Insulin production by the pancreatic B-cell involves a number of closely integrated steps. The only way to understand insulin production as a whole, and thus identify the potential defects associated with, or responsible for, diabetes, is to gain a better understanding of the molecular mechanism of these individual steps. This is the goal of the present study. For this it will be necessary to study B-cell subcellular fractions (Golgi subfractions, granule subpopulations, and lysosomes). Since subcellular fractionation demands larger amounts of tissue than are readily available using isolated islets, rat insulinoma cells will be used. The insulinoma cells will first be characterized in terms of proinsulin biosynthesis and conversion, and insulin release, to validate them as a model for B-cell function. In order to modify the density or other properties of the organelles of interest before subfractionation, thereby improving the resolution of the subfractionation procedure, the transport of products (including proinsulin) across the Golgi complex will be blocked with monensin or Tris. Proinsulin conversion to insulin will be blocked by incorporation of arginine and lysine analogs; this results in an associated block in the maturation of coated to mature uncoated granules (the clathrin-coated granule being the earliest immature form). The biochemical/ultrastructural transformations selected for detailed study and the issues raised are: 1) Proinsulin processing by the Golgi comlex: proinsulin must in some way be recognized by the Golgi complex and thereby concentrated in select Golgi regions for channeling towards, and ultimate packaging into, secretory granules. The recognition process could be receptor mediated. Putative proinsulin receptors on Golgi membranes will be studied. 2) Proinsulin conversion to isulin: the precise site of initiation of conversion in the Golgi complex will be localized, and the mechanism responsible for bringing proinsulin and the conversion enzyme into contact studied. 3) Granule heterogeneity: the functional significance of coated and uncoated granules will be studied, with particular reference to the mechanism responsible for the preferential release of newly synthesized insulin. 4) Degradation of insulin stores: the mechanism and regulation of this pathway will be studied. Crinophagy is the favored path but has not been confirmed biochemically. The impact of the insulin crystal on sensitivity of insulin to degradation within lysosomes will be evaluated.
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