Enhanced insulin gene expression by reduced intracellular glutathione level in insulin secreting cells MIN6.
Glutathione (GSH) participates in deoxidization and elimination of hydrogen peroxide and other reactive oxygen species, and plays an important part in the antioxidant system. To investigate the effect of GSH content on insulin gene expression, we utilized a stable transfectant, designated as ribo-MIN6 cells, which were stably transfected with the ribozyme of gamma-glutamylcysteine synthetase (gamma-GCS), exhibiting approximately 50% reduction of intracellular GSH content. We transiently transfected a luciferase expression vector driven by human preproinsulin gene promoter spanning from -1998 to +237 (pINS-1998/luc) and several deletion constructs into ribo-MIN6. Furthermore, transient transfection with ribozyme vector and pINS-1998/luc into wild-type MIN6 cells was also carried out. Luciferase activity was about 9-fold higher in ribo-MIN6 cells as compared to wild-type MIN6 cells. In the transient transfection of pINS-1998/luc with gamma-GCS ribozyme vector into wild-type MIN6 cells, the luciferase activity was increased in proportion to the added amounts of ribozyme vector. In transfection with deletion constructs, two major sites were found to be critical for insulin promoter activity. For the wild-type MIN6 cells, regions important for the promoter activity were also located at regions similar to those of ribo-MIN6 cells. Our results suggest that the suppression of intracellular GSH level might, in part, regulate the insulin gene expression.
Ozaki
M
M
First Department of Internal Medicine, Nagasaki University School of Medicine, Nagasaki, Japan.
Yamasaki
H
H
Kondo
H
H
Yamasaki
S
S
Fujita
N
N
Yamauchi
M D
MD
Abe
T
T
Yamakawa
K
K
Sera
Y
Y
Uotani
S
S
Kawasaki
E
E
Takino
H
H
Kijima
H
H
Kondo
T
T
Yamaguchi
Y
Y
Eguchi
K
K
D000818:Animals; D002460:Cell Line; D005786:Gene Expression Regulation; D005721:Glutamate-Cysteine Ligase / Q000378:metabolism; D005978:Glutathione / Q000378:metabolism*; D007150:Immunohistochemistry; D007328:Insulin / Q000235:genetics* / Q000378:metabolism*; D000078790:Insulin Secretion; D008156:Luciferases / Q000235:genetics / Q000378:metabolism; D051379:Mice; D011401:Promoter Regions, Genetic; D014162:Transfection
D007328:Insulin; D008156:Luciferases; D005721:Glutamate-Cysteine Ligase; D005978:Glutathione