UBC Theses and Dissertations
Removing, replacing, and processing proinsulin in beta-cells Ramzy, Adam Rehim
Diabetes affects over 425 million worldwide, costs billions, and causes morbidity and mortality for patients. Though insulin injections are lifesaving, insufficient β-cell mass and function leaves patients facing risks of chronic hyperglycemia and acute risks of hypoglycemia. Replacement of β-cells via transplantation of cadaveric islets is a functional cure but is limited by a paucity of donor tissue. If β-cell replacement or (re)generation therapies were abundantly available, they could be potential cures for diabetes. To this end, investigating β-cell development and function is worthwhile. In the current thesis, we first characterized the role of insulin on β-cell development and maturation by studying insulin knockout mice (Ins1-/-Ins2-/-). Though insulin was necessary for β-cell maturation, insulin replacement by islet transplantation but not insulin injection, supported maturation of endogenous β-cells. Second, we developed and characterized an adeno associated virus (AAV) carrying Cre recombinase regulated by an insulin promoter (AAV Ins1-Cre) for in vivo genetic manipulations. AAV Ins1-Cre produced efficient recombination in β-cells alongside off-target recombination, making it a useful tool when off-target effects are controlled for or deemed unimportant. Third, we assessed the viability of a gene therapy for the Ins1-/-Ins2-/- mouse model of monogenic diabetes. We delivered an insulin gene to β-cells (an Ins1 promoter driving human insulin (INS) or mouse insulin 1 (Ins1)) using AAV Ins1-INS or AAV Ins1-Ins1. Though the AAV delivered the insulin gene to β-cells, Ins1-/-Ins2-/- β-cells retained a processing defect leading to secretion of insulin’s precursor proinsulin. We created adult insulin knockout mice using AAV Ins1-Cre and failed to prevent onset of diabetes with AAV Ins1-Ins1. Finally, in Chapter 5 we assessed the production of mature insulin in human β-cells. Despite consensus on the role of prohormone convertase 2 (PC2) in proinsulin processing, we provide evidence that unlike mouse β-cells, human β-cells produce mature insulin without PC2. This thesis provides insight into the developmental impact of “removing insulin” from β-cells, assesses the viability of a gene therapy “replacing insulin” in β-cells, and revises a longstanding theory on the “processing of proinsulin” in human β-cells. These findings may guide development of gene- and cell- based therapies for diabetes.
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