Functional analysis of the glucose response element of the rat glucagon receptor gene in insulin-producing INS-1 cells

Abstract

Glucose stimulates the transcription of the glucagon receptor gene in hepatocytes and in pancreatic β cells. We recently identified a glucose response element in the immediate upstream non-coding region of the rat glucagon receptor gene. We previously showed that this DNA element is centered on a palindromic sequence of 19 nucleotides (termed as G box), containing two E boxes separated by three nucleotides. In the present study, we further characterized the DNA sequence requirements for the glucose induced expression of the rat glucagon receptor gene. Transfection study realized in the insulin-producing INS-1 cells revealed that a fragment of 49 nucleotides, centered on the G box, bears all the features required for the glucose activation. Mutations performed in the 5′-E box totally abolished the glucose responsiveness, whereas mutations or deletion of the 3′-E box only had a limited effect. Deletions performed upstream from the G box revealed that an accessory factor binding site, located in the region just upstream from the G box, is required for full stimulation by glucose. Finally, by using G box based probes in gel shift experiments, we demonstrated that USF1/USF2 transcription factors are part of the proteinic complex that binds to the glucose response element of the rat glucagon receptor gene promoter. In conclusion, in contrast to many other glucose regulated genes, only the 5′-E box appears to be a crucial DNA element for the glucose transcriptional effect. However, an accessory factor binding site located in the region just upstream from the G box is required for a complete stimulation by glucose.