The electrical, ionic, and secretory responses of pancreatic beta cells to glucose are mediated by the metabolism of the sugar. Glucokinase (GK) catalyzes the initial and rate limiting step in the utilization of glucose by the beta cell at physiologic glucose concentrations and is thought to play an important role in modulating insulin secretion. To study the function and regulation of GK in the beta cell further, we have characterized cDNAs encoding GK from both the liver and insulinoma tissue and have mapped the mapped the transcription units in both tissues. Our studies have shown there are two different promoters in a single GK gene. One promoter is expressed specifically in the liver and appears to be regulated by insulin while another promoter, located further upstream, appears active only in the beta cell. We are proposing to study the function and regulation of GK in insulinoma cell lines (Aim 1) and to examine the GK beta cell promoter using a fusion gene analysis (Aim 2).
In Aim 1 complimentary approaches will be used to test the function of GK as the beta cell """"""""glucose sensor"""""""". By altering the expression of GK in two different insulinoma cell lines we hope to alter the """"""""set point"""""""" of glucose-stimulated insulin secretion. RINm5F cells have normal glucose uptake but appear to lack a high-Km glucose phosphorylating activity and do not secrete insulin in response to glucose. Another cell line, beta-TC, shows high-Km glucose usage but is stimulated to secrete insulin by less than normal glucose concentrations. We will determine the effects of expressing GK in the RINm5f cells and the effects of expressing GK antisense RNA in the beta-TC cells.
In Aim 2 an analysis of the beta cell GK promoter will be undertaken using a fusion gene approach. Both a 5' deletional analysis and the use of internal deletion mutants should enable us to identify elements necessary for the beta cell-specific expression of GK. Elements important for the cell type expression of GK are likely to bind factors which are important for beta cell-specific gene expression. These factors will be studied further using mobility shift and methylation interference assays. Efforts to purify and clone one of these factors will be initiated.
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