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-/-Ins2f/f PdxCreER and Ins1-/-Ins2f/f mice administered AAV Ins1-Cre)]. Although 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 AAV Ins1-Ins1, but Ins1-/-Ins2-/- β-cells still had a processing defect of both replaced Ins1 and pro-islet amyloid polypeptide (proIAPP). 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 prohormone convertase 1/3 (PC1/3) and detected defective sorting of PC1/3 to glycogen-containing vacuoles and retention in the endoplasmic reticulum 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.

Insulin receptor (IR) insufficiency in β-cells leads to impaired insulin secretion and reduced β-cell hyperplasia in response to hyperglycemia. Selective IR deficiency in β-cells in later embryological development may lead to compensatory β-cell hyperplasia. Although these findings suggest insulin signaling on the β-cell is important for β-cell function, they are confounded by loss of signaling by the insulinlike growth factors through the IR. To determine whether insulin itself is necessary for β-cell development and maturation, we performed a characterization of pancreatic islets in mice with deletions of both nonallelic insulin genes (Ins1-/-Ins2-/-). We immunostained neonatal Ins1-/-Ins2-/- and Ins1+/+Ins2+/+ pancreata and performed quantitative polymerase chain reaction on isolated neonatal islets. Insulin-deficient islets had reduced expression of factors normally expressed in maturing β-cells, including muscoloaponeurotic fibrosarcoma oncogene homolog A, homeodomain transcription factor 6.1, and glucose transporter 2. Ins1-/-Ins2-/-β-cells expressed progenitor factors associated with stem cells or dedifferentiated β-cells, including v-myc avian myolocytomatosis viral oncogene lung carcinoma derived and homeobox protein NANOG. We replaced insulin by injection or islet transplantation to keep mice alive into adulthood to determine whether insulin replacement was sufficient for the completed maturation of insulin-deficient β-cells. Short-term insulin glargine (Lantus®) injections partially rescued the β-cell phenotype, whereas long-term replacement of insulin by isogenic islet transplantation supported the formation of more mature β-cells. Our findings suggest that tightly regulated glycemia, insulin species, or other islet factors are necessary for β-cell maturation.