IRS-2 mediates the antiapoptotic effect of insulin in neonatal hepatocytes

Hepatology. 2004 Dec;40(6):1285-94. doi: 10.1002/hep.20485.

Abstract

To assess the role of insulin action and inaction in the liver, immortalized hepatocyte cell lines have been generated from insulin receptor substrate (IRS)-2(-/-) and wild-type mice. Using this model, we have recently demonstrated that the lack of IRS-2 in neonatal hepatocytes resulted in insulin resistance. In the current study, we show that immortalized neonatal hepatocytes undergo apoptosis on serum withdrawal, with caspase-3 activation and DNA laddering occurring earlier in the absence of IRS-2. Insulin rescued wild-type hepatocytes from serum withdrawal-induced caspase-3 activation and DNA fragmentation in a dose-dependent manner, but it failed to rescue hepatocytes lacking IRS-2. In IRS-2(-/-) cells, insulin failed to phosphorylate Bad. Furthermore, in these cells, insulin was unable to translocate Foxo1 from the nucleus to the cytosol. Adenoviral infection of wild-type cells with constitutively active Foxo1 (ADA) induced caspase-8 and caspase-3 activities, proapoptotic gene expression, DNA laddering and apoptosis. Dominant negative Foxo1 regulated the whole pathway in an opposite manner. Prolonged insulin treatment (24 hours) increased expression of antiapoptotic genes (Bcl-xL), downregulated proapoptotic genes (Bim and nuclear Foxo1), and decreased caspase-3 activity in wild-type hepatocytes but not in IRS-2(-/-) cells. Infection of IRS-2(-/-) hepatocytes with adenovirus encoding IRS-2 reconstituted phosphatidylinositol 3-kinase (PI 3-kinase)/Akt/Foxo1 signaling, restored pro- and antiapoptotic gene expression, and decreased caspase-3 activity in response to insulin, thereby blocking apoptosis. In conclusion, IRS-2 signaling is specifically required through PIP3 generation to mediate the survival effects of insulin. Epidermal growth factor, via PIP3/Akt/Foxo1 phosphorylation, was able to rescue IRS-2(-/-) hepatocytes from serum withdrawal-induced apoptosis, modulating pro- and anti-apoptotic gene expression and downregulating caspase-3 activity.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Animals, Newborn
  • Apoptosis / drug effects
  • Apoptosis / physiology*
  • Apoptosis Regulatory Proteins
  • Bcl-2-Like Protein 11
  • Blood Proteins / pharmacology
  • Carrier Proteins / genetics
  • Epidermal Growth Factor / pharmacology
  • Female
  • Forkhead Box Protein O1
  • Forkhead Transcription Factors
  • Gene Expression
  • Hepatocytes / cytology
  • Hepatocytes / drug effects
  • Hepatocytes / physiology*
  • Hypoglycemic Agents / pharmacology*
  • Insulin / pharmacology*
  • Insulin Receptor Substrate Proteins
  • Intracellular Signaling Peptides and Proteins
  • Male
  • Membrane Proteins / genetics
  • Mice
  • Mice, Mutant Strains
  • Phosphatidylinositol 3-Kinases / metabolism
  • Phosphoproteins / genetics
  • Phosphoproteins / metabolism*
  • Pregnancy
  • Protein-Serine-Threonine Kinases / metabolism
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / metabolism
  • Proto-Oncogene Proteins c-akt
  • Proto-Oncogene Proteins c-bcl-2 / genetics
  • Signal Transduction / drug effects
  • Signal Transduction / physiology
  • Transcription Factors / genetics
  • Transcription Factors / metabolism
  • bcl-X Protein

Substances

  • Apoptosis Regulatory Proteins
  • Bcl-2-Like Protein 11
  • Bcl2l1 protein, mouse
  • Bcl2l11 protein, mouse
  • Blood Proteins
  • Carrier Proteins
  • Forkhead Box Protein O1
  • Forkhead Transcription Factors
  • Foxo1 protein, mouse
  • Hypoglycemic Agents
  • Insulin
  • Insulin Receptor Substrate Proteins
  • Intracellular Signaling Peptides and Proteins
  • Irs2 protein, mouse
  • Membrane Proteins
  • Phosphoproteins
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins c-bcl-2
  • Transcription Factors
  • bcl-X Protein
  • Epidermal Growth Factor
  • Phosphatidylinositol 3-Kinases
  • Protein-Serine-Threonine Kinases
  • Proto-Oncogene Proteins c-akt