The major focus of the proposed work will be the study of glucose-regulated insulin biosynthesis in insulinoma cells adapted to culture, and evaluation of the genes which mediate these responses in man. The hypothesis to be tested is that defects in these regulatory genes are responsible for at least some of the inherited susceptibility to NIDDM. There are 4 specific aims: 1) A fingerprinting method for mRNA analysis will be used to define glucose-responsive genes in mouse insulinoma (bTC6-F7) cells. These genes will be cloned and sequenced, and glucose regulation confirmed by RNA analysis. Human cDNAs and genomic clones homologous to the mouse genes will be isolated and characterized. Sequences of promoter regions may delineate common glucose-regulatory cis-elements. 2) The transcription factor Egr-1, an immediate early gene induced by growth factors and other stimuli, is known to regulate mammalian development and gene expression. Egr-1 is a homologue of an important yeast glucose regulatory protein. In preliminary experiments it was shown that Egr-1 is present in bTC6-F7 cells and that its transcription is regulated by glucose. Experiments will be conducted to determine the molecular steps in the process, and to define islet Egr-1 target genes. 3) Novel islet b-cell genes will be assessed as contributors to the inherited susceptibility to NIDDM. Candidates include human homologues of mouse glucose-regulated genes identified in Specific Aim 1 mammalian homologs of genes which are essential for glucose regulation of gene expression in yeast, and islet homeodomain genes. Approaches will be isolation and characterization of human cDNA and genomic clones, chormosomal mapping and identification of simple sequence repeat polymorphisms (SSRPs), linkage analysis in NIDDM families, and analysis of genomic DNA for mutations. 4) Mapping NIDDM susceptibility genes is complicated because of multifactorial etiology. An alternative strategy is to map genes involved in monogenic disorders of b-cell function. The hypothesis to be tested is that genes essential for normal islet b-cell function are mutated in these disorders. Defects in these genes may contribute to susceptibility to other forms of diabetes. The long-range goal of these studies is to provide a molecular genetic classification of NIDDM.
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