title: Differential effects of ethanol on insulin-signaling through the insulin receptor substrate-1.
abstract: Insulin stimulation increases cell proliferation and energy metabolism by activating the insulin receptor substrate I (IRS-1)-signaling pathways. This downstream signaling is mediated by interactions of specific tyrosyl phosphorylated (PY) IRS-1 motifs with SH2-containing molecules such as growth-factor receptor-bound protein 2 (Grb2) and Syp. Ethanol inhibits insulin-stimulated tyrosyl phosphorylation of IRS-1 and DNA synthesis. This study explores the roles of the Grb2- and Syp-binding motifs of IRS-1 in relation to the inhibitory effects of ethanol on insulin-stimulated DNA synthesis, proliferating cell nuclear antigen (PCNA) and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) expression, and activation of mitogen-activated protein kinase (MAPK), which is known to be essential for cell proliferation. NIH3T3 cells were stably transfected with wild-type IRS-1, or IRS-1 mutated at the Grb2 (IRS-1deltaGrb2), Syp (IRS-1deltaSyp), or Grb2 and Syp (IRS-1deltaGrb2deltaSyp)- binding sites. Cells transfected with IRS-1 had increased levels of DNA synthesis, PCNA, GAPDH, and activated MAPK. The IRS-1deltaGrb2 transfectants were highly responsive to insulin stimulation, achieving levels of GAPDH, PCNA, and activated MAPK that were higher than control. In contrast, the IRS-1deltaSyp and IRS-1deltaGrb2deltaSyp transfectants had reduced levels of DNA synthesis, PCNA, and activated MAPK. Ethanol exposure decreased insulin-stimulated DNA synthesis, PCNA, GAPDH, and activated MAPK levels in all clones, but the wild-type IRS-1 transfectants were relatively resistant, and the IRS-1deltaGrb2 transfectants were extraordinarily sensitive to these inhibitory effects of ethanol. The findings suggest that insulin-stimulated DNA synthesis and PCNA expression are mediated through the Syp-binding domain, whereas GAPDH expression and MAPK activation are modulated through both the Grb2 and Syp motifs of IRS-1. In addition, ethanol exposure may preferentially inhibit downstream signaling that requires interaction between Syp and PY-IRS-1.
authors.affiliation: MGH East Cancer Center, Department of Medicine, Massachusetts General Hospital, Boston, USA. delamonte@hotmail.com
authors.forename: S M
authors.initials: SM
authors.lastname: de la Monte
authors.forename: N
authors.initials: N
authors.lastname: Ganju
authors.forename: S
authors.initials: S
authors.lastname: Tanaka
authors.forename: K
authors.initials: K
authors.lastname: Banerjee
authors.forename: P J
authors.initials: PJ
authors.lastname: Karl
authors.forename: N V
authors.initials: NV
authors.lastname: Brown
authors.forename: J R
authors.initials: JR
authors.lastname: Wands
mesh_terms: D002455:Cell Division / Q000502:physiology; D002460:Cell Line; D004734:Energy Metabolism / Q000187:drug effects*; D000431:Ethanol / Q000494:pharmacology*; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases / Q000187:drug effects / Q000378:metabolism; D006801:Humans; D007328:Insulin / Q000502:physiology*; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins / Q000187:drug effects* / Q000502:physiology; D018809:Proliferating Cell Nuclear Antigen / Q000187:drug effects / Q000502:physiology; D011972:Receptor, Insulin / Q000187:drug effects* / Q000502:physiology
chemical_list: C526292:IRS1 protein, human; D007328:Insulin; D055504:Insulin Receptor Substrate Proteins; D010750:Phosphoproteins; D018809:Proliferating Cell Nuclear Antigen; D000431:Ethanol; D005987:Glyceraldehyde-3-Phosphate Dehydrogenases; D011972:Receptor, Insulin