title: Phagocyte-like NADPH oxidase generates ROS in INS 832/13 cells and rat islets: role of protein prenylation.
abstract: Recent evidence suggests that an acute increase in the generation of phagocyte-like NADPH-oxidase (Nox)-mediated reactive oxygen species (ROS) may be necessary for glucose-stimulated insulin secretion. Using rat islets and INS 832/13 cells, we tested the hypothesis that activation of specific G proteins is necessary for nutrient-mediated intracellular generation of ROS. Stimulation of β-cells with glucose or a mixture of mitochondrial fuels (mono-methylsuccinate plus α-ketoisocaproic acid) markedly elevated intracellular accumulation of ROS, which was attenuated by selective inhibitors of Nox (e.g., apocynin or diphenyleneiodonium chloride) or short interfering RNA-mediated knockdown of p47(phox), one of the subunits of Nox. Selective inhibitors of protein prenylation (FTI-277 or GGTI-2147) markedly inhibited nutrient-induced ROS generation, suggesting that activation of one (or more) prenylated small G proteins and/or γ-subunits of trimeric G proteins is involved in this signaling axis. Depletion of endogenous GTP levels with mycophenolic acid significantly reduced glucose-induced activation of Rac1 and ROS generation in these cells. Other immunosuppressants, like cyclosporine A or rapamycin, which do not deplete endogenous GTP levels, failed to affect glucose-induced ROS generation, suggesting that endogenous GTP is necessary for glucose-induced Nox activation and ROS generation. Treatment of INS 832/13 cells or rat islets with pertussis toxin (Ptx), which ADP ribosylates and inhibits inhibitory class of trimeric G proteins (i.e., G(i) or G(o)), significantly attenuated glucose-induced ROS generation in these cells, implicating activation of a Ptx-sensitive G protein in these signaling cascade. Together, our findings suggest a prenylated Ptx-sensitive signaling step couples Rac1 activation in the signaling steps necessary for glucose-mediated generation of ROS in the pancreatic β-cells.
authors.affiliation: Dept. of Pharmaceutical Sciences, Eugene Applebaum College of Pharmacy and Health Sciences, Wayne State Univ., Detroit, MI 48201, USA.
authors.forename: Ismail
authors.initials: I
authors.lastname: Syed
authors.forename: Chandrashekara N
authors.initials: CN
authors.lastname: Kyathanahalli
authors.forename: Anjaneyulu
authors.initials: A
authors.lastname: Kowluru
mesh_terms: D000818:Animals; D002460:Cell Line; D004791:Enzyme Inhibitors / Q000494:pharmacology; D019204:GTP-Binding Proteins / Q000378:metabolism*; D005947:Glucose / Q000378:metabolism; D006160:Guanosine Triphosphate / Q000378:metabolism; D007328:Insulin / Q000378:metabolism; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000201:enzymology*; D007515:Islets of Langerhans / Q000166:cytology / Q000187:drug effects / Q000201:enzymology*; D007651:Keto Acids / Q000378:metabolism; D008928:Mitochondria / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D018384:Oxidative Stress; D037342:Pertussis Toxin / Q000494:pharmacology; D017368:Protein Prenylation; D034622:RNA Interference; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D015398:Signal Transduction; D013386:Succinates / Q000378:metabolism; D046509:Tissue Culture Techniques; D020830:rac1 GTP-Binding Protein / Q000378:metabolism
chemical_list: D004791:Enzyme Inhibitors; D007328:Insulin; D007651:Keto Acids; D017382:Reactive Oxygen Species; D013386:Succinates; C013082:alpha-ketoisocaproic acid; D006160:Guanosine Triphosphate; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; D037342:Pertussis Toxin; D019204:GTP-Binding Proteins; C499134:Rac1 protein, rat; D020830:rac1 GTP-Binding Protein; D005947:Glucose; C041105:monomethyl succinate