The insulin/IGF-1 (insulin-like growth factor 1) signalling pathway promotes adipocyte differentiation via complex signalling networks. Here, using microarray analysis of brown preadipocytes that are derived from wild-type and insulin receptor substrate (Irs) knockout animals that exhibit progressively impaired differentiation, we define 374 genes/expressed-sequence tags whose expression in preadipocytes correlates with the ultimate ability of the cells to differentiate. Many of these genes, including preadipocyte factor-1 (Pref-1) and multiple members of the Wnt signalling pathway, are related to early adipogenic events. Necdin is also markedly increased in Irs knockout cells that cannot differentiate, and knockdown of necdin restores brown adipogenesis with downregulation of Pref-1 and Wnt10a expression. Insulin receptor substrate proteins regulate a necdin-E2F4 interaction that represses peroxisome-proliferator-activated receptor gamma (PPARgamma) transcription via a cyclic AMP response element binding protein (CREB)-dependent pathway. Together these define a key signalling network that is involved in brown preadipocyte determination.

The most widely distributed members of the family of insulin receptor substrate (IRS) proteins are IRS-1 and IRS-2. These proteins participate in insulin and insulin-like growth factor 1 signaling, as well as the actions of some cytokines, growth hormone, and prolactin. To more precisely define the specific role of IRS-1 in adipocyte biology, we established brown adipocyte cell lines from wild-type and IRS-1 knockout (KO) animals. Using differentiation protocols, both with and without insulin, preadipocyte cell lines derived from IRS-1 KO mice exhibited a marked decrease in differentiation and lipid accumulation (10 to 40%) compared to wild-type cells (90 to 100%). Furthermore, IRS-1 KO cells showed decreased expression of adipogenic marker proteins, such as peroxisome proliferator-activated receptor gamma (PPAR␥), CCAAT/enhancer-binding protein alpha (C/EBP␣), fatty acid synthase, uncoupling protein-1, and glucose transporter 4. The differentiation deficit in the KO cells could be reversed almost completely by retrovirus-mediated reexpression of IRS-1, PPAR␥, or C/EBP␣ but not the thiazolidinedione troglitazone. Phosphatidylinositol 3-kinase (PI 3-kinase) assays performed at various stages of the differentiation process revealed a strong and transient activation in IRS-1, IRS-2, and phosphotyrosine-associated PI 3-kinase in the wild-type cells, whereas the IRS-1 KO cells showed impaired phosphotyrosine-associated PI 3-kinase activation, all of which was associated with IRS-2. Akt phosphorylation was reduced in parallel with the total PI 3-kinase activity. Inhibition of PI 3-kinase with LY294002 blocked differentiation of wild-type cells. Thus, IRS-1 appears to be an important mediator of brown adipocyte maturation. Furthermore, this signaling molecule appears to exert its unique role in the differentiation process via activation of PI 3-kinase and its downstream target, Akt, and is upstream of the effects of PPAR␥ and C/EBP␣.Adipocytes play a central role in lipid homeostasis and the maintenance of energy balance in vertebrates (18). White adipose tissue is the primary site of storage of triglycerides and release of fatty acids in response to changing energy needs (12). Brown adipocytes, on the other hand, store smaller amounts of triglycerides and account for much of the basal thermogenic energy expenditure through the expression of uncoupling protein-1 (UCP-1) (19). Obesity, an excessive accumulation of white adipose tissue, occurs when energy intake by an individual exceeds the rate of energy expenditure, whereas brown adipocyte mass is highest in young mammals and disorders such as pheochromocytoma (27).Characterization of cell lines that progress from an undifferentiated progenitor state to mature white adipocytes has led to a good understanding of the factors involved in the adipogenic program. Among these factors, the transcription factors peroxisome proliferator-activated receptor gamma (PPAR␥) and CCAAT/enhancer-binding proteins (C/EBPs) appear to play a central role. PPAR␥ is highly enriched in adipos...

Insulin promotes adipocyte differentiation via a complex signaling network involving multiple insulin receptor substrates (IRSs). In cultured brown preadipocytes, expression of IRS-1 and IRS-2 mRNAs and proteins was at relatively high levels before and after differentiation into mature fat cells, while IRS-3 transcript was not detectable in preadipocytes but increased during the course of differentiation, and IRS-4 mRNA was barely detected in both states. To determine more precisely the roles of various IRS proteins in adipogenesis, we established and characterized brown preadipocyte cell lines from wild-type and IRS knockout (