Regulation of the promoter region of the human adiponutrin/PNPLA3 gene by glucose and insulin

The adiponutrin/PNPLA3 gene is highly expressed in adipose tissue and liver. Its expression is down-regulated by fasting and rapidly induced by refeeding a high carbohydrate diet. We aimed to determine whether the promoter region of adiponutrin is regulated by glucose and insulin. Endogenous adiponutrin mRNA was increased in mouse 3T3-L1 and human SGBS adipocytes and in human HepG2 cells cultured in 25 mM glucose compared to absence of glucose. A 3100 bp 5′-upstream region of the human adiponutrin gene was cloned into a luciferase reporter plasmid and used in transient transfection studies. Promoter activity was up-regulated by 25 mM glucose, 4.7-fold in HepG2 cells and 2-fold in CHO cells. The effect was shown in CHO cells to be concentration dependent and to depend on glucose metabolism as a non-metabolisable analogue was without effect. In CHO cells constitutively expressing human insulin receptor (CHO-IR), there was a concentration dependent increase of promoter activity by insulin in the presence of glucose. Cotransfection with an expression plasmid for upstream stimulatory factor 2 (USF2), increased promoter activity 1.6-fold in CHO-IR cells. The combined effect of insulin and USF2 (2.3-fold) was greater than the individual effects. Cotransfection of carbohydrate-response element binding protein did not elicit any induction of promoter activity. These results point to potential mechanisms for the observed in vivo nutritional regulation of adiponutrin expression and its up-regulation in fatty liver and by obesity.

Research highlights
► 25 mM glucose increased expression of adiponutrin mRNA in cell lines.
► Glucose increased promoter activity of 3100 bp 5′-upstream region of adiponutrin gene.
► Insulin and USF2 acted independently to increase adiponutrin promoter activity.
► ChREBP did not increase adiponutrin promoter activity in CHO cells.