title: Role of IRS-3 in the insulin signaling of IRS-1-deficient brown adipocytes.
abstract: Insulin receptor substrate-1 (IRS-1) plays an essential role in mediating the insulin signals that trigger mitogenesis, lipid synthesis, and uncoupling protein-1 gene expression in mouse brown adipocytes. Expression of IRS-3 is restricted mainly to white adipose tissue; expression of this IRS protein is virtually absent in brown adipocytes. We have tested the capacity of IRS-3 to mediate insulin actions in IRS-1-deficient brown adipocytes. Thus, we expressed exogenous IRS-3 in immortalized IRS-1-/- brown adipocytes at a level comparable with that of endogenous IRS-3 in white adipose tissue. Under these conditions, IRS-3 signaling in response to insulin was observed, as revealed by tyrosine phosphorylation of IRS-3, and the activation of phosphatidylinositol (PI) 3-kinase associated with this recombinant protein. However, although insulin promoted the association of Grb-2 with recombinant IRS-3 in IRS-1-/- cells, the exogenous expression of this IRS family member failed to activate p42/44 MAPK and mitogenesis in brown adipocytes lacking IRS-1. Downstream of PI 3-kinase, IRS-3 expression restored insulin-induced Akt phosphorylation, which is impaired by the lack of IRS-1 signaling. Whereas the generation of IRS-3 signals enhanced adipocyte determination and differentiation-dependent factor 1/sterol regulatory element-binding protein (ADD-1/SREBP-1c) and fatty acid synthase mRNA and protein expression, activation of this pathway was unable to reconstitute CCAAT/enhancer-binding protein alpha and uncoupling protein-1 transactivation and gene expression in response to insulin. Similar results were obtained following insulin-like growth factor-I stimulation. In brown adipocytes expressing the IRS-3F4 mutant, the association of the p85alpha regulatory subunit via Src homology 2 binding was lost, but insulin nevertheless induced PI 3-kinase activity and Akt phosphorylation in a wortmannin-dependent manner. In contrast, activation of IRS-3F4 signaling failed to restore the induction of ADD-1/SREBP-1c and fatty acid synthase gene expression in IRS-1-deficient brown adipocytes. These studies demonstrate that recombinant IRS-3 may reconstitute some, but not all, of the signals required for insulin action in brown adipocytes. Thus, our data further implicate a unique role for IRS-1 in triggering insulin action in brown adipocytes.
authors.affiliation: Departamento de Bioquímica y Biología Molecular/Instituto de Bioquímica, Centro Mixto Consejo Superior de Investigaciones Científicas/Universidad Complutense, Facultad de Farmacia, Universidad Complutense, Madrid 28040, Spain.
authors.forename: Mónica
authors.initials: M
authors.lastname: Arribas
authors.forename: Angela M
authors.initials: AM
authors.lastname: Valverde
authors.forename: Manuel
authors.initials: M
authors.lastname: Benito
mesh_terms: D017667:Adipocytes / Q000378:metabolism*; D002001:Adipose Tissue, Brown / Q000378:metabolism*; D000818:Animals; D015152:Blotting, Northern; D015153:Blotting, Western; D022762:CCAAT-Enhancer-Binding Proteins / Q000096:biosynthesis; D002454:Cell Differentiation; D015500:Chloramphenicol O-Acetyltransferase / Q000378:metabolism; D004268:DNA-Binding Proteins / Q000096:biosynthesis; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D007328:Insulin / Q000378:metabolism*; D055504:Insulin Receptor Substrate Proteins; D051379:Mice; D008928:Mitochondria / Q000378:metabolism; D019950:Mitogen-Activated Protein Kinase 1 / Q000378:metabolism; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D008954:Models, Biological; D019869:Phosphatidylinositol 3-Kinases / Q000378:metabolism; D010750:Phosphoproteins / Q000235:genetics* / Q000378:metabolism / Q000502:physiology*; D010766:Phosphorylation; D011233:Precipitin Tests; D012333:RNA, Messenger / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D012190:Retroviridae / Q000235:genetics; D015398:Signal Transduction; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D015533:Transcriptional Activation; D014162:Transfection; D014443:Tyrosine / Q000378:metabolism
chemical_list: D022762:CCAAT-Enhancer-Binding Proteins; D004268:DNA-Binding Proteins; D004791:Enzyme Inhibitors; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; C526295:Irs1 protein, mouse; C108872:Irs3 protein, mouse; D010750:Phosphoproteins; D012333:RNA, Messenger; D011994:Recombinant Proteins; C497388:Srebf1 protein, mouse; D051780:Sterol Regulatory Element Binding Protein 1; D014157:Transcription Factors; D014443:Tyrosine; D015500:Chloramphenicol O-Acetyltransferase; D019869:Phosphatidylinositol 3-Kinases; D019950:Mitogen-Activated Protein Kinase 1; D048052:Mitogen-Activated Protein Kinase 3; D020928:Mitogen-Activated Protein Kinases