and specific aims): The basic goal of this proposal is to develop a direct and reproducible means of studying gene expression in the intact rat pancreatic islet by the efficient introduction of hybrid reporter transgene whose activity can be measured based on the resolution of the individual islet cell. The objective of the study is to gain further understanding on the regulation of insulin biosynthesis by glucose. The experimental approach employs a series of adenovirus vectors that express a marker transgene, Green Fluorescent Protein(GFP), under the transcriptional control of the insulin as well as other islet cell gene promoter. The effects of glucose and other physiological parameters on insulin expression under the control of specific promoter constructs will be determined. GFP activity in the living islet will be correlated with other variables such as redox status, calcium and other metabolic or signal transduction intermediates. Characterized activities will be mapped to specific DNA regulatory elements using mutated insulin promoter. Furthermore, the effects of co-infecting genes for candidate regulatory proteins such as metabolic enzymes or transcription factors on insulin(GFP) expression will be tested. In order to test the hypothesis that the functional unit of insulin synthesis and secretion is the entire pancreatic islet and not the isolated beta cell, and that the beta cell population is functionally heterogeneous, the fluorescent marker approach will be used to re-examined many observations previously made using cultured insulioma cell lines or fetal islet cells, as well as to isolate and further analyze beta cell populations on the basis of individual function. Finally, these techniques will also be applied to the observation of the insulin promoter in islet from animal models of Type-II diabetes mellitus where beta cell function is known to be abnormal.
| Meyer, K; Irminger, J C; Moss, L G et al. (1998) Sorting human beta-cells consequent to targeted expression of green fluorescent protein. Diabetes 47:1974-7 |
| de Vargas, L M; Sobolewski, J; Siegel, R et al. (1997) Individual beta cells within the intact islet differentially respond to glucose. J Biol Chem 272:26573-7 |
