetreated rats. The reduction in insulin-induced Akt phosphorylation may help to explain the insulin resistance in liver and muscle of epinephrine-treated rats and in the liver of dexamethasone-treated rats.     

Dietary protein deprivation decreases the serine phosphorylation of insulin receptor substrate-1 in rat skeletal muscle

Evidence has shown that protein malnutrition tends to increase peripheral insulin sensitivity, but the molecular mechanism underlying this increase is not yet clear. Here we show that, in rat muscle, the state of insulin receptor (IR) substrate-1 (IRS-1), a pivotal component of the signaling pathway of the IR, changes drastically according to protein supply. After rats were fed a protein-free diet (PF) or a 12% casein diet for 1 week, their IR and IRS-1 states were analyzed by immunoblotting using various antibodies. PF slightly increased the amount of IR without affecting the state of IR tyrosine phosphorylation. In contrast, PF decreased the amount of IRS-1 and markedly increased phosphorylation of IRS-1 tyrosine residues after insulin injection. Moreover, IRS-1 in PF rats exhibited faster mobility in SDS-PAGE as well as far less phosphorylation of Ser612 and Ser307, indicating hypophosphorylation on its serine residues. Results of additional experiments using energy-restricted (pair-fed) rats and streptozotocin-induced diabetic rats suggest that dietary protein deficiency by itself alters serine phosphorylation of IRS-1, while the up-regulation of tyrosine phosphorylation requires other factors, such as a reduction in basal plasma insulin. The serine dephosphorylation followed by up-regulation of insulin-dependent IRS-1 tyrosine phosphorylation in skeletal muscle of PF rats in vivo is similar to a phenomenon observed in cultured cells under restriction of amino acids in the medium. With these findings, it could be inferred that the reduction of serine phosphorylation contributes to the sensitization of IRS-1 to IR tyrosine kinase under protein malnutrition.     

High leptin levels acutely inhibit insulin-stimulated glucose uptake without affecting glucose transporter 4 translocation in l6 rat skeletal muscle cells

Obesity is a major risk factor for the development of insulin resistance, characterized by impaired stimulation of glucose disposal into muscle. The mechanisms underlying insulin resistance are unknown. Here we examine the direct effect of leptin, the product of the obesity gene, on insulin-stimulated glucose uptake in cultured rat skeletal muscle cells. Preincubation of L6 myotubes with leptin (2 or 100 nM, 30 min) had no effect on basal glucose uptake but reduced insulin-stimulated glucose uptake. However, leptin had no effect on the insulin-induced gain in myc-tagged glucose transporter 4 (GLUT4) appearance at the cell surface of L6 myotubes. Preincubation of cells with leptin also had no effect on insulin-stimulated tyrosine phosphorylation of insulin receptor, IRS-1 and IRS-2, phosphatidylinositol 3-kinase activity, or Akt phosphorylation. We have previously shown that insulin regulates glucose uptake via a signaling pathway sensitive to inhibitors of p38 MAP kinase. Here, leptin pretreatment reduced the extent of insulin-stimulated p38 MAP kinase phosphorylation and phosphorylation of cAMP response element binder, a downstream effector of p38 MAP kinase. These results show that high leptin levels can directly reduce insulin-stimulated glucose uptake in L6 muscle cells despite normal GLUT4 translocation. The mechanism of this effect could involve inhibition of insulin-stimulated p38 MAP kinase and GLUT4 activation.     

