Hexosamines as mediators of nutrient sensing and regulation in diabetes

High concentrations of glucose induce insulin resistance, impair insulin secretion, and affect hepatic glucose production in a manner that mirrors Type 2 diabetes, and hexosamines mimic many of these effects. This has led to the hypothesis that cells use hexosamine flux as a glucose- and satiety-sensing pathway. The hexosamine hypothesis for glucose sensing has been validated by overexpressing the rate-limiting enzyme for hexosamine synthesis, glutamine: fructose-6-phosphate amidotransferase (GFA) in several tissues including muscle, liver, fat, and beta cells. With overexpression of GFA in transgenic animals, skeletal muscle becomes insulin resistant, the liver synthesizes excess fatty acid, and the beta cell secretes excess insulin leading to hyperinsulinemia. Thus, excess hexosamine flux leads to a coordinated response whereby fuel is shunted toward long-term storage, mirroring the "thrifty phenotype." Chronically, however, these same adaptive changes result ultimately in obesity, hyperlipidemia, beta cell failure, and Type 2 diabetes. These results suggest a mechanism by which chronic overnutrition leads to the phenotype of Type 2 diabetes.     

Overexpression of glutamine:fructose-6-phosphate amidotransferase in rat-1 fibroblasts enhances glucose-mediated glycogen accumulation via suppression of glycogen phosphorylase activity

The hexosamine biosynthesis pathway (HBP) mediates many of the adverse effects of excess glucose. We have shown previously that glucose down-regulates basal and insulin-stimulated glycogen synthase (GS) activity. Overexpression of the rate-limiting enzyme in the HBP, glutamine:fructose-6-phosphate amidotransferase (GFA), mimics these effects of high glucose and renders the cells more sensitive to glucose. Here we examine the role of the HBP in regulating cellular glycogen content. Glycogen content and glycogen phosphorylase (GP) activity were determined in Rat-1 fibroblasts that overexpress GFA. In both GFA and controls there was a dose-dependent increase in glycogen content (approximately 8-fold) in cells cultured in increasing glucose concentrations (1-20 mM). There was a shift to the left in the glucose dose-response curve for glycogen content in GFA cells (ED50 for glycogen content = 5.80+/-1.05 vs. 8.84+/-0.87 mM glucose, GFA vs. control). Inhibition of GFA reduced glycogen content by 28.4% in controls cultured in 20 mM glucose. In a dose-dependent manner, glucose resulted in a more than 35% decrease in GP activity in controls. GP activity in GFA cells was suppressed compared with that in controls, and there was no glucose-induced down-regulation of GP activity. Glucosamine and uridine mimicked the effects of glucose on glycogen content and GP activity. However, chronic overexpression of GFA is a unique model of hexosamine excess, as culturing control cells in low dose glucosamine (0.1-0.25 mM) did not suppress GP activity and did not eliminate the glucose-mediated down-regulation of GP activity. We conclude that increased flux through the HBP results in enhanced glycogen accumulation due to suppression of GP activity. These results demonstrate that the HBP is an important regulator of cellular glucose metabolism and supports its role as a cellular glucose/satiety sensor.     

High glucose and insulin in combination cause insulin receptor substrate-1 and -2 depletion and protein kinase B desensitisation in primary cultured rat adipocytes: possible implications for insulin resistance in type 2 diabetes

