Insulin Null β-cells Have a Prohormone Processing Defect That Is Not Reversed by AAV Rescue of Proinsulin Expression

Abstract

Up to 6% of diabetes has a monogenic cause including mutations in the insulin gene and patients are candidates for a gene therapy. Using a mouse model of permanent neonatal diabetes, we assessed the efficacy of an adeno-associated virus (AAV) mediated gene therapy. We used AAVs with a rat insulin 1 promoter (Ins1) regulating a human insulin gene (INS; AAV Ins1-INS) or native mouse insulin 1 (Ins1; AAV Ins-Ins1) to deliver an insulin gene to β-cells of constitutive insulin null mice (Ins1 -/-Ins2 -/-) and adult inducible insulin deficient mice (Ins1 -/-Ins2 f/f PdxCreER and Ins1 -/-Ins2 f/f mice administered AAV Ins1-Cre). Though AAV Ins1-INS could successfully infect and confer insulin expression to β-cells, insulin null β-cells had a prohormone processing defect. Secretion of abundant proinsulin transiently reversed diabetes. We reattempted therapy with an AAV carrying mouse Ins1, but Ins1 -/-Ins2 -/- β-cells still had a processing defect of both replaced Ins1 and pro-islet amyloid polypeptide. In adult inducible models, β-cells that lost insulin expression developed a processing defect that resulted in impaired proIAPP processing and elevated circulating proIAPP, and cells infected with AAV Ins1-Ins1 to rescue insulin expression secreted proinsulin. We assessed the subcellular localization of PC1/3 and detected defective sorting of PC1/3 to glycogen-containing vacuoles and retention in the ER as a potential mechanism underlying defective processing. We provide evidence that persistent production of endogenous proinsulin within β-cells is necessary for β-cells to be able to properly store and process proinsulin.

Loss of VPS41 triggers rapid insulin degradation and dysregulated autophagy in pancreatic beta cells

“…Pdx1 expression is essential for both the cellular development of insulin-positive beta-cells and maintenance of beta-cell identity[34]. It is also appreciated that consistent proinsulin production is necessary for maintenance of beta-cell identity; dual Ins1 and Ins2 genetic KO mice develop proinsulin processing defects that cannot be rescued with insulin gene re-expression[35], which we similarly observed in our attempts to re-express proinsulin in VPS41 KO INS1 cells (Figure 1E). Ultimately, we find that VPS41 deletion generates a chronically insulin-deficient beta-cell that eventually undergoes a dedifferentiation phenotype.…”