Article: article from journal or magazin.
Characterization of the murine high Km glucose transporter GLUT2 gene and its transcriptional regulation by glucose in a differentiated insulin-secreting cell line.
Journal of Biological Chemistry
In pancreatic beta-cells, the high Km 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 glucose 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 glucose at the transcriptional level. By run-on transcription assays we showed that glucose induced GLUT2 gene transcription 3-4-fold in INS-1 cells which was paralleled by a 1.7-2.3-fold increase in cytoplasmic GLUT2 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 translation 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 were identified in liver, intestine, kidney, and beta-cells mRNAs. Plasmids were constructed with the mouse promoter region linked to the reporter gene chloramphenicol acetyltransferase (CAT), and transiently and stably transfected in the INS-1 cells. Glucose induced a concentration-dependent increase in CAT activity which reached a maximum of 3.6-fold at 20 mM glucose. Similar CAT constructs made of the human GLUT2 promoter region and the CAT gene displayed the same glucose-dependent increase in transcriptional activity when transfected into INS-1 cells. Comparison of the mouse and human promoter regions revealed sequence identity restricted to a few stretches of sequences which suggests that the glucose responsive element(s) may be conserved in these common sequences.
Animals, Base Sequence, Biological Transport, Cell Differentiation, Cloning, Molecular, Codon, Initiator, Gene Expression Regulation, Neoplastic, Genes, Reporter, Glucose/metabolism, Glucose/pharmacology, Glucose Transporter Type 2, Insulin/secretion, Insulinoma, Islets of Langerhans/cytology, Islets of Langerhans/drug effects, Mice, Molecular Sequence Data, Monosaccharide Transport Proteins/biosynthesis, Monosaccharide Transport Proteins/genetics, Rats, Regulatory Sequences, Nucleic Acid/genetics, Sequence Homology, Nucleic Acid, Transcription, Genetic, Tumor Cells, Cultured
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