title: Insulin substrates 1 and 2 are corequired for activation of atypical protein kinase C and Cbl-dependent phosphatidylinositol 3-kinase during insulin action in immortalized brown adipocytes.
abstract: Phosphatidylinositol 3-kinase (PI3K)-dependent activation of atypical protein kinase C (aPKC) is required for insulin-stimulated glucose transport. Although insulin receptor substrate-1 (IRS-1) and IRS-2, among other factors, activate PI3K, there is little information on the relative roles of IRS-1and IRS-2 during aPKC activation by insulin action in specific cell types. Presently, we have used immortalized brown adipocytes in which either IRS-1 or IRS-2 has been knocked out by recombinant methods to examine IRS-1 and IRS-2 requirements for activation of aPKC. We have also used these adipocytes to see if IRS-1 and IRS-2 are required for activation of Cbl, which is required for insulin-stimulated glucose transport and has been found to function upstream of both PI3K/aPKC and Crk during thiazolidinedione action in 3T3/L1 adipocytes [Miura et al. (2003) Biochemistry 42, 14335]. In brown adipocytes in which either IRS-1 or IRS-2 was knocked out, insulin-induced increases in aPKC activity and glucose transport were markedly diminished. These effects of insulin on aPKC and glucose transport were fully restored by retroviral-mediated expression of IRS-1 or IRS-2 in their respective knockout cells. Knockout of IRS-1 or IRS-2 also inhibited insulin-induced increases in Cbl binding to the p85 subunit of PI3K, which, along with IRS-1/2, may be required for activation of PI3K, aPKC, and glucose transport during insulin action in 3T3/L1 adipocytes. These findings provide evidence that directly links both IRS-1 and IRS-2 to aPKC activation in immortalized brown adipocytes, and further suggest that IRS-1 and IRS-2 are required for the activation of Cbl/PI3K during insulin action in these cells.
authors.affiliation: Research Service, James A. Haley Veterans Hospital, and Department of Internal Medicine, University of South Florida College of Medicine, Tampa, Florida 33612, USA.
authors.forename: Atsushi
authors.initials: A
authors.lastname: Miura
authors.forename: Mini P
authors.initials: MP
authors.lastname: Sajan
authors.forename: Mary L
authors.initials: ML
authors.lastname: Standaert
authors.forename: Gautam
authors.initials: G
authors.lastname: Bandyopadhyay
authors.forename: C Ronald
authors.initials: CR
authors.lastname: Kahn
authors.forename: Robert V
authors.initials: RV
authors.lastname: Farese
mesh_terms: D064413:3-Phosphoinositide-Dependent Protein Kinases; D002001:Adipose Tissue, Brown / Q000166:cytology / Q000201:enzymology / Q000378:metabolism*; D000818:Animals; D002461:Cell Line, Transformed; D003847:Deoxyglucose / Q000378:metabolism; D004357:Drug Synergism; D004789:Enzyme Activation; D007328:Insulin / Q000494:pharmacology*; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes / Q000378:metabolism; D051379:Mice; D018345:Mice, Knockout; D050722:Oncogene Protein v-cbl; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism* / Q000502:physiology; D010750:Phosphoproteins / Q000172:deficiency / Q000235:genetics / Q000502:physiology*; D010766:Phosphorylation; D011493:Protein Kinase C / Q000378:metabolism* / Q000502:physiology; D017346:Protein Serine-Threonine Kinases / Q000378:metabolism / Q000502:physiology; D021122:Protein Subunits / Q000378:metabolism; D011518:Proto-Oncogene Proteins / Q000378:metabolism; D051057:Proto-Oncogene Proteins c-akt; D015682:Retroviridae Proteins, Oncogenic / Q000502:physiology*; D014316:Tritium / Q000378:metabolism; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta
chemical_list: D064413:3-Phosphoinositide-Dependent Protein Kinases; D003847:Deoxyglucose; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D047908:Intracellular Signaling Peptides and Proteins; D007527:Isoenzymes; D050722:Oncogene Protein v-cbl; D019869:Phosphatidylinositol 3-Kinases; D010750:Phosphoproteins; D011493:Protein Kinase C; D017346:Protein Serine-Threonine Kinases; D021122:Protein Subunits; D011518:Proto-Oncogene Proteins; D051057:Proto-Oncogene Proteins c-akt; D015682:Retroviridae Proteins, Oncogenic; D014316:Tritium; D000099293:Protein Kinase C-lambda; D000099221:Protein Kinase C zeta; C526295:Irs1 protein, mouse; C526298:Irs2 protein, mouse