anisomycin and Insulin-Resistance

Stress stimuli such as tumor necrosis factor (TNF) have been shown to induce insulin receptor substrate (IRS)-1 serine phosphorylation and insulin resistance by transactivation of ErbB receptors. We aimed at elucidating the potential role of p38 mitogen-activated protein kinase (p38MAPK) in mediating stress-induced ErbB receptors activation.. p38MAPK effect on ErbBs transactivation and insulin signaling was assessed in Fao or HepG2 cells, exposed to stress stimuli, and on metabolic parameters in ob/ob and C57/BL6 mice.. High-fat diet-fed mice and ob/ob mice exhibited elevated hepatic p38MAPK activation associated with glucose intolerance and hyperinsulinemia. Liver expression of dominant-negative (DN)-p38MAPKα in ob/ob mice reduced fasting insulin levels and improved glucose tolerance, whereas C57/BL6 mice overexpressing wild-type p38MAPKα exhibited enhanced IRS-1 serine phosphorylation and reduced insulin-stimulated IRS-1 tyrosine phosphorylation. Fao or HepG2 cells exposed to TNF, anisomycin, or sphingomyelinase demonstrated rapid transactivation of ErbB receptors leading to PI3-kinase/Akt activation and IRS-1 serine phosphorylation. p38MAPK inhibition either by SB203580, by small interfering RNA, or by DN-p38MAPKα decreased ErbB receptors transactivation and IRS-1 serine phosphorylation and partially restored insulin-stimulated IRS-1 tyrosine phosphorylation. When cells were incubated with specific ErbB receptors antagonists or in cells lacking ErbB receptors, anisomycin- and TNF-induced IRS-1 serine phosphorylation was attenuated, despite intact p38MAPK activation. The stress-induced p38MAPK activation leading to ErbB receptors transactivation was associated with intracellular reactive oxygen species generation and was attenuated by treatment with antioxidants.. Hepatic p38MAPK is activated following various stress stimuli. This event is upstream to ErbB receptors transactivation and plays an important role in stress-induced IRS-1 serine phosphorylation and insulin resistance.

Topics: Animals; Anisomycin; Cell Line, Tumor; Electrophoresis, Polyacrylamide Gel; Enzyme Inhibitors; Hep G2 Cells; Humans; Imidazoles; Immunoblotting; Immunoprecipitation; Insulin Receptor Substrate Proteins; Insulin Resistance; Male; Mice; Mice, Inbred C57BL; Mice, Obese; Models, Biological; p38 Mitogen-Activated Protein Kinases; Phosphorylation; Pyridines; Rats; Reactive Oxygen Species; Receptor, ErbB-2; Receptor, ErbB-3; Serine; Sphingomyelin Phosphodiesterase; Tumor Necrosis Factors

To investigate the role of c-Jun NH (2)-terminal kinase (JNk) in insulin resistance after burn and its mechanism.. Twenty-four Sprague-Dawley rats were randomized to control, burn and burn + anisomycin groups. The rats in control group received sham burn trauma, and burn and burn + anisomycin groups received 30% total body surface area (TBSA) full thickness burn injury. Anisomycin (5 mg/kg) together with 250 microl dimethyl sulfoxide (DMSO) was injected to the rats in anisomycin group intravenously, and only 250 microl DMSO in the other two groups. Euglycemic-hyperinsulinemic glucose clamps was performed 2 hours after the injection. The changes of phospho-serine 307, phospho-tyrosine of insulin receptor substrate (IRS)-1 and phospho-JNK in muscle tissues were determined and compared using immunoprecipitation and Western blot analysis or immunohistochemistry in the three groups.. The infusing rates of total 10% glucose (mg x kg(-1) x min(-1)) in control, burn and burn + anisomycin group were 12.3 +/- 0.4, 6.6 +/- 0.3, 6.5 +/- 0.4, respectively. The level of IRS-1 Serine 307 phosphorylation and phospho-JNK in muscle increased significantly, while insulin-induced tyrosine phosphorylation of IRS-1 decreased markedly after burn.. The activation of JNK elevates the level of IRS-1 phospho-serine 307 and might play a role in insulin resistance after burn in rats.