OBJECTIVE: The purpose of this study was to investigate the cellular effects of long-term exposure to high insulin and glucose levels on glucose transport and insulin signalling proteins. DESIGN AND METHODS: Rat adipocytes were cultured for 24 h in different glucose concentrations with 10(4) microU/ml of insulin or without insulin. After washing, (125)I-insulin binding, basal and acutely insulin-stimulated d-[(14)C]glucose uptake, and insulin signalling proteins and glucose transporter 4 (GLUT4) were assessed. RESULTS: High glucose (15 and 25 mmol/l) for 24 h induced a decrease in basal and insulin-stimulated glucose uptake compared with control cells incubated in low glucose (5 or 10 mmol/l). Twenty-four hours of insulin treatment decreased insulin binding capacity by approximately 40%, and shifted the dose-response curve for insulin's acute effect on glucose uptake 2- to 3-fold to the right. Twenty-four hours of insulin treatment reduced basal and insulin-stimulated glucose uptake only in the presence of high glucose (by approximately 30-50%). At high glucose, insulin receptor substrate-1 (IRS-1) expression was downregulated by approximately 20-50%, whereas IRS-2 was strongly upregulated by glucose levels of 10 mmol/l or more (by 100-400%). Insulin treatment amplified the suppression of IRS-1 when combined with high glucose and also IRS-2 expression was almost abolished. Twenty-four hours of treatment with high glucose or insulin, alone or in combination, shifted the dose-response curve for insulin's effect to acutely phosphorylate protein kinase B (PKB) to the right. Fifteen mmol/l glucose increased GLUT4 in cellular membranes (by approximately 140%) compared with 5 mmol/l but this was prevented by a high insulin concentration. CONCLUSIONS: Long-term exposure to high glucose per se decreases IRS-1 but increases IRS-2 content in rat adipocytes and it impairs glucose transport capacity. Treatment with high insulin downregulates insulin binding capacity and, when combined with high glucose, it produces a marked depletion of IRS-1 and -2 content together with an impaired sensitivity to insulin stimulation of PKB activity. These mechanisms may potentially contribute to insulin resistance in type 2 diabetes.     

Mechanism of hexosamine-induced insulin resistance in transgenic mice overexpressing glutamine:fructose-6-phosphate amidotransferase: decreased glucose transporter GLUT4 translocation and reversal by treatment with thiazolidinedione

Hexosamines have been hypothesized to mediate aspects of glucose sensing and toxic effects of hyperglycemia. For example, insulin resistance results when the rate-limiting enzyme for hexosamine synthesis, glutamine:fructose-6-phosphate amidotransferase (GFA), is overexpressed in muscle and adipose tissue of transgenic mice. The glucose infusion rates required to maintain euglycemia at insulin infusion rates of 0.5, 2, 15, and 20 mU/kg x min were 39-90% lower in such transgenic mice, compared with their control littermates (P < or = 0.01). No differences were observed in hepatic glucose output, serum insulin levels, or muscle ATP levels. Uptake of 2-deoxyglucose, measured under conditions of hyperinsulinemia, was significantly lower in transgenic hindlimb muscle, compared with controls (85.9 +/- 17.8 vs. 166.8 +/- 15.1 pmol deoxyglucose/g x min). The decrease in glucose uptake by transgenic muscle was associated with a disruption in the translocation of the insulin-stimulated glucose transporter GLUT4. Fractionation of muscle membranes on a discontinuous sucrose gradient revealed that insulin stimulation of control muscle led to a 28.8% increase in GLUT4 content in the 25% fraction and a 61.2% decrease in the 35% fraction. In transgenic muscle, the insulin-stimulated shifts in GLUT4 distribution were inhibited by over 70%. Treatment of the transgenic animals with the thiazolidinedione troglitazone completely reversed the defect in glucose disposal without changing GFA activity or the levels of uridine 5'-diphosphate-N-acetylglucosamine. Overexpression of GFA in skeletal muscle thus leads to defects in glucose transport similar to those seen in type 2 diabetes. These data support the hypothesis that excess glucose metabolism through the hexosamine pathway may be responsible for the diminished insulin sensitivity and defective glucose uptake that are seen with hyperglycemia.     

Impaired ('diabetic') insulin signaling and action occur in fat cells long before glucose intolerance--is insulin resistance initiated in the adipose tissue?

This review postulates and presents recent evidence that insulin resistance is initiated in the adipose tissue and also suggests that the adipose tissue may play a pivotal role in the induction of insulin resistance in the muscles and the liver. Marked impairments in insulin's intracellular signaling cascade are present in fat cells from type 2 diabetic patients, including reduced IRS-1 gene and protein expression, impaired insulin-stimulated PI3-kinase and PKB/Akt activities. In contrast, upstream insulin signaling in skeletal muscle from diabetic subjects only shows modest impairments and PKB/Akt activation in vivo by insulin appears normal. However, insulin-stimulated glucose transport and glycogen synthesis are markedly reduced.Similar marked impairments in insulin signaling, including reduced IRS-1 expression, impaired insulin-stimulated PI3-kinase and PKB/Akt activities are also seen in some (approximately 30%) normoglycemic individuals with genetic predisposition for type 2 diabetes. In addition, GLUT4 expression is markedly reduced in these cells, similar to what is seen in diabetic cells. The individuals with reduced cellular expression of IRS-1 and GLUT4 are also markedly insulin resistant and exhibit several characteristics of the Insulin Resistance Syndrome.Thus, a 'diabetic' pattern is seen in the fat cells also in normoglycemic subjects and this is associated with a marked insulin resistance in vivo. It is proposed that insulin resistance and/or its effectors is initiated in fat cells and that this may secondarily encompass other target tissues for insulin, including the impaired glucose transport in the muscles.     

