u-0126 and Insulin-Resistance

Iron overload is frequently observed in type 2 diabetes mellitus (DM2), but the underlying mechanisms remain unclear. We hypothesize that hepcidin may be directly regulated by insulin and play an important role in iron overload in DM2. We therefore examined the hepatic iron content, serum iron parameters, intestinal iron absorption, and liver hepcidin expression in rats treated with streptozotocin (STZ), which was given alone or after insulin resistance induced by a high-fat diet. The direct effect of insulin on hepcidin and its molecular mechanisms were furthermore determined in vitro in HepG2 cells. STZ administration caused a significant reduction in liver hepcidin level and a marked increase in intestinal iron absorption and serum and hepatic iron content. Insulin obviously upregulated hepcidin expression in HepG2 cells and enhanced signal transducer and activator of transcription 3 protein synthesis and DNA binding activity. The effect of insulin on hepcidin disappeared when the signal transducer and activator of transcription 3 pathway was blocked and could be partially inhibited by U0126. In conclusion, the current study suggests that hepcidin can be directly regulated by insulin, and the suppressed liver hepcidin synthesis may be an important reason for the iron overload in DM2.

Topics: Animals; Butadienes; Diabetes Mellitus, Experimental; Enzyme Inhibitors; Hepcidins; Insulin; Insulin Resistance; Iron; Iron Overload; Liver; Male; Nitriles; Rats; Rats, Sprague-Dawley; Signal Transduction; STAT3 Transcription Factor

Skeletal muscle insulin resistance in type 2 diabetes is associated with a shift from oxidative to glycolytic metabolism in myofibers. However, whether this metabolic switch is detrimental or adaptive for metabolic homeostasis has not been resolved. We recently demonstrated that the Baf60c/Deptor pathway promotes glycolytic metabolism in the muscle and protects mice from diet-induced insulin resistance. However, the nature of the signals that impinge on this pathway and the role of Baf60c in glucose homeostasis in the severe insulin-resistant state remain unknown. Here we show that expression of Baf60c and Deptor was downregulated in skeletal muscle in obesity, accompanied by extracellular signal-related kinase (ERK) activation. In cultured myotubes, inhibition of ERK, but not Jun NH2-terminal kinase and IκB kinase, blocked the downregulation of Baf60c and Deptor by the proinflammatory cytokine tumor necrosis factor-α. Treatment of obese mice with the ERK inhibitor U0126 rescued Baf60c and Deptor expression in skeletal muscle and lowered blood glucose. Transgenic rescue of Baf60c in skeletal muscle restored Deptor expression and Akt phosphorylation and ameliorated insulin resistance in ob/ob mice. This study identifies the Baf60c/Deptor pathway as a target of proinflammatory signaling in skeletal muscle that may link meta-inflammation to skeletal myofiber metabolism and insulin resistance.

Topics: Animals; Butadienes; Chromosomal Proteins, Non-Histone; Down-Regulation; Enzyme Inhibitors; Glucose; Glucose Tolerance Test; Homeostasis; I-kappa B Kinase; Inflammation; Insulin; Insulin Resistance; Intracellular Signaling Peptides and Proteins; Mice; Muscle Fibers, Skeletal; Muscle Proteins; Muscle, Skeletal; Nitriles; Obesity; Signal Transduction; Tumor Necrosis Factor-alpha

Lipid and protein tyrosine phosphatase, phosphatase and tension homologue (PTEN), is a widely known negative regulator of insulin/phosphoinositide 3-kinase signaling. Down-regulation of PTEN is thus widely documented to ameliorate insulin resistance in peripheral tissues such as skeletal muscle and adipose. However, not much is known about its exact role in neuronal insulin signaling and insulin resistance. Moreover, alterations of PTEN in neuronal systems have led to discovery of several unexpected outcomes, including in the neurodegenerative disorder Alzheimer's disease (AD), which is increasingly being recognized as a brain-specific form of diabetes. In addition, contrary to expectations, its neuron-specific deletion in mice resulted in development of diet-sensitive obesity. The present study shows that PTEN, paradoxically, positively regulates neuronal insulin signaling and glucose uptake. Its down-regulation exacerbates neuronal insulin resistance. The positive role of PTEN in neuronal insulin signaling is likely due to its protein phosphatase actions, which prevents the activation of focal adhesion kinase (FAK) and extracellular signal-regulated kinase (ERK), the kinases critically involved in neuronal energy impairment and neurodegeneration. Results suggest that PTEN acting through FAK, the direct protein substrate of PTEN, prevents ERK activation. Our findings provide an explanation for unexpected outcomes reported earlier with PTEN alterations in neuronal systems and also suggest a novel molecular pathway linking neuronal insulin resistance and AD, the two pathophysiological states demonstrated to be closely linked.