Molecular basis for the insulinomimetic effects of C-peptide

AIMS/HYPOTHESIS: C-peptide, released by the beta-cells of pancreatic islets, elicits salutary responses in Type I (insulin-dependent) diabetes mellitus but the molecular mechanisms behind these effects are not known. We assessed whether synthetic rat C-peptide stimulates insulin-like cellular effects in a classic insulin target tissue. METHODS: To clarify the molecular mechanisms involved in several insulinomimetic actions, we investigated the effect of C-peptide on the insulin signalling pathway in rat skeletal muscle cells. We used L6 myoblasts and myocytes to measure the effects of C-peptide or insulin or both on glycogen synthesis and amino acid uptake. We also studied the effects of C-peptide on insulin receptor autophosphorylation, its tyrosine kinase activity, phosphorylation of IRS-1, PI 3-kinase, Akt, p90Rsk, MAPK, and GSK3 in these cells. RESULTS: In L6 cells, physiological concentrations of C-peptide (0.3-3 nmol/l) significantly activated insulin receptor tyrosine kinase, IRS-1 tyrosine phosphorylation, PI 3-kinase activity, MAPK phosphorylation, p90Rsk, and GSK3 phosphorylation. A scrambled C-peptide sequence - the control - showed no effects. Wortmannin blocked C-peptide-induced glycogen synthesis while pertussis toxin had no effect. Only submaximal insulin concentrations (up to 10 nmol/l) combined with submaximal C-peptide concentrations led to additive effects. CONCLUSION/INTERPRETATION: C-peptide added to the maximal insulin dose (100 nmol/l) did not increase the effect of insulin alone. We thus conclude that the same signalling elements are used by both ligands. However, the lack of Akt activation by C-peptide and the bell-shaped dose response induced by C-peptide indicate that C-peptide has some effects by another distinct mechanism. We speculate that C-peptide could modulate the metabolic effects of insulin by enhancing them at low hormone concentrations and dampening them at high hormone concentrations.     

Cytosolic protein tyrosine phosphatase-epsilon is a negative regulator of insulin signaling in skeletal muscle

Whereas positive regulatory events triggered by insulin binding to insulin receptor (IR) have been well documented, the mechanism by which the activated IR is returned to the basal status is not completely understood. Recently studies focused on the involvement of protein tyrosine phosphatases (PTPs) and how they might influence IR signaling. In this study, we examined the possibility that cytosolic PTPepsilon (cytPTPepsilon) is involved in IR signaling. Studies were performed on L6 skeletal muscle cells. cytPTPepsilon was overexpressed by using pBABE retroviral expression vectors. In addition, we inhibited cytPTPepsilon by RNA silencing. We found that insulin induced rapid association of cytPTPepsilon with IR. Interestingly, this association appeared to occur in the plasma membrane and on stimulation with insulin the two proteins internalized together. Moreover, it appeared that almost all internalized IR was associated with cytPTPepsilon. We found that knockdown of cytPTPepsilon by RNA silencing increased insulin-induced tyrosine phosphorylation of IR and IR substrate (IRS)-1 as well as phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Moreover, overexpression of wild-type cytPTPepsilon reduced insulin-induced tyrosine phosphorylation of IR, IRS-1, and phosphorylation of protein kinase B and glycogen synthase kinase-3 and insulin-induced stimulation of glucose uptake. Finally, insulin-induced tyrosine phosphorylation of IR and IRS-1 was greater in skeletal muscle from mice lacking the cytPTPepsilon gene than that from wild-type control animals. We conclude that cytPTPepsilon serves as another major candidate negative regulator of IR signaling in skeletal muscle.     

Modulation of insulin-stimulated glycogen synthesis by Src Homology Phosphatase 2

We have examined the requirement of the protein tyrosine phosphatase Src Homology Phosphatase 2 (SHP2) for insulin-stimulated glycogen synthesis. To this end, 3T3L1 fibroblasts were stably transfected with either wild type or a catalytically inactive C463A-mutant of SHP2, and analysed for insulin-induced glycogen synthesis, tyrosine phosphorylation of the insulin receptor and IRS-1, and activation of phosphatidylinositol 3'-kinase (PI 3'-kinase). Glycogen synthesis was stimulated 9.1+/-0.9-fold by insulin in untransfected cells. In cells expressing the dominant-negative C463A-SHP2 mutant, the stimulation of glycogen synthesis by insulin was strongly enhanced (18.7+/-2.7-fold stimulation), while this response was impaired in cells overexpressing wild-type SHP2 (6.6+/-1.1-fold stimulation). When exploring the early post-receptor signalling pathways that contribute to glycogen synthesis, we found that insulin stimulated the tyrosine phosphorylation of IRS-1, and the activation of IRS-1-associated PI 3'-kinase more strongly in C463A-SHP2 expressing 3T3L1-cells (18.1+/-4.7-fold) than in parental 3T3L1 cells (6.8+/-0.5-fold). In 3T3L1 cells overexpressing wild-type SHP2, the insulin stimulation of IRS-1 tyrosine phosphorylation and the activation of PI 3'-kinase (4.5+/-1.0-fold) were impaired. An enhanced activity of SHP2 leads to negative modulation of insulin signalling by reducing the tyrosine phosphorylation of IRS-1 and the concomitant activation of PI 3'-kinase. This results in an impaired ability of insulin to stimulate glycogen synthesis.     

