Subcellular localization of insulin receptor substrate family proteins associated with phosphatidylinositol 3-kinase activity and alterations in lipolysis in primary mouse adipocytes from IRS-1 null mice

To clarify the roles of insulin receptor substrate (IRS) family proteins in phosphatidylinositol (PI) 3-kinase activation and insulin actions in adipo cytes, we investigated the intracellular localization of IRS family protein s and PI 3-kinase activation in response to insulin by fractionation of mou se adipocytes from wild-type and IRS-1 null mice. In adipocytes from wild-t ype mice, tyrosine-phosphorylated IRS-1 and IRS-2, which were found to asso ciate with PI 3-kinase in response to insulin, were detected in the plasma membrane (PM) and low-density microsome (LDM) fractions. By contrast, tyros ine-phosphorylated IRS-3 (pp60), which was found to associate with PI 3-kin ase, was predominantly localized in the PM fraction. In adipocytes from IRS -1-null mice, insulin-stimulated PI 3-kinase activity in anti-phosphotyrosi ne (alpha PY) immunoprecipitates in the LDM fraction was almost exclusively mediated via IRS-2 and was reduced to 25%;however, insulin-stimulated PI 3 -kinase activity in the PM fraction was primarily mediated via IRS-3 and wa s reduced to 60%. To determine the potential functional impact of the disti nct subcellular localization of IRSs and associating PI 3-kinase activity o n adipocyte-specific metabolic actions, we examined lipolysis in IRS-1 null mice. The level of isoproterenol-induced lipolysis was increased 5.1-fold in adipocytes from IRS-1 null mice as compared with wild-type mice. Moreove r, hormone-sensitive Lipase (HSL) protein was increased 4.3-fold in adipocy tes from IRS-1-null mice compared with wild-type mice, and HSL mRNA express ion was also increased. The antilipolytic effect of insulin in IRS-1 null a dipocytes, however, was comparable to that in wild-type mice. Thus, discord ance between these two insulin actions as well as the transcriptional and t ranslational effect (HSL mRNA and protein regulation) and the PM effect (an tilipolysis) of insulin may be explained by distinct roles of both PI 3-kin ase activity associated with IRS-1/IRS-2 and PI 3-kinase activity associate d with IRS-3 in insulin actions related to their subcellular localization.