Topics: Alzheimer Disease; Amyloid beta-Protein Precursor; Animals; Butadienes; Cell Line, Tumor; Deoxyglucose; Enzyme Activation; Extracellular Signal-Regulated MAP Kinases; Focal Adhesion Kinase 1; Gene Knockdown Techniques; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Inositol Polyphosphate 5-Phosphatases; Insulin; Insulin Resistance; MAP Kinase Signaling System; Mice; Neurons; Nitriles; Phosphatidylinositol 3-Kinases; Phosphatidylinositols; Phosphoric Monoester Hydrolases; Phosphorylation; Primary Cell Culture; Protein Processing, Post-Translational; Proto-Oncogene Proteins c-akt; PTEN Phosphohydrolase; RNA Interference

Plasma C-reactive protein (CRP) concentration is increased in the metabolic syndrome, which consists of a cluster of cardiovascular disease risk factors, including insulin resistance. It is not known, however, whether CRP is merely a marker of accompanying inflammation or whether it contributes causally to insulin resistance. The objective of this study is to investigate the role that CRP may play in the development of insulin resistance. We examined the effect of single-dose intravenous administration of purified human (h)CRP on insulin sensitivity in Sprague-Dawley rats using the euglycemic, hyperinsulinemic clamp technique. hCRP was associated with impaired insulin suppression of endogenous glucose production with no reduction in peripheral tissue glucose uptake, suggesting that hCRP mediated insulin resistance in the liver but not extrahepatic tissues. We further assessed components of the insulin signaling pathway and mitogen-activated protein kinases (MAPKs) in the liver. Liver tissues derived from hCRP-treated rats showed reduced insulin-stimulated insulin receptor substrate (IRS) tyrosine phosphorylation, IRS/phosphatidylinositol 3-kinase (PI3K) association, and Akt phosphorylation, consistent with hCRP-induced impairment of hepatic insulin signaling. Furthermore, hCRP enhanced phosphorylation of extracellular signal-regulated kinase (ERK)1/2 and p38 MAPK as well as IRS-1 Ser(612) . Finally, we observed in primary cultured rat hepatocytes that U0126 (a selective inhibitor of MAPK/ERK kinase1/2) corrected hCRP-induced impairment of insulin signaling.. hCRP plays an active role in inducing hepatic insulin resistance in the rat, at least in part by activating ERK1/2, with downstream impairment in the insulin signaling pathway.

Topics: Adiponectin; Animals; Butadienes; C-Reactive Protein; Glucose Clamp Technique; Humans; Imidazoles; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Interleukin-6; Leptin; Liver; Male; Mitogen-Activated Protein Kinases; Nitriles; Phosphorylation; Pyridines; Rats; Rats, Sprague-Dawley; Signal Transduction; Tumor Necrosis Factor-alpha

Patients with diabetes are at higher risk of stroke and experience increased morbidity and mortality after stroke. We hypothesized that cortical neurons develop insulin resistance, which decreases neuroprotection via circulating insulin and insulin-like growth factor-I (IGF-I). Acute insulin treatment of primary embryonic cortical neurons activated insulin signaling including phosphorylation of the insulin receptor, extracellular signal-regulated kinase (ERK), Akt, p70S6K, and glycogen synthase kinase-3β (GSK-3β). To mimic insulin resistance, cortical neurons were chronically treated with 25 mM glucose, 0.2 mM palmitic acid (PA), or 20 nM insulin before acute exposure to 20 nM insulin. Cortical neurons pretreated with insulin, but not glucose or PA, exhibited blunted phosphorylation of Akt, p70S6K, and GSK-3β with no change detected in ERK. Inhibition of the phosphatidylinositol 3-kinase (PI3-K) pathway during insulin pretreatment restored acute insulin-mediated Akt phosphorylation. Cortical neurons in adult BKS-db/db mice exhibited higher basal Akt phosphorylation than BKS-db(+) mice and did not respond to insulin. Our results indicate that prolonged hyperinsulinemia leads to insulin resistance in cortical neurons. Decreased sensitivity to neuroprotective ligands may explain the increased neuronal damage reported in both experimental models of diabetes and diabetic patients after ischemia-reperfusion injury.

