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Contribution of distal promoter elements to transcriptional regulation of glucose-dependent insulinotropic polypeptide in intestinal STC-1 cells Yu, Irene Lok Yan

Abstract

Diabetes mellitus is a group of chronic metabolic disorders that are characterized by high blood glucose resulting from a lack of or insufficient secretion of insulin, which is a source of medical and financial burden to more than 285 million people worldwide. Current treatments for diabetes include lifestyle modifications, medication, and insulin therapy, but these treatments do not save patients from diabetic complications including blindness, limb amputations, circulatory disorders, and increased risk of developing kidney failure, cardiovascular diseases, and neuropathies. Glucose-dependent insulinotropic polypeptide (GIP) is a gastrointestinal hormone that plays an integral role in the finely-tuned secretion of insulin following a meal, and the cells that express GIP have demonstrated potential for being a target for insulin gene therapy. Understanding how the GIP gene is regulated will provide insights into the defining characteristics of GIP-expressing cells and how these can be harnessed for therapy. In the present study, two enhancer cis-regulatory elements which accounted for 40-65% of GIP promoter activity were identified in a previously uncharacterized well-conserved region of the distal 5’ upstream rat GIP promoter by a series of luciferase reporter studies. Pax6 and Pdx1, two transcription factors that have been previously shown to be important for GIP expression, were shown to bind at these sites using electrophoretic mobility shift assays, mutational analysis, and chromatin immunoprecipitation. The development of a fluorescence-based isolation technique for primary GIP-expressing cells was documented. Cell numbers (20,000 – 35,000) were purified for the isolation of RNA in sufficient quantity and quality (80-140 ng, and RNA integrity number = 6.8-7.9, respectively) for microarray. The feasibility of isolating primary GIP-expressing cells presents a model which would allow for non-biased screening for the identification of additional trans-regulatory elements which may act at well-established and newly characterized cis-regulatory elements.

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