Essential Role of Insulin Receptor Substrate 1 (IRS-1) and IRS-2 in Adipocyte Differentiation

Abstract

To investigate the role of insulin receptor substrate 1 (IRS-1) and IRS-2, the two ubiquitously expressed IRS proteins, in adipocyte differentiation, we established embryonic fibroblast cells with four different genotypes, i.e., wild-type, IRS-1 deficient (IRS-1^−/−), IRS-2 deficient (IRS-2^−/−), and IRS-1 IRS-2 double deficient (IRS-1^−/−IRS-2^−/−), from mouse embryos of the corresponding genotypes. The abilities of IRS-1^−/− cells and IRS-2^−/− cells to differentiate into adipocytes are approximately 60 and 15%, respectively, lower than that of wild-type cells, at day 8 after induction and, surprisingly, IRS-1^−/− IRS-2^−/− cells have no ability to differentiate into adipocytes. The expression of CCAAT/enhancer binding protein α (C/EBPα) and peroxisome proliferator-activated receptor γ (PPARγ) is severely decreased in IRS-1^−/−IRS-2^−/− cells at both the mRNA and the protein level, and the mRNAs of lipoprotein lipase and adipocyte fatty acid binding protein are severely decreased in IRS-1^−/−IRS-2^−/− cells. Phosphatidylinositol 3-kinase (PI 3-kinase) activity that increases during adipocyte differentiation is almost completely abolished in IRS-1^−/−IRS-2^−/− cells. Treatment of wild-type cells with a PI 3-kinase inhibitor, LY294002, markedly decreases the expression of C/EBPα and PPARγ, a result which is associated with a complete block of adipocyte differentiation. Moreover, histologic analysis of IRS-1^−/− IRS-2^−/− double-knockout mice 8 h after birth reveals severe reduction in white adipose tissue mass. Our results suggest that IRS-1 and IRS-2 play a crucial role in the upregulation of the C/EBPα and PPARγ expression and adipocyte differentiation.

ACKNOWLEDGMENTS

We thank B. M. Spiegelman and N. Yahagi for providing PPARγ2 expression vector and 36B4 cDNA probe, respectively, and E. Yoshida-Nagata and H. Chiyonobu for excellent technical assistance.

This work was supported by a grant-in-aid for creative basic research (10NP0201) from the Ministry of Education, Science, Sports, and Culture of Japan (to T. Kadowaki) and by health science research grants (Research on Human Genome and Gene Therapy) from the Ministry of Health and Welfare (to T. Kadowaki).