Bioactives of Artemisia dracunculus L enhance cellular insulin signaling in primary human skeletal muscle culture

An alcoholic extract of Artemisia dracunculus L (PMI 5011) has been shown to decrease glucose and improve insulin levels in animal models, suggesting an ability to enhance insulin sensitivity. We sought to assess the cellular mechanism by which this botanical affects carbohydrate metabolism in primary human skeletal muscle culture. We measured basal and insulin-stimulated glucose uptake, glycogen accumulation, phosphoinositide 3 (PI-3) kinase activity, and Akt phosphorylation in primary skeletal muscle culture from subjects with type 2 diabetes mellitus incubated with or without various concentrations of PMI 5011. We also analyzed the abundance of insulin receptor signaling proteins, for example, IRS-1, IRS-2, and PI-3 kinase. Glucose uptake was significantly increased in the presence of increasing concentrations of PMI 5011. In addition, glycogen accumulation, observed to be decreased with increasing free fatty acid levels, was partially restored with PMI 5011. PMI 5011 treatment did not appear to significantly affect protein abundance for IRS-1, IRS-2, PI-3 kinase, Akt, insulin receptor, or Glut-4. However, PMI 5011 significantly decreased levels of a specific protein tyrosine phosphatase, that is, PTP1B. Time course studies confirmed that protein abundance of PTP1B decreases in the presence of PMI 5011. The cellular mechanism of action to explain the effects by which an alcoholic extract of A dracunculus L improves carbohydrate metabolism on a clinical level may be secondary to enhancing insulin receptor signaling and modulating levels of a specific protein tyrosine phosphatase, that is, PTP1B.     

Vanadate and rapamycin synergistically enhance insulin-stimulated glucose uptake

Tyrosine dephosphorylation, serine phosphorylation, and proteasomal degradation of insulin receptor substrates (IRSs) are implicated in the negative regulation of insulin action. Here we show that simultaneous inhibition of IRS-1 tyrosine dephosphorylation and proteasomal degradation synergistically augments insulin-responsive glucose uptake. L6 skeletal muscle cells (L6 cells) were treated with inhibitors of protein-tyrosine phosphatases, proteasomal degradation, and mammalian target of rapamycin (mTOR), and the effects of insulin on glucose uptake, IRS-1 tyrosine phosphorylation, phosphatidylinositol (PI) 3-kinase activity, and IRS-1 mass were examined. Pretreatment of L6 cells with sodium orthovanadate (Na(3)VO(4)) plus the mTOR inhibitor rapamycin caused a 5-fold increase in insulin-responsive glucose uptake at 2 hours when compared to insulin alone. Evaluation of IRS-1 associated PI 3-kinase activity, IRS-1-associated p85 mass, and IRS-1 tyrosine phosphorylation showed that 2 hours after insulin addition they were reduced by 70% from maximal activity. Likewise, IRS-1 mass was reduced by 50%. When L6 cells were pretreated with Na(3)VO(4) plus the proteasome inhibitor MG-132 or the mTOR inhibitor rapamycin prior to insulin addition, IRS-1 mass loss as well as IRS-1/PI-3 kinase complex decay was blocked at 2 hours and PI 3-kinase activity was increased 2.5-fold and 4-fold, respectively, over insulin alone. Finally, treatment of L6 cells with subtherapeutic amounts of vanadyl sulfate and rapamycin induced a synergistic 3-fold increase in insulin-induced glucose uptake at 2 hours. These findings indicate that vanadium and rapamycin synergize to enhance glucose uptake by preventing IRS-1 mass loss and IRS-1/PI 3-kinase complex decay and may offer a new approach to enhance glucose transport in diabetes.     

1,25-Dihydroxyvitamin D improved the free fatty-acid-induced insulin resistance in cultured C2C12 cells

