Functional expression of insulin receptor substrate-1 is required for insulin-stimulated mitogenic signaling.

To examine the role of the insulin receptor substrate-1 (IRS-1) in mediating insulin biological responsiveness, we generated Chinese hamster ovary cell lines expressing antisense IRS-1 RNA. These cells displayed morphological alterations as well as markedly reduced growth rates compared to the parental cells. Furthermore, the antisense IRS-1 cell lines had decreased insulin-stimulated IRS-1 tyrosine phosphorylation, reduced phosphatidylinositol 3-kinase activation, and decreased thymidine incorporation relative to the parental cell line. Insulin-dependent transcriptional regulation of a serum response element/luciferase reporter construct (SRE-Luc) was also reduced in the antisense IRS-1-expressing cell lines. However, co-transfection with a plasmid directing the expression of rat IRS-1 fully restored insulin-stimulated SRE-Luc activity in the IRS-1 antisense cell lines. Thus, the inhibition in insulin signaling was a specific effect of decreased IRS-1 tyrosine phosphorylation. Taken together, these data demonstrate that insulin regulation of mitogenic signaling requires the functional expression of IRS-1 and documents its central importance in the insulin intracellular signaling pathway.