软脂酸诱导HepG2细胞胰岛素抵抗及花生四烯酸防治作用的机制研究

目的 探讨高浓度软脂酸(PA)诱导HepG2细胞胰岛素抵抗(IR)的机制及花生四烯酸(AA)对IR的防治作用。方法 (1)用高浓度软脂酸(PA)或10^-7mol／L高胰岛素(HI)培养HepG2细胞建立具有IR的细胞模型，测定培养液中葡萄糖含量及细胞内糖原含量作为鉴定指标；(2)用Western blot检测胞内糖原合酶(GS)和蛋白激酶B(PKB)蛋白水平；(3)用磷脂酰肌醇3激酶(P13K)抑制剂Wortmannin(WT)探讨其对胰岛素信号通路的影响；(4)观察AA是否对PA引起的IR有防治作用。结果 (1)0．20mmol／L PA或川培养HepG2细胞36h后，培养液中葡萄糖含量极显著增高，细胞内糖原含量极显著减少；(2)高浓度PA使磷酸化的PKB(P-Ser473)蛋白水平显著减少，磷酸化的糖原合酶(P-Ser641 GS)蛋白水平极显著增加；(3)WT使对照组GS活性及胞内糖原含量极显著减少，HI组和PA组胞内糖原含量均无统计学差异，但各实验组PKB活性都极显著减少；(4)PA AA组培养液中葡萄糖含量显著低于PA组，GS和PKB活性及胞内糖原含量显著增加。结论 高浓度PA或HI培养HepG2细胞能够诱导IR，其机制可能是其引起胰岛素信号传递途径中自PKB下游到GS之间的信号通路受阻所致。AA能改善PA引起的IR。     

Combined thiazolidinedione–metformin treatment synergistically improves insulin signalling to insulin receptor substrate-1-dependent phosphatidylinositol 3-kinase, atypical protein kinase C and protein kinase B/Akt in human diabetic muscle

Aims/hypotheses  Insulin-stimulated glucose transport in muscle is impaired in type 2 diabetes, presumably reflecting reduced activation of atypical protein kinase C (aPKC) and protein kinase B (PKB/Akt). As previously shown, reductions in aPKC activation are seen at sub-maximal and maximal insulin stimulation, reductions in PKB activation are best seen at sub-maximal insulin stimulation and aPKC reductions at maximal insulin are partly improved by thiazolidinedione or metformin treatment. However, effects of combined thiazolidinedione–metformin treatment on aPKC or PKB activation by sub-maximal and maximal insulin are unknown. Methods  Type 2 diabetic patients were examined before and 5 to 6 weeks after combined thiazolidinedione–metformin therapy for activation of muscle aPKC and PKBβ and their upstream activators, the insulin receptor (IR) and IRS-1-associated phosphatidylinositol 3-kinase (PI3K), during euglycaemic–hyperinsulinaemic clamp studies conducted with sub-maximal (400–500 pmol/l) and maximal (1400 pmol/l) insulin concentrations. Results  Following combined thiazolidinedione–metformin therapy, increases in glucose disposal and increases in sub-maximal and maximal insulin-induced activities of all four muscle signalling factors, IR, IRS-1-dependent PI3K (IRS-1/PI3K), aPKC and PKBβ, were observed. Increases in PKBβ enzyme activity were accompanied by increases in phosphorylation of PKB and its substrate, AS160, which is needed for glucose transport. Despite improved aPKC activity, muscle aPKC levels, which are diminished in type 2 diabetes, were not altered. Conclusions/interpretation  Combined thiazolidinedione–metformin treatment markedly improves sub-maximal and maximal insulin signalling to IR, IRS-1/PI3K, aPKC and PKBβ in type 2 diabetic muscle. These improvements exceed those previously reported after treatment with either agent alone.     