Topics: Animals; Blotting, Western; Butadienes; Cells, Cultured; Cerebral Cortex; Chromones; Enzyme Inhibitors; Female; Glycogen Synthase Kinase 3; Glycogen Synthase Kinase 3 beta; Immunohistochemistry; Insulin; Insulin Resistance; Insulin-Like Growth Factor I; Morpholines; Neurons; Nitriles; Phosphorylation; Pregnancy; Proto-Oncogene Proteins c-akt; Rats; Rats, Sprague-Dawley; Ribosomal Protein S6 Kinases, 70-kDa; Signal Transduction

Increased plasma concentrations of apolipoprotein B100 often present in patients with insulin resistance and confer increased risk for the development of atherosclerosis. Naturally occurring polyphenolic compounds including flavonoids have antiatherogenic properties. The aim of the current study was to evaluate the effect of the polymethoxylated flavonoid nobiletin on lipoprotein secretion in cultured human hepatoma cells (HepG2) and in a mouse model of insulin resistance and atherosclerosis.. Lipoprotein secretion was determined in HepG2 cells incubated with nobiletin or insulin. mRNA abundance was evaluated by quantitative real-time PCR, and Western blotting was used to demonstrate activation of cell signaling pathways. In LDL receptor-deficient mice (Ldlr(-/-)) fed a Western diet supplemented with nobiletin, metabolic parameters, gene expression, fatty acid oxidation, glucose homeostasis, and energy expenditure were documented. Atherosclerosis was quantitated by histological analysis.. In HepG2 cells, activation of mitogen-activated protein kinase-extracellular signal-related kinase signaling by nobiletin or insulin increased LDLR and decreased MTP and DGAT1/2 mRNA, resulting in marked inhibition of apoB100 secretion. Nobiletin, unlike insulin, did not induce phosphorylation of the insulin receptor or insulin receptor substrate-1 and did not stimulate lipogenesis. In fat-fed Ldlr(-/-) mice, nobiletin attenuated dyslipidemia through a reduction in VLDL-triglyceride (TG) secretion. Nobiletin prevented hepatic TG accumulation, increased expression of Pgc1α and Cpt1α, and enhanced fatty acid β-oxidation. Nobiletin did not activate any peroxisome proliferator-activated receptor (PPAR), indicating that the metabolic effects were PPAR independent. Nobiletin increased hepatic and peripheral insulin sensitivity and glucose tolerance and dramatically attenuated atherosclerosis in the aortic sinus.. Nobiletin provides insight into treatments for dyslipidemia and atherosclerosis associated with insulin-resistant states.

Topics: Animals; Atherosclerosis; Butadienes; Diet; Dyslipidemias; Electrophoresis, Polyacrylamide Gel; Energy Metabolism; Enzyme Inhibitors; Flavones; Hep G2 Cells; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Lipoproteins, VLDL; Male; MAP Kinase Kinase 1; MAP Kinase Kinase 2; Mice; Mice, Mutant Strains; Nitriles; Phosphorylation; Receptor, Insulin; Reverse Transcriptase Polymerase Chain Reaction; Signal Transduction; Triglycerides

Insulin resistance and hyperinsulinemia are major causes of cardiovascular morbidity and mortality. Matrix metalloproteinases (MMPs), highly expressed in activated mononuclear cells in vulnerable atherosclerotic lesions, are the main proteolytic enzymes controlling plaque stability. The aim of this study was to investigate the regulation of monocyte MMP-9 by insulin.. Stimulation of MMP-9 expression by insulin was time- and concentration-dependent in human monocytic THP-1 cells. Inhibition of insulin receptor (IR) maturation via inhibition of its activating convertase furin with the pharmacological furin-inhibitor decanoyl-RVKR-chloromethylketone, as well as blocking of IGF-1R function with a IGF-1R blocking antibody, demonstrated that insulin mediates increases in MMP-9 via IR activation. Inhibition of insulin's "metabolic" phosphatidylinositol 3-kinase signaling with wortmannin (50 nmol/L) or LY294002 (2.5 micromol/L) did not prevent insulin-dependent MMP-9 induction. In contrast inhibition of insulin's "mitogenic" Ras-Raf-mitogen-activated protein kinase-kinase pathways with PD98059 (15 micromol/L) or U0126 (2 micromol/L) inhibited insulin-induced MMP-9 gelatinolytic activity in THP-1 cells. Likewise, PD98059 inhibited insulin augmented MMP-9 levels in primary human monocytes, whereas wortmannin had no effect.. This study demonstrates that insulin can induce MMP-9 via mitogenic signaling pathways in monocytes, whereas phosphatidylinositol 3-kinase-dependent signaling, typically altered in insulin resistance, is not required. Induction of MMP-9 by insulin may potentially contribute to a pro-inflammatory state and the increased cardiovascular morbidity and mortality in type 2 diabetics.

