Cloning and sequencing of the 5′ region of the human glucose‐6‐phosphatase gene: transcriptional regulation by cAMP, insulin and glucocorticoids in H4IIE hepatoma cells

Abstract

We have cloned and sequenced the first 1.2 kb of the 5' region of the human glucose-6-phosphatase gene. Transfection of H4IIE hepatoma cells with the 1.2 kb fragment fused to a luciferase reporter gene demonstrated both basal and hormone responsive luciferase activity. Dexamethasone increased and insulin decreased luciferase activity. Insulin and dibutyryl cyclic AMP both significantly decreased activity in the presence of de gamethasone.

Identification of a cAMP response element within the glucose- 6-phosphatase hydrolytic subunit gene promoter which is involved in the transcriptional regulation by cAMP and glucocorticoids in H4IIE hepatoma cells

“…H4IIE cells were transfected using 10 µg of reporter-gene construct and 2 µg of pSV-β-galactosidase control vector per dish as described elsewhere [8]. Following transfection (18 h) the cells were shocked by 15 % glycerol for 2 min and subsequently incubated for 4 h in DMEM.…”

“…HepG2 cells were transfected in similar way, except that only 5 µg of reporter-gene vector and, in some experiments, 1 µg of an expression vector for the glucocorticoid receptor were transfected with the pSV-β-galactosidase control vector per dish. The luciferase activities were normalized by measuring the β-galactosidase activities in the extracts [8]. Each series of experiments was performed at least three times with at least two different plasmid preparations, and each transfection was performed in triplicate.…”

“…The 5h-flanking region of the human G6Pase gene up to nt k3920 relative to the transcription start site was obtained from a previously described cosmid clone [8] and sequenced in both directions by a combination of automated fluorescence sequencing and manual sequencing. The k3920\j57 fragment was cloned into the SacI\XhoI sites of the reporter-gene plasmid pGL3 basic.…”

“…The k3920\j57 fragment was cloned into the SacI\XhoI sites of the reporter-gene plasmid pGL3 basic. The construction of the reporter-gene construct containing the k1227\j57 fragment has been described previously [8]. Deletion mutants were created by exonuclease III (erase-a-base, Promega) or, in the case of the constructs containing the promoter fragments k180\j4, k161\j4, k150\j4 and k63\j4, by PCR using primers with nested XhoI and SacI restriction sites for subsequent cloning into pGL3 basic.…”

“…Glucagon (acting via cAMP) and glucocorticoids increase G6Pase activity by inducing G6Pase gene expression in the liver during starvation [4][5][6][7]. The recent cloning of the promoter regions of the human [8], rat [9] and mouse [10] G6Pase genes has made it possible to study the molecular basis of control of G6Pase gene expression. An insulin response element has recently been described in the G6Pase promoter and shown to be highly similar to one in the phosphoenolpyruvate carboxykinase (PEPCK) gene promoter [10].…”