Delays in insulin signaling towards glucose disposal in human skeletal muscle

We explored whether the delay that occurs between a rise in plasma insulin and the increase of glucose disposal occurs before, at, or downstream of steps that are believed to be part of the insulin signaling cascade. Skeletal muscle biopsies were obtained from 16 nondiabetic subjects before, and 20 and 180 min after plasma insulin levels had been augmented in euglycemic hyperinsulinemic glucose clamps. Although plasma insulin had reached 98% of its final concentration within 10 min, insulin receptor kinase (IRK) activity, p85 associated with insulin receptor substrate-1 (IRS-1), IRS-1-associated phosphatidylinositol 3-kinase (PI3K) activity, and Thr(308)-protein kinase B (PKB) phosphorylation in the muscle biopsies at 20 min had reached only 60, 48, 34 and 47% respectively of those at 180 min. This suggests a delay before the level of IRK and little or no delay between IRK and PKB activation. The observation that glycogen synthase activity and glucose disposal at 20 min had both only reached 25% of the respective values at 180 min suggests an additional delay downstream of the investigated signaling steps.     

Rosiglitazone, insulin treatment, and fasting correct defective activation of protein kinase C-zeta/lambda by insulin in vastus lateralis muscles and adipocytes of diabetic rats

Atypical protein kinases C (PKCs), zeta and lambda, and protein kinase B (PKB) are thought to function downstream of phosphatidylinositol 3-kinase (PI 3-kinase) and regulate glucose transport during insulin action in skeletal muscle and adipocytes. Insulin-stimulated glucose transport is defective in type II diabetes mellitus, and this defect is ameliorated by thiazolidinediones and lowering of blood glucose by chronic insulin therapy or short-term fasting. Presently, we evaluated the effects of these insulin-sensitizing modalities on the activation of insulin receptor substrate-1 (IRS-1)-dependent PI 3-kinase, PKC-zeta/lambda, and PKB in vastus lateralis skeletal muscles and adipocytes of nondiabetic and Goto-Kakizaki (GK) diabetic rats. Insulin provoked rapid increases in the activity of PI 3-kinase, PKC-zeta/lambda, and PKB in muscles and adipocytes of nondiabetic rats, but increases in IRS-1-dependent PI 3-kinase and PKC-zeta/lambda, but not PKB, activity were substantially diminished in GK muscles and adipocytes. Rosiglitazone treatment for 10-14 days, 10-day insulin treatment, and 60-h fasting reversed defects in PKC-zeta/lambda activation in GK muscles and adipocytes and increased glucose transport in GK adipocytes, without necessarily increasing IRS-1-dependent PI 3-kinase or PKB activation. Our findings suggest that insulin-sensitizing modalities, viz. thiazolidinediones, chronic insulin treatment, and short-term fasting, similarly improve defects in insulin-stimulated glucose transport at least partly by correcting defects in insulin-induced activation of PKC-zeta/lambda.     

UDP-N-acetylglucosamine transferase and glutamine: fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues

Summary Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc β-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using ³H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2–4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14–36-fold higher in submandibular gland and 5–18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 ± 0.3 nmol/mg · min, which was not different from that in normal subjects (3.3 ± 0.4 nmol/mg · min). A 180-min intravenous insulin infusion (40 mU/m²· min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions. [Diabetologia (1997) 40: 76–81]     

The phosphatidylinositol (PI)-5-phosphate 4-kinase type II enzyme controls insulin signaling by regulating PI-3,4,5-trisphosphate degradation

