Abstract P218: Identification of One Retinoic Acid Responsive Element in Glucokinase Gene Promoter

Abstract

Introduction: Studies have shown that vitamin A (VA) status affected hepatic glycogen content and body lipid levels in rats. The active metabolite of VA, retinoic acid (RA), regulates gene expression via activation of both retinoic acid receptor (RAR) and retinoid X receptor (RXR) bound to retinoic acid responsive element (RARE). We have reported that retinoids synergized with insulin to induce glucokinase gene ( Gck ) in primary rat hepatocyes. Hypothesis: We hypothesize that the rat promoter of Gck contains RARE(s). Methods: To identify the RARE(s) in the Gck promoter, we have made (1) a series of luciferase reporter gene constructs containing shortened DNA fragments of Gck promoter; (2) expression vectors containing cDNAs of rat RARα, RXRα and RXRγ; (3) linker-scan mutant reporter constructs covering 0.2 kb of the proximal Gck promoter region and a 20 bp region which disrupt the RA response. The responses of these reporter constructs to RA and the effects of the co-transfection of RAR and RXR on their activation were analyzed in primary hepatocytes and INS-1 insulinoma cells using dual luciferase assay. Results: (1) RA and RA + insulin, not insulin, activated the reporter gene constructs containing 1, 0.8, 0.6, 0.4, and 0.2 kb of Gck promoter in primary hepatocytes (from 1.7- to 2.6-fold). (2) RA alone did not activate Gck promoter constructs in INS-1 cells. However, co-transfection of RAR with reporter gene constructs restored and improved RA response of them in INS-1 cells and primary hepatocytes, respectively. (3) Two consecutive linker-scan mutant reporter constructs covering about 20 bp of the proximal region of Gck promoter, but not the adjacent ones, completely lost the response to RA. (4) One of the five linker-scan mutant reporter constructs covering this 20 bp region completed disrupted the RA response, demonstrating the identification of a RARE. Conclusions: RA induces the activation of Gck promoter constructs in primary hepatocytes. INS-1 cells are a useful tool for analyzing liver Gck promoter activation by RA with co-transfection of RAR/RXR. For the first time, one RARE is identified in the proximal region of rat Gck promoter. Further studies are needed to determine the roles of this identified RARE in mediating Gck expression in responses to hormonal and nutritional stimuli. Funding(This research has received full or partial funding support from the American Heart Association, National Center)