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To examine the impact of homozygous genetic disruption of insulin receptor substrate (IRS)-1 (IRS-1−/−) or IRS-2 (IRS-2−/−) on basal and insulin-stimulated carbohydrate and lipid metabolism in vivo, we infused 18-h fasted mice (wild-type (WT), IRS-1−/−, and IRS-2−/−) with [3-3H]glucose and [2H5]glycerol and assessed rates of glucose and glycerol turnover under basal (0–90 min) and hyperinsulinemic-euglycemic clamp (90–210 min; 5 mmglucose, and 5 milliunits of insulin·kg−1·min−1) conditions. Both IRS-1−/−and IRS-2−/−mice were insulin-resistant as reflected by markedly impaired insulin-stimulated whole-body glucose utilization compared with WT mice. Insulin resistance in the IRS-1−/−mice could be ascribed mainly to decreased insulin-stimulated peripheral glucose metabolism. In contrast, IRS-2−/−mice displayed multiple defects in insulin-mediated carbohydrate metabolism as reflected by (i) decreased peripheral glucose utilization, (ii) decreased suppression of endogenous glucose production, and (iii) decreased hepatic glycogen synthesis. Additionally, IRS-2−/−mice also showed marked insulin resistance in adipose tissue as reflected by reduced suppression of plasma free fatty acid concentrations and glycerol turnover during the hyperinsulinemic-euglycemic clamp. These data suggest important tissue-specific roles for IRS-1 and IRS-2 in mediating the effect of insulin on carbohydrate and lipid metabolismin vivoin mice. IRS-1 appears to have its major role in muscle, whereas IRS-2 appears to impact on liver, muscle, and adipose tissue.

Phosphorylation and subsequent activation of p70 S6 kinase (S6K) are events that are highly conserved in the cellular response to mitogens. The neuropeptide bombesin, which is a potent mitogen for Swiss 3T3 cells, stimulated a time- and dose-dependent activation of p70S6Kas determined by gel mobility shift and immune complex kinase assays. This effect was inhibited by the immunosuppressant rapamycin at 10 nM, which also completely abolished bombesin-stimulated DNA synthesis at identical concentrations. In striking contrast, the combination of bombesin and insulin in synergy stimulated maximum DNA synthesis in these cells despite persistent inhibition of p70S6Kby rapamycin throughout G1. These results indicate that activation of p70S6Kis not required for the transition of quiescent cells to the S phase of the cell cycle. The inhibitory effects of rapamycin on bombesin-stimulated cell cycle progression did not involve accumulation of the cyclin-dependent kinase inhibitor p27kip1but a striking inhibition of the expression of cyclin D1. This effect was circumvented by bombesin with insulin, suggesting that a rapamycin-insensitive pathway stimulated by this combination leads to cyclin D1 expression. Thus, these findings, dissociating the mitogenic effects of bombesin in synergy with insulin from activation of p70S6K, support the hypothesis that this kinase is a component of one of the parallel pathways that can lead to DNA synthesis rather than an obligatory point of convergence in mitogenic signaling.