Phosphatidylinositol-5-phosphate (PI-5-P) is a newly identified phosphoinositide with characteristics of a signaling lipid but no known cellular function. PI-5-P levels are controlled by the type II PI-5-P 4-kinases (PIP4K IIs), a family of kinases that converts PI-5-P into phosphatidylinositol-4,5-bisphosphate (PI-4,5-P2). The PI-5-P pathway is an alternative route for PI-4,5-P2 synthesis as the bulk of this lipid is generated by the canonical pathway in which phosphatidylinositol-4-phosphate (PI-4-P) is the intermediate. Here we examined the effect of activation of the PI-5-P pathway on phosphoinositide 3-kinase (PI3K) signaling by expressing PIP4K II beta in cells that lack this enzyme. Although PIP4K II generates PI-4,5-P2, a substrate for PI3K, expression of this enzyme reduced rather than increased phosphatidylinositol-3,4,5-trisphosphate (PI-3,4,5-P3) levels in cells stimulated with insulin or cells expressing activated PI3K. This reduction in PI-3,4,5-P3 levels resulted in decreased activation of the downstream protein kinase, Akt/PKB. Consistent with these results, expression of IpgD, a bacterial phosphatase that converts PI-4,5-P2 to PI-5-P, resulted in Akt activation, and this effect was partially reversed by PIP4K II beta. PIP4K II beta expression did not impair insulin-dependent association of PI3K with insulin receptor substrate 1 (IRS1) but abbreviated Akt activation, indicating that PIP4K II regulates PI-3,4,5-P3 degradation rather than synthesis. These data support a model in which the PI-5-P pathway controls insulin signaling that leads to Akt activation by regulating a PI-3,4,5-P3 phosphatase.     

RO 31-8220 activates c-Jun N-terminal kinase and glycogen synthase in rat adipocytes and L6 myotubes. Comparison to actions of insulin

The activation of c-Jun N-terminal kinase (JNK) by insulin and anisomycin has been reported to result in increases in glycogen synthase (GS) activity in rat skeletal muscle (Moxham et al., J Biol Chem, 1996, 271:30765-30773). In addition, the protein kinase C (PKC) inhibitor, RO 31-8220, has been reported to activate JNK in rat-1 fibroblasts (Beltman et al., J Biol Chem, 1996, 271:27018-27024). Presently, we found that the RO 31-8220, as well as insulin, activated JNK and GS and stimulated glucose incorporation into glycogen in rat adipocytes and L6 myotubes. In contrast to activation of JNK, RO 31-8220 inhibited extracellular response kinases 1 and 2 (ERK1/2) and had no significant effects on protein kinase B (PKB). Stimulatory effects of RO 31-8220 on JNK and glycogen metabolism were not explained by PKC inhibition, as other PKC inhibitors were without effect on glucose incorporation into glycogen and have no effect on JNK (Beltman et al., J Biol Chem, 1996, 271:27018). Insulin, on the other hand, activated JNK, as well as PKB and ERK1/2. However, stimulatory effects of insulin on GS and glucose incorporation into glycogen appeared to be fully intact and additive to those of RO 31-8220, despite the fact that insulin did not provoke additive increases in JNK activity above those observed with RO 31-8220 alone. Our findings suggest that JNK serves to activate GS during the action of RO 31-8220 in rat adipocytes and L6 myotubes; insulin, on the other hand, appears to activate GS largely independently of JNK.     

游离脂肪酸诱导3T3-L1脂肪细胞胰岛素抵抗的分子机制

目的研究游离脂肪酸（FFA）对3T3-L1脂肪细胞IKKβ及胰岛素信号转导蛋白的影响,探讨FFA诱导胰岛素抵抗（IR）的分子机制。方法诱导成熟的3T3-L1脂肪细胞与0.3-1.0mmol/L的软脂酸（PA）培养6-24h,以2-脱氧-〔^3H〕-D-葡萄糖摄入法观察葡萄糖的转运率,用Western blot检测IKKβ蛋白、IKKβ Ser181磷酸化、IRS-1蛋白、IRS-1 Ser307磷酸化、PI3Kp85蛋白及GluT4蛋白的表达。结果0.3-1.0mmol/LPA作用6-24h后,3T3-L1脂肪细胞的葡萄糖消耗明显减少,同时,Western blot显示,PA对IKKβ及GluT4蛋白的表达无明显影响,却能明显增加IKKβ Ser181及IRS-1 Ser307磷酸化,同时减少IRS-1蛋白和PI3Kp85蛋白的表达。结论FFA可以诱导IR,其分子机制可能与FFA激活IKKβ,使IRS-1丝氨酸残基磷酸化增加而酪氨酸残基磷酸化减少,进而使其下游的PI-3Kp85蛋白表达减少抑制葡萄糖转运有关。     

Leptin activates PI-3 kinase in C2C12 myotubes via janus kinase-2 (JAK-2) and insulin receptor substrate-2 (IRS-2) dependent pathways

