Christopher J. Rhodes Ph.D.

Regulation of Insulin Production

Insulin is initially synthesized as a precursor molecule, preproinsulin, exclusively in pancreatic islet ß-cells. The signal peptide on preproinsulin is cleaved co-translationally to yield newly synthesized proinsulin enabling its entrance into ß-cell's secretory pathway. Later on in the secretory pathway, as a new secretory granule is forming (known as ß-granules – the intracellular compartment of the ß-cell in which insulin is stored), proinsulin is cleaved by limited proteolysis at two specific sites on the molecule (marked by dibasic amino acids) to yield C-peptide and insulin with its A- and B-chain correctly aligned.

Under normal circumstances, the pancreatic ß-cell retains a remarkable state where nutrient-stimulated insulin secreted is rapidly replenished by a parallel upregulation of proinsulin biosynthesis, so that intracellular stores of insulin are maintained at optimal levels. The predominant regulation of proinsulin biosynthesis is mediated at the translational level, however the molecular mechanism behind this nutrient-induced control has been elusive. It should be noted that this is a specific regulation of proinsulin biosynthesis above that of general protein synthesis in the ß-cell. Recently, we have shown that there is a key cis-element found in the 5'-untranslated region of preproinsulin mRNA (named ppIGE for preproinsulin glucose element (pronounced pig-E)) that is required for glucose-induced translational control of proinsulin biosynthesis. There is an islet ß-cell protein that specifically binds to ppIGE (named ppIGE-BP) in a glucose-dependent manner, for which the laboratory has a concerted effort to identify. Once identified, an examination of the post-translational modification of ppIGE-BP and its interaction with ppIGE will be key to a better understanding of the translational regulation of proinsulin biosynthesis both in normal and diabetic situations. Proinsulin biosynthesis is coordinated with that of a subset of ~50 proteins which are also packaged into a ß-granule. This includes the endopeptidases, PC1/3 and PC2, that convert proinsulin to C-peptide and insulin and provides a mechanism whereby the tools for proinsulin processing are regulated in parallel to their substrate. Intriguingly, the ppIGE is also found in the 5'-untranslated regions of PC1/3 and PC2 and are thus their biosynthesis is likely controlled by a similar means to that of proinsulin.

Regulation of ß-Granule Trafficking

 This involves controlled increases in secondary signals, such as [Ca2+]i, that emanate as a consequence of increased glucose metabolism and are key for triggering insulin exocytosis. _{A series of electron micrographs depicting insulin exocytosis}However, at the cell biology level, the mechanism of insulin secretion in the ß-cell is complex involving transport of ß-granules from an intracellular storage pool to specific-regions on the plasma membrane (conceptually known as "active zones of exocytosis"); docking and priming of a ß-granule adjacent to the plasma membrane; and then a Ca2+-triggered fusion of the ß-granule and the plasma membranes to mark the final exocytotic step and release of the ß-granule contents (including insulin and C-peptide) into the circulation.

The mechanistic links as to how secondary signals regulate such ß-granule trafficking in the ß-cell is relatively undefined. _{Two beta-cells in culture where new (green), middle-aged (yellow) and old (red) B-granules have been fluorescently "tagged".}We are currently investigating molecular mechanisms of directing ß-granules to "active zones of exocytosis" in the ß-cell. Together with Drs. Lou Philipson and Michael Roe we are also generating a series of novel fluorescent reagents specifically direct to ß-granules that will enable the monitoring of ß-granule transport and exocytosis in real time.

There are ~10,000 ß-granules per ß-cell, but, surprisingly, relatively few undergo exocytosis. A ß-granule has a half life of ~3-5 days depending on the insulin secretory demand. Aged ß-granules are retired by intracellular degradation mechanisms such as autophagy, which the laboratory is currently investigating.

Under certain metabolic circumstances, including the pathogenesis of type-2 diabetes, an imbalance between insulin production and secretion arises, and autophagy is upregulated to maintain ß-granule numbers at optimal levels. _{A B-granule entering a multigranular body degradation vacuole by microautophagy.} However, if this upregulation of autophagy is chronic, then autophagic-mediated ß-cell death can occur, contributing to the loss of ß-cells that marks the onset of type-2 diabetes. The laboratory is currently investigating this concept.