Topics: Androstadienes; Antibodies, Blocking; Butadienes; Calcium-Calmodulin-Dependent Protein Kinases; Cell Line, Tumor; Dose-Response Relationship, Drug; Enzyme Activation; Flavonoids; Humans; Hypoglycemic Agents; Insulin; Insulin Resistance; Matrix Metalloproteinase 9; Mitogen-Activated Protein Kinase Kinases; Monocytes; Nitriles; Phospholipases A; Receptor, Insulin; Stimulation, Chemical; Time Factors; Wortmannin

Increase in the expression of leukocyte antigen-related (LAR) protein causes insulin resistance, an important contributor to atherosclerosis. However, the function of LAR in atherosclerosis is not known. To address whether LAR is important in the response of vascular cells to atherogenic stimuli, we investigated cell proliferation, migration, and insulin-like growth factor-1 receptor (IGF-1R) signaling in wild-type and LAR(-/-) mouse vascular smooth muscle cells (VSMC) treated with IGF-1. Absence of LAR significantly enhanced proliferation and migration of VSMC compared with wild-type cells after IGF-1 treatment. U0126 and LY249002, specific inhibitors of MAPK/ERK kinase (MEK) and phosphoinositide 3-kinase, respectively, inhibited IGF-1-induced DNA synthesis and migration in both wild-type and LAR(-/-) VSMC. IGF-1 markedly enhanced IGF-1R phosphorylation in both wild-type and LAR(-/-) VSMC, but the phosphorylation was 90% higher in knock-out cells compared with wild-type cells. Absence of LAR enhanced phosphorylation of insulin receptor substrate-1 and insulin receptor substrate-1-associated phosphoinositide 3-kinase activity in VSMC treated with IGF-1. IGF-1-induced phosphorylation of ERK1/2 also increased significantly in LAR(-/-) VSMC compared with wild-type cells. Furthermore, LAR directly binds to IGF-1R in glutathione S-transferase-LAR pull-down and IGF-1R immunoprecipitation experiments and recombinant LAR dephosphorylates IGF-1R in vitro. Neointima formation in response to arterial injury and IGF-1R phosphorylation in neointima increased significantly in LAR(-/-) mice compared with wild-type mice. A significant decrease in body weight, fasting insulin, and IGF-1 levels were observed in LAR(-/-) mice compared with wild-type mice. Together, these data indicate that LAR regulates IGF-1R signaling in VSMC and dysregulation of this phosphatase may lead to VSMC hyperplasia.

Topics: Animals; Arteries; Atherosclerosis; Butadienes; Cell Movement; Cell Proliferation; Cells, Cultured; Enzyme Inhibitors; Humans; Hyperplasia; Insulin Resistance; Insulin-Like Growth Factor I; Male; MAP Kinase Kinase Kinases; MAP Kinase Signaling System; Mice; Mice, Knockout; Nitriles; Phosphatidylinositol 3-Kinases; Phosphoinositide-3 Kinase Inhibitors; Phosphorylation; Protein Binding; Protein Processing, Post-Translational; Protein Tyrosine Phosphatase, Non-Receptor Type 1; Protein Tyrosine Phosphatases; Receptor-Like Protein Tyrosine Phosphatases, Class 4; Receptor, IGF Type 1; Receptors, Cell Surface; Tunica Media

