title: IRS-2 mediates the antiapoptotic effect of insulin in neonatal hepatocytes.
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.
authors.affiliation: Instituto de Bioquímica/Departamento de Bioquímica y Biología Molecular II, Centro Mixto CSIC/UCM, Facultad de Farmacia, Universidad Complutense, Madrid, Spain. valverde@farm.ucm.es
authors.forename: Angela M
authors.initials: AM
authors.lastname: Valverde
authors.forename: Isabel
authors.initials: I
authors.lastname: Fabregat
authors.forename: Deborah J
authors.initials: DJ
authors.lastname: Burks
authors.forename: Morris F
authors.initials: MF
authors.lastname: White
authors.forename: Manuel
authors.initials: M
authors.lastname: Benito
mesh_terms: D000818:Animals; D000831:Animals, Newborn; D017209:Apoptosis / Q000187:drug effects / Q000502:physiology*; D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D001798:Blood Proteins / Q000494:pharmacology; D002352:Carrier Proteins / Q000235:genetics; D004815:Epidermal Growth Factor / Q000494:pharmacology; D005260:Female; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D015870:Gene Expression; D022781:Hepatocytes / Q000166:cytology / Q000187:drug effects / Q000502:physiology*; D007004:Hypoglycemic Agents / Q000494:pharmacology*; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008297:Male; D008565:Membrane Proteins / Q000235:genetics; D051379:Mice; D008817:Mice, Mutant Strains; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics / Q000378:metabolism*; D011247:Pregnancy; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2 / Q000235:genetics; D015398:Signal Transduction / Q000187:drug effects / Q000502:physiology; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D051020:bcl-X Protein
chemical_list: D051017:Apoptosis Regulatory Proteins; D000072224:Bcl-2-Like Protein 11; D001798:Blood Proteins; D002352:Carrier Proteins; D004815:Epidermal Growth Factor; D000071161:Forkhead Box Protein O1; D051858:Forkhead Transcription Factors; D007004:Hypoglycemic Agents; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D008565:Membrane Proteins; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D017346:Protein Serine-Threonine Kinases; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D019253:Proto-Oncogene Proteins c-bcl-2; D014157:Transcription Factors; D051020:bcl-X Protein; C494812:Bcl2l1 protein, mouse; C000606776:Bcl2l11 protein, mouse; C467200:Foxo1 protein, mouse; C526298:Irs2 protein, mouse