Differential effects of ethanol on insulin-signaling through the insulin receptor substrate-1

Insulin stimulation increases cell proliferation and energy metabolism by a ctivating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosp horylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-fa ctor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stim ulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explo res the roles of the Grb2- and Syp-binding motifs of IRS-I in relation to t he inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proli ferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydro genase (GAPDH) expression, and activation of mitogen-activated protein kina se (MAPK), which is known to be essential for cell proliferation. NIH3T3 ce lls were stably transfected with wild-type IRS-1, or IRS-1 mutated at the G rb2 (IRS-1 Delta Grb2), Syp (IRS-1 Delta Syp), or Grb2 and Syp (IRS-1 Delta Grb2 Delta Syp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1 Delta Grb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. I n contrast, the IRS-1 Delta Syp and IRS-1 Delta Grb2 Delta Syp transfectant s had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol ex posure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activat ed MAPK levels in all clones, but the wild-type IRS-1 transfectants were re latively resistant, and the IRS-1 Delta Grb2 transfectants were extraordina rily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated thr ough the Syp-binding domain, whereas GAPDH expression and MAPK activation a re modulated through both the Grb2 and Syp motifs of IRS-1. In addition. et hanol exposure may preferentially inhibit downstream signaling that require s interaction between Syp and PY-IRS-1.