Topics: Adaptor Proteins, Signal Transducing; Animals; Anisomycin; Anti-Bacterial Agents; Blotting, Western; Burns; Dimethyl Sulfoxide; Disease Models, Animal; Female; Glucose Clamp Technique; Immunohistochemistry; Injections, Intravenous; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Male; Muscles; Phosphorylation; Random Allocation; Rats; Rats, Sprague-Dawley; Serine; Tyrosine

The cellular pathways involved in the impairment of insulin signaling by cellular stress, triggered by the inflammatory cytokine tumor necrosis factor-alpha (TNF) or by translational inhibitors like cycloheximide and anisomycin were studied. Similar to TNF, cycloheximide and anisomycin stimulated serine phosphorylation of IRS-1 and IRS-2, reduced their ability to interact with the insulin receptor, inhibited the insulin-induced tyrosine phosphorylation of IRS proteins, and diminished their association with phosphatidylinositol 3-kinase (PI3K). These defects were partially reversed by wortmannin and LY294002, indicating that a PI3K-regulated step is critical for the impairment of insulin signaling by cellular stress. Induction of cellular stress resulted in complex formation between PI3K and ErbB2/ErbB3 and enhanced PI3K activity, implicating ErbB proteins as downstream effectors of stress-induced insulin resistance. Indeed, stimulation of ErbB2/ErbB3 by NDFbeta1, the ErbB3 ligand, inhibited IRS protein tyrosine phosphorylation and recruitment of downstream effectors. Specific inhibitors of the ErbB2 tyrosine kinase abrogated the activation of ErbB2/ErbB3 and in parallel prevented the reduction in IRS protein functions. Taken together, our results suggest a novel mechanism by which cellular stress induces cross-talk between two different signaling pathways. Stress-dependent transactivation of ErbB2/ErbB3 receptors triggers a PI3K cascade that induces serine phosphorylation of IRS proteins culminating in insulin resistance.

Topics: Anisomycin; Cycloheximide; Genes, erbB; Genes, erbB-2; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Intracellular Signaling Peptides and Proteins; MAP Kinase Signaling System; Neuregulin-1; Phosphatidylinositol 3-Kinases; Phosphoproteins; Serine; Threonine; Transcriptional Activation; Tumor Necrosis Factor-alpha; Tyrosine

Serine/threonine phosphorylation of IRS-1 might inhibit insulin signaling, but the relevant phosphorylation sites are difficult to identify in cultured cells and to validate in isolated tissues. Recently, we discovered that recombinant NH2-terminal Jun kinase phosphorylates IRS-1 at Ser307, which inhibits insulin-stimulated tyrosine phosphorylation of IRS-1. To monitor phosphorylation of Ser307 in various cell and tissue backgrounds, we prepared a phosphospecific polyclonal antibody designated alphapSer307. This antibody revealed that TNF-alpha, IGF-1, or insulin stimulated phosphorylation of IRS-1 at Ser307 in 3T3-L1 preadipocytes and adipocytes. Insulin injected into mice or rats also stimulated phosphorylation of Ser307 on IRS-1 immunoprecipitated from muscle; moreover, Ser307 was phosphorylated in human muscle during the hyperinsulinemic euglycemic clamp. Experiments in 3T3-L1 preadipocytes and adipocytes revealed that insulin-stimulated phosphorylation of Ser307 was inhibited by LY294002 or wortmannin, whereas TNF-alpha-stimulated phosphorylation was inhibited by PD98059. Thus, distinct kinase pathways might converge at Ser307 to mediate feedback or heterologous inhibition of IRS-1 signaling to counterregulate the insulin response.

Topics: Animals; Anisomycin; CHO Cells; Cricetinae; Insulin; Insulin Antagonists; Insulin Resistance; Insulin-Like Growth Factor I; MAP Kinase Kinase 1; Mitogen-Activated Protein Kinase Kinases; Phosphatidylinositol 3-Kinases; Phosphorylation; Protein Serine-Threonine Kinases; Receptor, Insulin; Serine; Signal Transduction; Tumor Necrosis Factor-alpha; Tyrosine

Tumor necrosis factor alpha (TNFalpha) inhibits insulin action, in part, through serine phosphorylation of IRS proteins; however, the phosphorylation sites that mediate the inhibition are unknown. TNFalpha promotes multipotential signal transduction cascades, including the activation of the Jun NH(2)-terminal kinase (JNK). Endogenous JNK associates with IRS-1 in Chinese hamster ovary cells. Anisomycin, a strong activator of JNK in these cells, stimulates the activity of JNK bound to IRS-1 and inhibits the insulin-stimulated tyrosine phosphorylation of IRS-1. Serine 307 is a major site of JNK phosphorylation in IRS-1. Mutation of serine 307 to alanine eliminates phosphorylation of IRS-1 by JNK and abrogates the inhibitory effect of TNFalpha on insulin-stimulated tyrosine phosphorylation of IRS-1. These results suggest that phosphorylation of serine 307 might mediate, at least partially, the inhibitory effect of proinflammatory cytokines like TNFalpha on IRS-1 function.

