Phagocyte-like NADPH oxidase generates ROS in INS 832/13 cells and rat islets: role of protein prenylation

Am J Physiol Regul Integr Comp Physiol. 2011 Mar;300(3):R756-62. doi: 10.1152/ajpregu.00786.2010. Epub 2011 Jan 12.

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Cell Line
  • Enzyme Inhibitors / pharmacology
  • GTP-Binding Proteins / metabolism*
  • Glucose / metabolism
  • Guanosine Triphosphate / metabolism
  • Insulin / metabolism
  • Insulin-Secreting Cells / drug effects
  • Insulin-Secreting Cells / enzymology*
  • Islets of Langerhans / cytology
  • Islets of Langerhans / drug effects
  • Islets of Langerhans / enzymology*
  • Keto Acids / metabolism
  • Mitochondria / metabolism
  • NADPH Oxidases / antagonists & inhibitors
  • NADPH Oxidases / genetics
  • NADPH Oxidases / metabolism*
  • Oxidative Stress*
  • Pertussis Toxin / pharmacology
  • Protein Prenylation
  • RNA Interference
  • Rats
  • Rats, Sprague-Dawley
  • Reactive Oxygen Species / metabolism*
  • Signal Transduction
  • Succinates / metabolism
  • Tissue Culture Techniques
  • rac1 GTP-Binding Protein / metabolism

Substances

  • Enzyme Inhibitors
  • Insulin
  • Keto Acids
  • Reactive Oxygen Species
  • Succinates
  • alpha-ketoisocaproic acid
  • Guanosine Triphosphate
  • NADPH Oxidases
  • neutrophil cytosolic factor 1
  • Pertussis Toxin
  • GTP-Binding Proteins
  • Rac1 protein, rat
  • rac1 GTP-Binding Protein
  • Glucose
  • monomethyl succinate