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J. Biol. Chem., Vol. 278, Issue 46, 45189-45199, November 14, 2003

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Role of IRS-3 in the Insulin Signaling of IRS-1-deficient Brown Adipocytes^*

Mónica Arribas, Angela M. Valverde, and Manuel Benito

From the 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

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 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 p85 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.

Received for publication, February 4, 2003 , and in revised form, August 26, 2003.

^* This work was supported by Grant SAF 2001/1302 from the Ministerio de Ciencia y Tecnologia, Spain and by Red de Grupos en Diabetes Mellitus, Ministerio de Sanidad, Spain. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Contributed equally to this work.

To whom correspondence should be addressed: Departamento de Bioquímica y Biología Molecular, Centro Mixto CSIC/UCM, Facultad de Farmacia, Ciudad Universitaria, Madrid 28040, Spain. Tel.: 34-91-3941777; Fax: 34-91-3941779; E-mail: benito{at}farm.ucm.es.

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