Two independent cis-acting insulin response elements (IREs) in the gene encoding glyceraldehyde-3-phosphate dehydrogenase [D-glyceraldehyde-3-phosphate: NAD+ oxidoreductase (phosphorylating), EC 1.2.1.12], designated IRE-A and IRE-B, are sufficient to direct insulininducible gene expression. Using the electrophoretic mobility shift assay, a 4-fold increase in the amount of IRE-A DNA bound to nuclear proteins was detected when extracts isolated from insulin-stimulated differentiated 3T3-L1 cells or from the liver of rats refed a high-carbohydrate/low-fat diet after a 72-hr fast were compared to control nuclear extracts. The points of contact between protein and IRE-A DNA may represent a sequence recognized by at least one class of insulinsensitive transcription factor(s).The interaction of insulin with its cell surface receptor initiates changes in the activity and cellular content of metabolic enzymes that promote energy storage and cell growth. Although many genes have been described recently to be regulated by insulin at the level of transcription, the molecular mechanism by which insulin mediates its effects is unknown. In an ongoing attempt to elucidate the signal transduction pathway of insulin action on gene expression, we have attempted to define cis-acting sequences that mediate the effect of insulin on gene transcription and work backward to define the mechanism by which the trans-acting factors that interact with these sequences are regulated.We previously isolated an insulin-responsive gene for glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (1) and showed that the expression of a hybrid reporter gene containing bases -488 to + 21 of the human gene for GAPDH and the coding region of the gene encoding chloramphenicol acetyltransferase (CAT), designated plasmid HGAPDH-CAT, was increased 3-fold in insulin-treated differentiated 3T3-L1 cells and 5-fold in H35 hepatoma cells that were stably transfected with this hybrid gene (2). An increase in HGAPDH-CAT mRNA was detected within 4 hr after insulin exposure and was maintained overnight, a pattern that parallels the time course of GAPDH mRNA induction (3).These results indicated that the human gene for GAPDH contained a cis-acting element capable of conferring insulin inducibility to a marker gene. As it appears that binding of cellular transcription factors to inducible cis-acting elements is required for function, we used the gel shift assay to detect nuclear factors that bind this element in a specific manner. This paper describes the regulation of an insulin-inducible factor detected in vitro with the differentiated 3T3-L1 cultured cell line and in vivo in the liver of fasted rats refed a high-carbohydrate/low-fat diet, a model in which circulating insulin levels are high and many metabolic enzymes required to promote energy storage are induced.
Chronic benzodiazepine administration has been associated with alterations in binding and function at the GABAA receptor. To evaluate effects of chronic benzodiazepine exposure on messenger RNA (mRNA) concentrations for several GABAA receptor subunits, we treated mice with lorazepam, 2 mg kg1-daily for 1-28 days and evaluated mRNAs for the alphal and gamma2 subunits by Northern hybridization. In cerebral cortex, concentrations of mRNA for the alphal and gamma2 subunits were unchanged from vehicle or control after 1-10days of lorazepam. However, after 14days of treatment mRNA concentrations for both subunits decreased to approximately 50% of control values and remained decreased at 28 days. In contrast, no significant alterations were observed for either subunit mRNA in hippocampus or cerebellum over the same time course. Alterations in mRNAs in cortex occur after the development of tolerance and receptor downregulation in this model.
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