Transcriptional regulation of glucose-6-phosphatase catalytic subunit promoter by insulin and glucose in the carnivorous fish, Sparus aurata

Abstract

Increase in glucose-6-phosphatase catalytic subunit (G6Pase, G6pc) transcription enhances hepatic glucose production in non-insulin-dependent diabetes mellitus (NIDDM). The fact that carnivorous fish is an alternative model to study NIDDM led us to clone and characterise the first G6pc promoter region reported for fish and non-mammalian animals. The 5'-flanking region of G6pc from gilthead sea bream (Sparus aurata) was isolated by chromosome walking. With SMART RACE-PCR, the transcription start site was located 106 base pairs (bp) upstream of the translational start. Transfection analysis in HepG2 cells located a functional promoter in the 850 bp 5'-flanking isolated fragment (positions -770 to +80 relative to the transcription start). Sequential 5'-deletion analysis of the promoter fragment revealed that a core functional promoter for basal transcription is comprised within the 190 bp upstream of the transcription start site. In vivo, glucose and insulin reduced G6Pase mRNA levels in the fish liver. Transfection experiments in HepG2 cells showed that insulin repressed S. aurata G6pc under high-glucose conditions. Synergistic activation of piscine G6pc promoter was induced by cotransfection with expression plasmids for hepatocyte nuclear factor-4alpha (HNF-4alpha) and peroxisome proliferator-activated receptor-gamma coactivator-1 (PGC-1alpha). No direct relationship was found between PGC-1alpha coactivation of HNF-4alpha transactivation and the repressive effect of insulin. Interestingly, insulin hardly affected G6pc promoter activity in the absence of glucose, suggesting that a reduced capacity of insulin-dependent repression of piscine G6pc may lead to insulin resistance in carnivorous fish.