CHARACTERIZATION OF THE MURINE HIGH K(M) GLUCOSE-TRANSPORTER GLUT2 GENE AND ITS TRANSCRIPTIONAL REGULATION BY GLUCOSE IN A DIFFERENTIATED INSULIN-SECRETING CELL-LINE

In pancreatic beta-cells, high K-m glucose transporter GLUT2 catalyzes the first step in glucose-induced insulin secretion by glucose uptake . Expression of the transporter has been reported to be modulated by g lucose either at the protein or mRNA levels. In this study we used the differentiated insulinoma cell Line INS-1 which expresses high levels of GLUT2 and show that the expression of GLUT2 is regulated by glucos e at the transcriptional level. By run-on transcription assays we show ed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cel ls which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT 2 mRNA levels. To determine whether glucose regulatory sequences were present in the promoter region of GLUT2, we cloned and characterized a 1.4-kilobase region of mouse genomic DNA located 5' of the translatio n initiation site. By RNase protection assays and primer extension, we determined that multiple transcription initiation sites were present at positions -55, -64, and -115 from the first coding ATG and which we re identified in liver, intestine, kidney, and beta-cells mRNAs. Plasm ids were constructed with the mouse promoter region linked to the repo rter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentratio n dependent increase in CAT activity which reached a maximum of 3.6-fo ld at 20 mM glucose. Similar CAT constructs made of the human GLUT2 pr omoter region and the CAT gene displayed the same glucose-dependent in crease in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence i dentity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common se quences.