Insulin resistance in polycystic ovary syndrome (PCOS) results from a postbinding defect in signaling. Insulin receptor and insulin receptor substrate (IRS)-1 serine hyperphosphorylation by an unidentified kinase(s) contributes to this defect. We investigated whether insulin resistance is selective, affecting metabolic but not mitogenic pathways, in skeletal muscle as it is in cultured skin fibroblasts in PCOS. Extracellular signal-regulated kinase (ERK)1/2 activation was increased in skeletal muscle tissue and in cultured myotubes basally and in response to insulin in women with PCOS compared with control women. Mitogen-activated/extracellular signal-regulated kinase kinase (MEK)1/2 was also activated in PCOS, whereas p38 mitogen-activated protein kinase phosphorylation and signaling from the insulin receptor to Grb2 was similar in both groups. The activity of p21Ras was decreased and Raf-1 abundance increased in PCOS, suggesting that altered mitogenic signaling began at this level. MEK1/2 inhibition reduced IRS-1 Ser312 phosphorylation and increased IRS-1 association with the p85 subunit of phosphatidylinositol 3-kinase in both groups. We conclude that in PCOS skeletal muscle, 1) mitogenic signaling is enhanced in vivo and in culture, 2) ERK1/2 activation inhibits association of IRS-1 with p85 via IRS-1 Ser312 phosphorylation, and 3) ERK1/2 activation may play a role in normal feedback of insulin signaling and contribute to resistance to insulin's metabolic actions in PCOS.

Topics: Adult; Butadienes; Cells, Cultured; Extracellular Signal-Regulated MAP Kinases; Female; Humans; Insulin Receptor Substrate Proteins; Insulin Resistance; MAP Kinase Signaling System; Muscle, Skeletal; Nitriles; p38 Mitogen-Activated Protein Kinases; Phosphoproteins; Phosphorylation; Polycystic Ovary Syndrome; Proto-Oncogene Proteins c-raf; Proto-Oncogene Proteins p21(ras)

We previously demonstrated that trans-10, cis-12 conjugated linoleic acid (CLA) reduced the triglyceride content of human adipocytes by activating mitogen-activated protein kinase kinase/extracellular signal-related kinase (MEK/ERK) signaling via interleukins (IL) 6 and 8. However, the upstream mechanism is unknown. Here we show that CLA increased (>or=6 h) the secretion of IL-6 and IL-8 in cultures containing both differentiated adipocytes and stromal vascular (SV) cells, non-differentiated SV cells, and adipose tissue explants. CLA isomer-specific induction of IL-6 and tumor necrosis factor-alpha was associated with the activation of nuclear factor kappaB (NFkappaB) as evidenced by 1) phosphorylation of IkappaBalpha, IkappaBalpha kinase, and NFkappaB p65, 2) IkappaBalpha degradation, and 3) nuclear translocation of NFkappaB. Pretreatment with selective NFkappaB inhibitors and the MEK/ERK inhibitor U0126 blocked CLA-mediated IL-6 gene expression. Trans-10, cis-12 CLA suppression of insulin-stimulated glucose uptake at 24 h was associated with decreased total and plasma membrane glucose transporter 4 proteins. Inhibition of NFkappaB activation or depletion of NFkappaB by RNA interference using small interfering NFkappaB p65 attenuated CLA suppression of glucose transporter 4 and peroxisome proliferator-activated receptor gamma proteins and glucose uptake. Collectively, these data demonstrate for the first time that trans-10, cis-12 CLA promotes NFkappaB activation and subsequent induction of IL-6, which are at least in part responsible for trans-10, cis-12 CLA-mediated suppression of peroxisome proliferator-activated receptor gamma target gene expression and insulin sensitivity in mature human adipocytes.

Topics: Active Transport, Cell Nucleus; Adipocytes; Butadienes; Cell Differentiation; Cell Membrane; Cell Nucleus; Cells, Cultured; Cytokines; Cytoplasm; Deoxyglucose; Enzyme Inhibitors; Enzyme-Linked Immunosorbent Assay; Fatty Acids; Glucose; Glucose Transporter Type 4; Humans; Insulin; Insulin Receptor Substrate Proteins; Insulin Resistance; Interleukin-6; Interleukin-8; Linoleic Acids, Conjugated; MAP Kinase Kinase Kinases; Microscopy, Fluorescence; Microscopy, Phase-Contrast; Mitogen-Activated Protein Kinase 1; Mitogen-Activated Protein Kinase 3; Models, Biological; NF-kappa B; Nitriles; Phosphoproteins; Phosphorylation; PPAR gamma; Reverse Transcriptase Polymerase Chain Reaction; RNA Interference; RNA, Small Interfering; Time Factors; Transcription Factor RelA; Transfection; Triglycerides; Tumor Necrosis Factor-alpha