Does NAD(P)H oxidase-derived H2O2 participate in hypotonicity-induced insulin release by activating VRAC in β-cells?

Pflugers Arch. 2012 Feb;463(2):377-90. doi: 10.1007/s00424-011-1047-x. Epub 2011 Nov 18.


NAD(P)H oxidase (NOX)-derived H(2)O(2) was recently proposed to act, in several cells, as the signal mediating the activation of volume-regulated anion channels (VRAC) under a variety of physiological conditions. The present study aims at investigating whether a similar situation prevails in insulin-secreting BRIN-BD11 and rat β-cells. Exogenous H(2)O(2) (100 to 200 μM) at basal glucose concentration (1.1 to 2.8 mM) stimulated insulin secretion. The inhibitor of VRAC, 5-nitro-2-(3-phenylpropylamino)-benzoate (NPPB) inhibited the secretory response to exogenous H(2)O(2). In patch clamp experiments, exogenous H(2)O(2) was observed to stimulate NPPB-sensitive anion channel activity, which induced cell membrane depolarization. Exposure of the BRIN-BD11 cells to a hypotonic medium caused a detectable increase in intracellular level of reactive oxygen species (ROS) that was abolished by diphenyleneiodonium chloride (DPI), a universal NOX inhibitor. NOX inhibitors such as DPI and plumbagin nearly totally inhibited insulin release provoked by exposure of the BRIN-BD11 cells to a hypotonic medium. Preincubation with two other drugs also abolished hypotonicity-induced insulin release and reduced basal insulin output: 1) N-acetyl-L-cysteine (NAC), a glutathione precursor that serves as general antioxidant and 2) betulinic acid a compound that almost totally abolished NOX4 expression. As NPPB, each of these inhibitors (DPI, plumbagin, preincubation with NAC or betulinic acid) strongly reduced the volume regulatory decrease observed following a hypotonic shock, providing an independent proof that VRAC activation is mediated by H(2)O(2). Taken together, these data suggest that NOX-derived H(2)O(2) plays a key role in the insulin secretory response of BRIN-BD11 and native β-cells to extracellular hypotonicity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Acetylcysteine / pharmacology
  • Animals
  • Cells, Cultured
  • Glucose / pharmacology
  • Hydrogen Peroxide / metabolism*
  • Hydrogen Peroxide / pharmacology*
  • Hypotonic Solutions
  • Insulin / metabolism*
  • Insulin-Secreting Cells / cytology
  • Insulin-Secreting Cells / drug effects*
  • Insulin-Secreting Cells / metabolism*
  • Models, Animal
  • NADPH Oxidases / metabolism*
  • Nitrobenzoates / pharmacology
  • Onium Compounds / pharmacology
  • Patch-Clamp Techniques
  • Pentacyclic Triterpenes
  • Rats
  • Rats, Wistar
  • Reactive Oxygen Species / metabolism
  • Triterpenes / pharmacology
  • Voltage-Dependent Anion Channels / metabolism*


  • Hypotonic Solutions
  • Insulin
  • Nitrobenzoates
  • Onium Compounds
  • Pentacyclic Triterpenes
  • Reactive Oxygen Species
  • Triterpenes
  • Voltage-Dependent Anion Channels
  • 5-nitro-2-(3-phenylpropylamino)benzoic acid
  • betulinic acid
  • diphenyleneiodonium
  • Hydrogen Peroxide
  • NADPH Oxidases
  • Glucose
  • Acetylcysteine