title: Insulin receptor substrate-4 signaling in quiescent rat hepatocytes and in regenerating rat liver.
abstract: This study was designed to characterize insulin receptor substrate-4 (IRS-4) in isolated rat hepatocytes and to examine its role in liver regeneration. Subcellular fractionation revealed that 85% of IRS-4 is located at isolated hepatocyte plasma membranes. The distribution of IRS-4 among intracellular compartments remained unchanged in insulin-stimulated cells. Two bands corresponding to 145 and 138 kd were observed in immunoblotting experiments. Immunoprecipitation of hepatocyte lysates with a highly specific antibody against IRS-4 led to an insulin and insulin-like growth factor 1 (IGF-1)-dependent increase in phosphotyrosine residues of the 145-kd band. IRS-4 was found to be associated with Src homology 2 (SH2) domain-containing proteins (phosphatidylinositol 3-kinase [PI 3-kinase] and Src homology phosphatase [SHP-2]) and with protein kinase C zeta (PKC zeta). Insulin and IGF-1 elicited a rapid and dose-dependent binding of these 3 proteins to IRS-4. These data suggest that IRS-4 is insulin-/IGF-1-activated by phosphorylation and not by translocation, inducing the recruitment of SH2 domain-containing proteins and PKC zeta to the membrane. To evaluate the possible role of IRS-4 in liver regeneration, we also examined this system after partial hepatectomy (PH). One day after PH, IRS-1 expression increased, consistent with a stimulatory role in the regenerative process, whereas it decreased 7 days after liver resection. This drastic IRS-1 depletion occurred at the expense of increased IRS-2 and IRS-4 expression 7 days after PH. In addition, at this period of time after surgery, the in vivo insulin stimulation of remnant rat livers showed an increase in IRS-4/PI 3-kinase association. Given that 1 and 7 days after PH isolated hepatocytes responded similarly to insulin in terms of induced cell proliferation, a compensatory role is proposed for IRS-2/4 induction. In conclusion, IRS-4 is activated by insulin and IGF-1-like IRS-1 in rat hepatocytes, and the induced expression of IRS-4 is a compensatory mechanism that plays a role in conditions of liver regeneration.
authors.affiliation: Unidad de Toxicología Molecular Hepática, Departamento de Bioquímica y Biología Molecular, Universidad de Alcalá, Alcalá de Henares, Spain.
authors.forename: Oscar
authors.initials: O
authors.lastname: Escribano
authors.forename: María Dolores
authors.initials: MD
authors.lastname: Fernández-Moreno
authors.forename: José Antonio
authors.initials: JA
authors.lastname: Zueco
authors.forename: Cesar
authors.initials: C
authors.lastname: Menor
authors.forename: Jesus
authors.initials: J
authors.lastname: Fueyo
authors.forename: Rosa María
authors.initials: RM
authors.lastname: Ropero
authors.forename: Inés
authors.initials: I
authors.lastname: Diaz-Laviada
authors.forename: Irene D
authors.initials: ID
authors.lastname: Román
authors.forename: Luis G
authors.initials: LG
authors.lastname: Guijarro
mesh_terms: D000818:Animals; D002455:Cell Division / Q000187:drug effects; D022781:Hepatocytes / Q000166:cytology / Q000378:metabolism*; D007328:Insulin / Q000494:pharmacology; D055504:Insulin Receptor Substrate Proteins; D007334:Insulin-Like Growth Factor I / Q000494:pharmacology; D047908:Intracellular Signaling Peptides and Proteins; D008115:Liver Regeneration / Q000502:physiology*; D008297:Male; D010750:Phosphoproteins / Q000378:metabolism*; D010766:Phosphorylation / Q000187:drug effects; D011233:Precipitin Tests; D051381:Rats; D017208:Rats, Wistar; D011972:Receptor, Insulin / Q000378:metabolism; D015398:Signal Transduction; D013347:Subcellular Fractions / Q000378:metabolism; D014018:Tissue Distribution
chemical_list: C491532:IRS4 protein, rat; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; C526296:Irs1 protein, rat; C526299:Irs2 protein, rat; D010750:Phosphoproteins; D007334:Insulin-Like Growth Factor I; D011972:Receptor, Insulin