Translocation of IRS-1 to Cytosol Attenuates Insulin-Stimulated Glucose Transport without Affecting PI3-Kinase Activity

ABSTRACT

IRS-1 is a major substrate for insulin receptor tyrosine kinase. It is reported that intracellular translocation of serine-phosphorylated IRS-1 from low density microsome (LDM) fraction to cytosol attenuates its ability to transmit insulin signaling to the downstream molecules. In this study, we examined which insulin signal and action were affected by translocation of IRS-1 in 3T3-L1 adipocytes. Adenovirus-mediated overexpression of constitutively active PI3-kinase (p110CAAX) induces translocation of IRS-1 to cytosol without increasing IRS-1tyrosine phosphorylation. IRS-1 protein localized in cytosol fraction in p110CAAX-expressing cells maintained the ability to be tyrosine-phosphorylated by short term insulin treatment. Long term treatment with insulin for 4 to 8 h decreased tyrosine phosphorylation of IRS-1, PI3- kinase activity, Akt phosphorylation and glucose uptake by second stimulation with insulin. Pretreatment with rapamycin, a specific mTOR inhibitor, increased the protein level of IRS-1 in LDM fraction and restored the attenuated insulin signaling and glucose uptake after long term insulin treatment. On the other hand, pretreatment with lactacystin, a specific proteasomal inhibitor, increased the protein level of serine-phosphorylated IRS-1 in cytosol fraction. In this condition, insulin signaling from IRS-1 to Akt was restored, but glucose uptake was not. Taken together, we conclude that localization of IRS-1 in LDM fraction is necessary for insulin-stimulated glucose uptake, while IRS-1, once serinephosphorylated and translocated to cytosol, fails to stimulate glucose uptake despite its intact ability to be tyrosine phosphorylated and to transmit insulin signaling to Akt level.