h-89 and Insulin-Resistance

Oxidative stress plays an important role in the development of beta-cell dysfunction and insulin resistance, two major pathophysiological abnormalities of type 2 diabetes. Expression levels of antioxidant enzymes in beta cells are very low, rendering them more susceptible to damage caused by reactive oxygen species (ROS). Although the antioxidant effects of glucagon-like peptide-1 (GLP-1) and its analogs have been previously reported, the exact mechanisms involved are still unclear. In this study, we demonstrated that GLP-1 was able to effectively inhibit oxidative stress and cell death of INS-1E beta cells induced by the pro-oxidant tert-butyl hydroperoxide (tert-BOOH). Incubation with GLP-1 enhanced cellular levels of glutathione and the activity of its related enzymes, glutathione-peroxidase (GPx) and -reductase (GR) in beta cells. However, inhibition of ERK, but not of the PI3K/AKT pathway abolished, at least in part, the antioxidant effect of GLP-1. Moreover, ERK activation seems to be protein kinase A (PKA)-dependent because inhibition of PKA with H-89 was sufficient to block the GLP-1-derived protective effect on beta cells. GLP-1 likewise increased the synthesis of GR and favored the translocation of the nuclear transcription factor erythroid 2p45-related factor (Nrf2), a transcription factor implicated in the expression of several antioxidant/detoxificant enzymes. Glucose-stimulated insulin secretion was also preserved in beta-cells challenged with tert-BOOH but pre-treated with GLP-1, probably through the down-regulation of the mitochondrial uncoupling-protein2 (UCP2). Thus, our results provide additional mechanisms of action of GLP-1 to prevent oxidative damage in beta cells through the modulation of signaling pathways involved in antioxidant enzyme regulation.

Topics: Animals; Antioxidants; Cyclic AMP-Dependent Protein Kinases; Diabetes Mellitus, Type 2; Extracellular Signal-Regulated MAP Kinases; Glucagon-Like Peptide 1; Glucose; Glutathione; Glutathione Reductase; Humans; Insulin; Insulin Resistance; Insulin Secretion; Insulin-Secreting Cells; Isoquinolines; NF-E2-Related Factor 2; Oxidative Stress; Rats; Reactive Oxygen Species; Sulfonamides; tert-Butylhydroperoxide; Uncoupling Protein 2

Exendin-4 (Ex-4) is a glucagon-like peptide-1 receptor (GLP-1R) agonist that has been used as a drug injected subcutaneously for treatment of type 2 diabetes. Many studies have revealed molecular targets of Ex-4, but its influence on adipokines has not been determined. Our study showed that Ex-4 induced secretion of adiponectin into the culture medium of 3T3-L1 adipocytes. This effect of Ex-4 is due to increased adiponectin mRNA level through the GLP-1R. Both forskolin and 3-isobutyl-1-methylxanthine (IBMX), which may finally elevate cyclic adenosine monophosphate (cAMP) concentration, prevented the induction of adiponectin expression by Ex-4. Moreover, H89, a protein kinase A inhibitor, blocked the effect of Ex-4 on adiponectin. On the other hand, Ex-4 decreased the mRNA levels of inflammatory adipokines. The results indicate that Ex-4 directly promotes adiponectin secretion via the protein kinase A pathway in 3T3-L1 adipocytes and may ameliorate insulin resistance.

Topics: 1-Methyl-3-isobutylxanthine; 3T3-L1 Cells; Adipocytes; Adipokines; Adiponectin; Animals; Colforsin; Cyclic AMP-Dependent Protein Kinases; Exenatide; Glucagon-Like Peptide 1; Glucagon-Like Peptide-1 Receptor; Inflammation; Insulin Resistance; Isoquinolines; Mice; Peptides; Phosphodiesterase Inhibitors; Protein Kinase Inhibitors; Receptors, Glucagon; RNA, Messenger; Sulfonamides; Venoms

Homocysteine is an intermediate in the sulfur amino acid metabolism. Recent studies suggested that there might be links between hyperhomocysteinemia and insulin resistance. In the present study, we investigated the effect of homocysteine on glucose metabolism. We demonstrated that the levels of insulin were significantly higher in mice with hyperhomocysteinemia than those in the normal mice after administration of glucose. The effect of insulin on glucose output was significantly blocked in the homocysteine-treated hepatocytes. In addition, the expression of phosphoenolpyruvate carboxykinase (PEPCK) gene was elevated in the liver of mice with hyperhomocysteinemia and primary mouse hepatocytes treated with homocysteine. The action of homocysteine was suppressed by H89, a protein kinase A (PKA) inhibitor. Thus, hyperhomocysteinemia may be considered as a risk factor that contributes to the development of insulin resistance with respect to elevated glucose output and upregulation of PEPCK, probably via the PKA pathway. Our study provides a novel mechanistic explanation for the development of insulin resistance in hyperhomocysteinemia.

Topics: Animals; Blotting, Western; Cyclic AMP-Dependent Protein Kinases; Gene Expression Regulation, Enzymologic; Glucose; Hepatocytes; Homocysteine; Hyperhomocysteinemia; Insulin Resistance; Isoquinolines; Mice; Mice, Inbred BALB C; Phosphoenolpyruvate Carboxykinase (ATP); Protein Kinase Inhibitors; Reverse Transcriptase Polymerase Chain Reaction; Sulfonamides

Cyclic nucleotide phosphodiesterase (PDE) 3B plays an important role in the antilipolytic action of insulin and, thereby, the release of fatty acids from adipocytes. Increased concentrations of circulating fatty acids as a result of elevated or unrestrained lipolysis cause insulin resistance. The lipolytic action of tumor necrosis factor (TNF)-alpha is thought to be one of the mechanisms by which TNF-alpha induces insulin resistance. Ceramide is the suggested second messenger of TNF-alpha action, and in this study, we used 3T3-L1 adipocytes to investigate the effects of C(2)-ceramide (a short-chain ceramide analog) on the expression and regulation of PDE3B and lipolysis. Incubation of adipocytes with 100 micromol/l C(2)-ceramide (N-acetyl-sphingosine) resulted in a time-dependent decrease of PDE3B activity, accompanied by decreased PDE3B protein expression. C(2)-ceramide, in a time- and dose-dependent manner, stimulated lipolysis, an effect that was blocked by H-89, an inhibitor of protein kinase A. These ceramide effects were prevented by 20 micromol/l troglitazone, an antidiabetic drug. In addition to downregulation of PDE3B, the antilipolytic action of insulin was decreased by ceramide treatment. These results, together with data from other studies on PDE3B and lipolysis in diabetic humans and animals, suggest a novel pathway by which ceramide induces insulin resistance. Furthermore, PDE3B is demonstrated to be a target for troglitazone action in adipocytes.

Topics: 3',5'-Cyclic-AMP Phosphodiesterases; 3T3 Cells; Adipocytes; Animals; Blotting, Western; Chromans; Cyclic AMP-Dependent Protein Kinases; Cyclic Nucleotide Phosphodiesterases, Type 3; Drug Interactions; Enzyme Inhibitors; Insulin; Insulin Resistance; Isoquinolines; Lipolysis; Mice; Receptors, Cytoplasmic and Nuclear; Sphingosine; Sulfonamides; Thiazoles; Thiazolidinediones; Transcription Factors; Troglitazone; Tumor Necrosis Factor-alpha