Summary We have recently shown that leptin mimicks insulin effects on glucose transport and glycogen synthesis through a phosphatidylinositol-3 (PI) kinase dependent pathway in C₂C₁₂ myotubes. The aim of the present study was to identify the signalling path from the leptin receptor to the PI-3 kinase. We stimulated C₂C₁₂ myotubes with insulin (100 nmol/l, 5 min) or leptin (0.62 nmol/l, 10 min) and determined PI-3 kinase activity in immunoprecipitates with specific non-crossreacting antibodies against insulin-receptor substrate (IRS 1/IRS 2) and against janus kinase (JAK 1 and JAK 2). While insulin-stimulated PI-3 kinase activity is detected in IRS-1 and IRS-2 immunoprecipitates, leptin-stimulated PI-3 kinase activity is found only in IRS-2 immunoprecipitates, suggesting that the leptin signal to PI-3 kinase occurs via IRS-2 and not IRS-1. Leptin-, but not insulin-stimulated PI-3 kinase activity is also detected in immunoprecipitates with antibodies against JAK-2, but not JAK-1. The data suggest that JAK-2 and IRS-2 couple the leptin signalling pathway to the insulin signalling chain. Since we have also detected leptin-stimulated tyrosine phosphorylation of JAK-2 and IRS-2 in C₂C₁₂ myotubes it can be assumed that leptin activates JAK-2 which induces tyrosine phosphorylation of IRS-2 leading to activation of PI-3 kinase. As we could not detect the long leptin receptor isoform in C₂C₁₂ myotubes we conclude that this signalling pathway is activated by a short leptin receptor isoform. [Diabetologia (1997) 40: 1358–1362] Received: 4 August 1997     

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.     

C-reactive protein induces phosphorylation of insulin receptor substrate-1 on Ser<Superscript>307</Superscript> and Ser<Superscript>612</Superscript> in L6 myocytes,thereby impairing the insulin signalling pathway that promotes glucose transport

Aims/hypothesis C-reactive protein (CRP) is associated with insulin resistance and predicts development of type 2 diabetes. However, it is unknown whether CRP directly affects insulin signalling action. To this aim, we determined the effects of human recombinant CRP (hrCRP) on insulin signalling involved in glucose transport in L6 myotubes. Materials and methods L6 myotubes were exposed to endotoxin-free hrCRP and insulin-stimulated activation of signal molecules, glucose uptake and glycogen synthesis were assessed. Results We found that hrCRP stimulates both c-Jun N-terminal kinase (JNK) and extracellular signal-regulated kinase (ERK)1/2 activity. These effects were paralleled by a concomitant increase in IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹², respectively. The stimulatory effects of hrCRP on IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² were partially reversed by treatment with specific JNK and ERK1/2 inhibitors, respectively. Exposure of L6 myotubes to hrCRP reduced insulin-stimulated phosphorylation of IRS-1 at Tyr⁶³², a site essential for engaging p85 subunit of phosphatidylinositol-3 kinase (PI-3K), protein kinase B (Akt) activation and glycogen synthase kinase-3 (GSK-3) phosphorylation. These events were accompanied by a decrease in insulin-stimulated glucose transporter (GLUT) 4 translocation to the plasma membrane, glucose uptake and glucose incorporation into glycogen. The inhibitory effects of hrCRP on insulin signalling and insulin-stimulated GLUT4 translocation were reversed by treatment with JNK inhibitor I and the mitogen-activated protein kinase inhibitor, PD98059. Conclusions/interpretation Our data suggest that hrCRP may cause insulin resistance by increasing IRS-1 phosphorylation at Ser³⁰⁷ and Ser⁶¹² via JNK and ERK1/2, respectively, leading to impaired insulin-stimulated glucose uptake, GLUT4 translocation, and glycogen synthesis mediated by the IRS-1/PI-3K/Akt/GSK-3 pathway.     

