cAMP prevents the glucose-mediated stimulation of GLUT2 gene transcription in hepatocytes.
Glucose homoeostasis necessitates the presence in the liver of the high Km glucose transporter GLUT2. In hepatocytes, we and others have demonstrated that glucose stimulates GLUT2 gene expression in vivo and in vitro. This effect is transcriptionally regulated and requires glucose metabolism within the hepatocytes. In this report, we further characterized the cis-elements of the murine GLUT2 promoter, which confers glucose responsiveness on a reporter gene coding the chloramphenicol acetyl transferase (CAT) gene. 5'-Deletions of the murine GLUT2 promoter linked to the CAT reporter gene were transfected into a GLUT2 expressing hepatoma cell line (mhAT3F) and into primary cultured rat hepatocytes, and subsequently incubated at low and high glucose concentrations. Glucose stimulates gene transcription in a manner similar to that observed for the endogenous GLUT2 mRNA in both cell types; the -1308 to -212 bp region of the promoter contains the glucose-responsive elements. Furthermore, the -1308 to -338 bp region of the promoter contains repressor elements when tested in an heterologous thymidine kinase promoter. The glucose-induced GLUT2 mRNA accumulation was decreased by dibutyryl-cAMP both in mhAT3F cells and in primary hepatocytes. A putative cAMP-responsive element (CRE) is localized at the -1074/-1068 bp region of the promoter. The inhibitory effect of cAMP on GLUT2 gene expression was observed in hepatocytes transfected with constructs containing this CRE (-1308/+49 bp fragment), as well as with constructs not containing the consensus CRE (-312/+49 bp fragment). This suggests that the inhibitory effect of cAMP is not mediated by the putative binding site located in the repressor fragment of the GLUT2 promoter. Taken together, these data demonstrate that the elements conferring glucose and cAMP responsiveness on the GLUT2 gene are located within the -312/+49 region of the promoter.
Rencurel
F
F
Centre de Recherche sur l'Endocrinologie Moléculaire et le Développement, C.N.R.S. UPR 1511, Meudon-Bellevue, France.
Waeber
G
G
Bonny
C
C
Antoine
B
B
Maulard
P
P
Girard
J
J
Leturque
A
A
D000818:Animals; D003994:Bucladesine / Q000494:pharmacology; D006528:Carcinoma, Hepatocellular / Q000378:metabolism; D002460:Cell Line; D000242:Cyclic AMP / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D004347:Drug Interactions; D005260:Female; D005786:Gene Expression Regulation / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology*; D051273:Glucose Transporter Type 2; D008099:Liver / Q000166:cytology / Q000187:drug effects*; D008113:Liver Neoplasms / Q000378:metabolism; D009004:Monosaccharide Transport Proteins / Q000096:biosynthesis* / Q000235:genetics; D011401:Promoter Regions, Genetic; D051381:Rats; D017208:Rats, Wistar; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017384:Sequence Deletion; D014158:Transcription, Genetic / Q000187:drug effects*; D014407:Tumor Cells, Cultured
D051273:Glucose Transporter Type 2; D009004:Monosaccharide Transport Proteins; D011993:Recombinant Fusion Proteins; D003994:Bucladesine; D000242:Cyclic AMP; D005947:Glucose