Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha

Authors: YANG, JIANQI - Case Western Reserve University (CWRU); KONG, XIAOYING - Case Western Reserve University (CWRU); MARTINS-SANTOS, MARIA EMILIA - Case Western Reserve University (CWRU); ALEMAN, GABRIELA - Case Western Reserve University (CWRU); CHACO, ERNESTINE - Case Western Reserve University (CWRU); Liu, Ge - George; WU, SHWU-YUAN - University Of Texas Southwestern Medical Center; SAMOLS, DAVID - Case Western Reserve University (CWRU); HAKIMI, PARVIN - Case Western Reserve University (CWRU); CHIANG, CHENG-MING - University Of Texas Southwestern Medical Center; HANSON, RICHARD - Case Western Reserve University (CWRU)

Submitted to: Journal of Biological Chemistry
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/21/2009
Publication Date: 8/3/2009
Citation: Yang, J., Kong, X., Martins-Santos, M.S., Aleman, G., Chaco, E., Liu, G., Wu, S., Samols, D., Hakimi, P., Chiang, C., Hanson, R.W. 2009. Activation of SIRT1 by resveratrol represses transcription of the gene for the cytosolic form of phosphoenolpyruvate carboxykinase (GTP) by deacetylating hepatic nuclear factor 4alpha. Journal of Biological Chemistry. 284(40):27042-27053.

Interpretive Summary: PEPCK-C is a key enzyme in both hepatic and renal gluconeogenesis and in glyceroneogenesis in many mammalian tissues. Its transcription has medical and economical significance, as PEPCK-C is the key enzyme in the control of hepatic glucose output and is thus a potential target for the regulation of blood glucose in human health and animal production. Many of the regulatory elements have been identified in its promoter. In our earlier comparative genomics study, we identified a novel and evolutionarily conserved binding site for HNF4a in its promoter using the software program TFLOC. In this paper, we confirmed this site is functional using experimental approaches including the luciferase reporter assay and the gel-shift assay. Small molecules classified as SIRT1 activator such as isonicotinamide (IsoNAM), were found to repress the expression of PEPCK-C in both the test and control cell lines. We further investigated the mechanism and found that IsoNAM inhibits the expression of PEPCK-C via deacetylation of HNF4a by SIRT1. This study provides a new way of controlling the expression of PEPCK-C using small molecules.

Technical Abstract: The cytosolic isoform of phosphoenolpyruvate carboxykinase (GTP) (PEPCK-C) is a key enzyme of gluconeogenesis and glyceroneogenesis. While this enzyme is often over-expressed in diabetes and obesity, studies showed that decrease in its expression results in lessening the diseases condition in animal models. In this study, a group of small chemical molecules including isonicotinamide (IsoNAM), resveratrol, fiestin, and butein, which are classified as SIRT1 activator, were found to repress the expression of Pck1, the rodent gene for PEPCK-C. Using an in silico prediction method in combination with the luciferase reporter assay and the gel-shift assay, a novel and evolutionarily conserved binding site for HNF4a, located at -272/-252 of the Pck1 promoter, was identified to be critical for IsoNAM-induced repression of Pck1. Treatment of IsoNAM rendered a time-dependent decrease in the acetylated nuclear HNF4a in the HepG2 cells. In accordance, IsoNAM did not inhibit the expression of Pck1 in the CHO cell, a known HNF4a-null cell line; and transient expression of HNF4a in CHO cells re-established IsoNAM-induced inhibition of Pck1. Results from gel-shift assay further demonstrated that an attenuated inaction between HNF4a and the DNA binding site after the treatment of IsoNAM. Over-expression of SIRT1 resulted in a repression of Pck1 expression. We conclude that IsoNAM inhibits the expression of Pck1 via deacetylation of HNF4a by SIRT1. This study provides a new way of controlling the expression of PEPCK-C using small molecules.