Chronic ethanol intake impairs insulin signaling in rats by disrupting Akt association with the cell membrane: Role of TRB3 in inhibition of Akt/protein kinase B activation

Chronic and excessive alcohol consumption is an important and modifiable risk factor for type 2 diabetes. We previously reported elevations in hepatic Class 1 alcohol dehydrogenase (ADH) expression in ethanol-fed rats correspondent with reduced levels of mature, nuclear sterol-regulatory element-binding protein-1 (SREBP-1), an insulin-induced transcriptional repressor of the ADH gene. In this report, we have studied the effects of insulin and ethanol on ADH gene expression in a highly differentiated rat hepatoma cell line (FGC-4), as well as the in vivo effects of chronic intake of an ethanol-containing diet on hepatic insulin signaling. Insulin inhibited ADH gene expression, and this was abolished by LY294002 (a phosphatidylinositol 3-kinase inhibitor) and small interfering RNA knockdown of SREBP-1. Chronic ethanol intake led to decreased phosphorylation of Akt (protein kinase B) at Thr³⁰⁸, increased phosphorylation of Akt at Ser⁴⁷³, and decreased phosphorylation of glycogen synthase kinase-3β (a downstream effector of Akt). Hepatic membrane-associated Akt content was decreased and cytosolic Akt content was increased in rats fed an ethanol-containing diet. Thus, disruptive effects of ethanol on insulin signaling occurred via impaired phosphorylation of Akt at Thr³⁰⁸. TRB3, a negative regulator of Akt, was induced in liver of ethanol-fed rats. In ethanol-treated FGC-4 cells, small interfering RNA knockdown of TRB3 increased membrane-associated Akt and the phosphorylation of Akt at Thr³⁰⁸. Our results suggest that ethanol induces TRB3, which, through binding to the pleckstrin homology domain of Akt, prevents its plasma membrane association, Akt-Thr³⁰⁸ phosphorylation, and subsequent Akt-mediated signaling. Ethanol inhibition of insulin signaling reduces nuclear SREBP accumulation and results in disinhibition of Class 1 ADH transcription.