Characterization of the human skeletal muscle glycogen synthase gene (GYS1) promoter.

Abstract

Impaired activation of the human skeletal muscle glycogen synthase by insulin is typical for type 2 diabetic patients. Regulation of glycogen synthase occurs mainly by phosphorylation/dephoshorylation but little is known whether there also is transcriptional regulation. Therefore we studied transcriptional regulation of the human skeletal muscle glycogen synthase gene (GYS1) and evaluated the effects of insulin and forskolin on the promoter activity. Seven promoter fragments were expressed in C2C12 myoblasts and myotubes and in HEK293 cells, and the luciferase assay was used to determine transcriptional activity. The highest luciferase activity, 350-fold of the promoterless vector, was obtained with nucleotides -692 to +59 in myotubes (P < 0.001), while the nucleotides -250 to +59 provided the highest, 45-fold, activity in the HEK293 cells (P < 0.001). Longer promoter constructs (nucleotides -971, -1707 and -2158 to +59, respectively) had low promoter activity in both cell types. Forskolin treatment for 24 h resulted in approximately 30% decreased promoter activity in myotubes (P < 0.05). Insulin treatment for 0.5-3 h did not increase GYS1 promoter activity; instead the activity was slightly but significantly decreased after 24 h in myotubes (P < 0.005). From our results we conclude that basal GYS1 promoter activity is obtained from the first 250 nucleotides of the promoter, while the nucleotides -692 to -544 seem to be responsible for muscle-specific expression, and nucleotides -971 to -692 for negative regulation. In myotubes, the GYS1 promoter was sensitive to negative regulation by forskolin, whereas insulin did not increase GYS1 transcription.