Insulin-like growth factor-1 potentiates platelet activation via the IRS/PI3Kalpha pathway

As insulin-like growth factor-1 (IGF-1) is present in the alpha granules of platelets and its receptor is expressed on the platelet surface, it may contribute to the amplification of platelet responses and pathogenesis of cardiovascular disease. The functional and signaling pathways that are involved in IGF-1 modulation of platelet function, however, are presently unknown. Here, I report that IGF-1 stimulation of platelets results in dose-dependent phosphorylation of the IGF receptor in the range of 1 to 100 nM. Phosphorylation of the IGF receptor is rapid and sustained, with maximal phosphorylation reached within 1 minute. Furthermore, IGF-1 stimulates tyrosine phosphorylation of insulin receptor substrate-1 (IRS-1) and IRS-2 and their association with the p85 subunit of phosphoinositide-3 kinase (PI3K). IGF-1-stimulated tyrosine phosphorylation of IRS-1 and IRS-2 and subsequent p85 binding is transient and precedes phosphorylation of protein kinase B (PKB) on Ser473. PAR-1-mediated platelet aggregation is potentiated by IGF-1 and this potentiation, together with PKB phosphorylation, is abolished by the PI3Kalpha inhibitors PI-103 and PIK-75. Importantly, the IGF receptor inhibitor NVP-AEW541 and the neutralization antibody alphaIR3 inhibit SFLLRN-stimulated aggregation, implicating IGF-1 in autocrine regulation of platelet function. These results demonstrate that IGF-1 activates the IGF receptor/IRS/PI3K/PKB pathway, and that PI3Kalpha is essential for the potentiatory effect of IGF-1 on platelet responses.     

Fatty acid-induced insulin resistance: decreased muscle PI3K activation but unchanged Akt phosphorylation.

The mechanisms by which elevated plasma nonesterified fatty acid (NEFA) levels induce skeletal muscle insulin resistance remain unclear. A NEFA-induced defect in the activation of PI3K, which plays a key role in insulin's stimulation of glucose transport, has been invoked. We sought to examine the effects of elevated plasma NEFA (approximately 1 mmol/liter) on muscle PI3K activity, insulin receptor substrate (IRS)-1 (important for activation of PI3K), and Akt, which is downstream of PI3K and activated by phosphorylation on serine and threonine in a PI3K-dependent manner. Ten normal men [age, 37 +/- 9 yr (mean +/- SD); body mass index, 25.2 +/- 3.8 kg/m(2)] underwent two 5-h hyperinsulinemic (80 mU/m(2) x min) euglycemic clamps with basal and end of clamp biopsies of the vastus lateralis muscle. Plasma NEFAs were increased in one study by infusion of 20% Intralipid (1 ml/min) and heparin (900 U/h) throughout and for 2.5 h beforehand. Skeletal muscle protein levels were quantified by Western blotting. Elevated plasma NEFA reduced whole-body insulin-stimulated glucose disposal by 24% (42.1 +/- 4.0 vs. 54.8 +/- 3.6 micromol/kg x min; P < 0.001). Basal muscle IRS-1 was the same in the two studies. IRS-1 levels decreased by 40% in the control glucose clamps (P < 0.005), but did not change during the Intralipid study. Total tyrosine phosphorylated IRS-1 increased by 29% during the control clamps (P < 0.05), but by only 18% (NS) during the Intralipid studies. Total levels of p85alpha subunit of PI3K and Akt were not influenced by plasma NEFA levels either in the basal state or during the glucose clamps. The insulin-induced increase in IRS-1-associated PI3K activity was impaired by elevated NEFA, so that activity at the end of the clamps with Intralipid was 35% lower than in the control clamps (P < 0.05). The percentage reduction in PI3K activation correlated with the reduction in insulin-stimulated glucose disappearance rate that was induced by elevated NEFA (r = 0.70; P < 0.05). Basal P-ser- and P-thr-Akt levels were very low and unaffected by NEFA levels. The glucose clamps resulted in a marked increase in P-ser and P-thr Akt levels. Despite the decrease in PI3K in the Intralipid study, no defect in Akt phosphorylation was found. In summary, NEFA-induced insulin resistance is associated with an impairment of IRS-1 tyrosine phosphorylation and IRS-1-associated PI3K activation. Down-regulation of IRS-1 levels is also impaired. The NEFA-induced defect in muscle glucose uptake appears to be a consequence of a defect in the insulin-signaling pathway leading to impaired PI3K activation. This in turn may lead to impaired glucose transport through an Akt-independent pathway because Akt phosphorylation was unaffected by elevated NEFA levels.     

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.