BACKGROUND: Epidemiological evidence has indicated that vitamin D deficiency increased the risk of insulin resistance in metabolic syndrome. The present study was conducted to test the hypothesis that 1,25-dihydroxyvitamin D may improve the free fatty-acid (FFA)-induced insulin resistance in muscle cells. METHOD: The insulin resistance of muscle cell model was established by treatment of FFA in differentiated C2C12 cells. Glucose uptake of C2C12 myotubes was analysed by the 3H-labelled 2-deoxyglucose uptake assay. The diameter of myotubes was measured under the condition of glutaraldehyde-induced autofluorescense. Tyrosine phosphorylated insulin receptor substrate 1 (IRS-1) was measured by immunoprecipitation. Serine phosphorylated IRS-1 and protein kinase B (Akt), extracellular signal-related kinase (ERK), c-Jun amino-terminal kinases (JNK) as well as their phosphorylated form were analysed by Western blots. RESULTS: Compared with a vehicle-treated group, FFA treatment in myotubes was associated with 70.6% reduction in insulin-mediated uptake of glucose, a five-fold increase in serine phosphorylation of IRS-1, 76.9% decrease in tyrosine phosphorylation of IRS-1 and 81.8% decrease in phosphorylation of Akt. Supplement of 1,25-dihydroxyvitamin D improved the FFA-induced inhibition of glucose uptake in a dose- dependent (p < 0.001) and time-dependent manner (p < 0.01). This was accompanied by increase in tyrosine phosphorylation of IRS-1 and phosphorylated Akt and decrease in serine phosphorylation of IRS-1 (p < 0.001). 1,25-Dihydroxyvitamin D also inhibited the FFA-induced reduction in myotube diameter by 35.3% (p < 0.001). JNK phosphorylation was reduced by 126.7% with treatment of 1,25-dihydroxyvitamin D (p < 0.001). 1,25-Dihydroxyvitamin D had no effect on FFA-induced ERK phosphorylation (p = 0.84). CONCLUSION: 1,25-Dihydroxyvitamin D improved the FFA-induced insulin resistance in muscle cells. Copyright (c) 2008 John Wiley & Sons, Ltd.     

Phosphorylation of insulin receptor substrate 1 by glycogen synthase kinase 3 impairs insulin action

The phosphorylation of insulin receptor substrate 1 (IRS-1) on tyrosine residues by the insulin receptor (IR) tyrosine kinase is involved in most of the biological responses of insulin. IRS-1 mediates insulin signaling by recruiting SH2 proteins through its multiple tyrosine phosphorylation sites. The phosphorylation of IRS-1 on serine/threonine residues also occurs in cells; however, the particular protein kinase(s) promoting this type of phosphorylation are unknown. Here we report that glycogen synthase kinase 3 (GSK-3) is capable of phosphorylating IRS-1 and that this modification converts IRS-1 into an inhibitor of IR tyrosine kinase activity in vitro. Expression of wild-type GSK-3 or an “unregulated” mutant of the kinase (S9A) in CHO cells overexpressing IRS-1 and IR, resulted in increased serine phosphorylation levels of IRS-1, suggesting that IRS-1 is a cellular target of GSK-3. Furthermore, insulin-induced tyrosine phosphorylation of IRS-1 and IR was markedly suppressed in cells expressing wild-type or the S9A mutant, indicating that expression of GSK-3 impairs IR tyrosine kinase activity. Taken together, our studies suggest a new role for GSK-3 in attenuating insulin signaling via its phosphorylation of IRS-1 and may provide new insight into mechanisms important in insulin resistance.     

Stearoyl CoA desaturase 1 is elevated in obesity but protects against fatty acid-induced skeletal muscle insulin resistance in vitro

Aims/hypothesis Stearoyl CoA desaturase 1 (SCD1) is implicated in mediating obesity and insulin resistance. Paradoxically, SCD1 converts saturated fatty acids, the lipid species implicated in mediating insulin resistance, to monounsaturated fatty acids. The aim of the present study was to assess the molecular mechanisms that implicate SCD1 in the aetiology of fatty acid-induced insulin resistance.Methods SCD1 protein was transiently decreased or increased in rat L6 skeletal muscle myotubes using SCD1 short interfering RNA (siRNA) or liposome-mediated transfection of pcDNA3.1/Hygro-mSCD1, respectively.Results Reducing SCD1 protein resulted in marked esterification of exogenous fatty acids into diacylglycerol (DAG) and ceramide. Insulin-stimulated Akt activity and phosphorylation and 2-deoxyglucose uptake were reduced with SCD1 siRNA. Exposure of L6 myotubes to palmitate abolished insulin-stimulated glucose uptake in both control and SCD1 siRNA myotubes. Overexpression of SCD1 resulted in triacylglycerol esterification but attenuated ceramide and DAG accumulation and protected myotubes from fatty acid-induced insulin resistance.Conclusions/interpretation SCD1 protects from cellular toxicity in L6 myotubes by preventing excessive accumulation of bioactive lipid metabolites.Electronic supplementary material Supplementary material is available for this article at and is accessible to authorised users.