Date of Award
Master of Science
Dr. Susan R. Stapleton
Dr. Leonard Beuving
Dr. Leonard Ginsberg
Masters Thesis-Open Access
The expression of several genes has been shown to be either positively or negatively regulated by insulin at the transcriptional level, however no single insulin responsive DNA element has yet been identified. The transcription factors responsible for stimulation or inhibition of gene expression by insulin also remain to be identified. In recent years, expression of the gene for glucose-6-phosphate dehydrogenase (G6PDH) in primary rat hepatocytes has been found to be influenced by hormones such as insulin, dexamethosone, and glucagon. The molecular mechanism(s) however, underlying hormonal regulation of G6PDH gene transcription remains to be elucidated. The focus of this study was to characterize the insulin responsive elements which exhibit positive regulation of the glucose-6-phosphate dehydrogenase gene by insulin.
In this study, a segment (+57 to -878) of the rat G6PDH promoter region was cloned into a plasmid containing the luciferase reporter gene. Serial 5' deletions were used to define minimal sequences required for insulin regulation. The results suggest that the insulin responsive element(s) within the G6PDH promoter may be located in the region from -130 to -50. Gel mobility shift assays using the sequence from -130 to -39 as a probe revealed nuclear factor(s) from primary rat hepatocytes that complexed with this sequence. Competition for the nuclear factor(s) using unlabeled probe, determined that this DNA-protein interaction is specific. Deoxyribonuclease I (DNase I) footprinting revealed a protected region spanning from -126 to -99.
Holmen, "Identification of an Insulin-Responsive Element in the Rat Glucose-6-Phosphate Dehydrogenase Gene" (1995). Master's Theses. 4504.