Technical Abstract: Insulin receptor substrate (IRS) proteins are key mediators to the actions of both IGF-I and insulin. In cell culture models loss of inhibition or loss of IRS-1 expression increases the rates of apoptosis while overexpression of IRS-1 or -2 has been linked to protection from apoptosis. In the mammary gland, the expression of both proteins is dramatically upregulated with lactation and subsequently downregulated with involution. Previous work in our laboratory with transgenic mice that overexpress des(1-3)IGF-I in the mammary gland led us to the hypothesis that mammary cell apoptosis during involution would be inhibited by transgenic overexpression of IRS-1 or -2. To test this hypothesis, transgene constructs were engineered to overexpress HA-tagged IRS-1 or -2 in the mammary gland using the MMTV-LTR. Natural mammary involution was then compared between nontrangenic and MMTV-IRS-1 mice or nontransgenic and MMTV-IRS-2 mice in two separate experiments by analyzing mammary tissue wet weight, morphology, and apoptosis on days 16, 18, 20 and 22 postpartum. Analysis of transgene expression by western blotting either for the HA epitope or for IRS demonstrated that both transgenes were highly expressed during lactation. On day 16 postpartum, IRS-1 and IRS-2 were 10 and 15-fold overexpressed respectively. For both transgenes the expression declined from day 16 through 22, but remained significantly elevated over the levels of the endogenous proteins. Interestingly, despite the fact that the overexpressed IRS proteins were constitutively tyrosine phosphorylated, only minimal changes were seen in downstream Akt and ERK1/2 phosphorylation. When averaged over all genotypes and across both experiments, mammary gland wet weight was 0.4+/-0.02 g (mean±sem) on day 16 and decreased (P<0.0001) to 0.2 +/- 0.02 g by day 20 postpartum. Mammary gland apoptosis averaged over all genotypes on day 16 postpartum was 0.2 +/- 0.4 and 0.7 +/-0.6% in two separate experiments. By day 20 postpartum, mammary gland apoptosis averaged over all genotypes increased (P<0.05) to 2.2 +/- 0.4 and 3.4 +/- 0.6% in two separate experiments. Neither the overexpression of IRS-1 nor the overexpression IRS-2 affected mammary gland wet weight or apoptosis during natural involution. These results support the conclusion that overexpression of IRS-1 or -2 in and of themselves is insufficient to inhibit natural mammary gland involution. This project supported by NIH/NCI CA94118 to AVL.