Topics: Amino Acid Sequence; Animals; Anisomycin; CHO Cells; Cricetinae; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Mice; Mitogen-Activated Protein Kinases; Molecular Sequence Data; Phosphoproteins; Phosphorylation; Protein Binding; Receptor, Insulin; Recombinant Proteins; Serine; Signal Transduction; Tumor Necrosis Factor-alpha

Tumor necrosis factor-alpha (TNFalpha) has been implicated as a contributing mediator of insulin resistance observed in pathophysiological conditions such as obesity, cancer-induced cachexia, and bacterial infections. Previous studies have demonstrated that TNFalpha confers insulin resistance by promoting phosphorylation of serine residues on insulin receptor substrate 1 (IRS-1), thereby diminishing subsequent insulin-induced tyrosine phosphorylation of IRS-1. However, little is known about which signaling molecules are involved in this process in adipocytes and about the temporal sequence of events that ultimately leads to TNFalpha-stimulated IRS-1 serine phosphorylation. In this study, we demonstrate that specific inhibitors of the MAP kinase kinase (MEK)1/2-p42/44 mitogen-activated protein (MAP) kinase pathway restore insulin signaling to normal levels despite the presence of TNFalpha. Additional experiments show that MEK1/2 activity is required for TNFalpha-induced IRS-1 serine phosphorylation, thereby suggesting a mechanism by which these inhibitors restore insulin signaling. We observe that TNFalpha requires 2.5-4 h to markedly reduce insulin-triggered tyrosine phosphorylation of IRS-1 in 3T3-L1 adipocytes. Although TNFalpha activates p42/44 MAP kinase, maximal stimulation is observed within 10-30 min. To our surprise, p42/44 activity returns to basal levels well before IRS-1 serine phosphorylation and insulin resistance are observed. These activation kinetics suggest a mechanism of p42/44 action more complicated than a direct phosphorylation of IRS-1 triggered by the early spike of TNFalpha-induced p42/44 activity. Chronic TNFalpha treatment (>> 72 h) causes adipocyte dedifferentiation, as evidenced by the loss of triglycerides and down-regulation of adipocyte-specific markers. We observe that this longer term TNFalpha-mediated dedifferentiation effect utilizes alternative, p42/44 MAP kinase-independent intracellular pathways. This study suggests that TNFalpha-mediated insulin resistance, but not adipocyte dedifferentiation, is mediated by the MEK1/2-p42/44 MAP kinase pathway.

Topics: 3T3 Cells; Adipocytes; Animals; Anisomycin; Cell Differentiation; Enzyme Inhibitors; Epidermal Growth Factor; Flavonoids; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Kinetics; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Mitogen-Activated Protein Kinase Kinases; Mitogen-Activated Protein Kinases; Phosphoproteins; Phosphoserine; Phosphotyrosine; Protein Serine-Threonine Kinases; Protein-Tyrosine Kinases; Signal Transduction; Tumor Necrosis Factor-alpha

Deficiency of the G-protein subunit Galphai2 impairs insulin action (Moxham, C. M., and Malbon, C. C. (1996) Nature 379, 840-844). By using the promoter for the phosphoenolpyruvate carboxykinase gene, conditional, tissue-specific expression of the constitutively active mutant form (Q205L) of Galphai2 was achieved in mice harboring the transgene. Expression of Q205L Galphai2 was detected in skeletal muscle, liver, and adipose tissue of transgenic mice. Whereas the Galphai2-deficient mice displayed blunted insulin action, the Q205L Galphai2-expressing mice displayed enhanced insulin-like effects. Glycogen synthase in skeletal muscle was found to be activated in Q205L Galphai2-expressing mice, in the absence of the administration of insulin. Analysis of members of mitogen-activated protein kinase family revealed that both c-Jun N-terminal kinase and p38 are constitutively activated in vivo in the mice that express the Q205L Galphai2. ERK1,2, in contrast, are unaffected in the Q205L Galphai2-expressing mice. Insulin, like expression of Q205L Galphai2, activates both p38 and c-Jun N-terminal kinases as well as glycogen synthase. Activation of c-Jun N-terminal and p38 kinases in vivo with anisomycin, however, was insufficient to activate glycogen synthase. Much like Galphai2 deficiency provokes insulin resistance, expression of Q205L constitutively active Galphai2 mimics insulin action in vivo, sharing with insulin the activation of two mitogen-activated protein kinase members, p38 and c-Jun N-terminal kinases.

Topics: Animals; Anisomycin; Calcium-Calmodulin-Dependent Protein Kinases; Enzyme Activation; Glycogen Synthase; GTP-Binding Protein alpha Subunit, Gi2; GTP-Binding Protein alpha Subunits, Gi-Go; GTP-Binding Proteins; Insulin; Insulin Resistance; JNK Mitogen-Activated Protein Kinases; Mice; Mice, Transgenic; Mitogen-Activated Protein Kinases; Muscle, Skeletal; p38 Mitogen-Activated Protein Kinases; Protein Synthesis Inhibitors; Proto-Oncogene Proteins