β-Cells respond to metabolic demand with functional compensation, followed by decompensation and failure, but it is not clear what controls this progression. We examined if anti-apoptotic Bcl-xL prevents β-cell decompensation under chronic glucose excess.

Autophagy can help protect stressed β-cells. We investigated if disruption of Transcription Factor EB (Tfeb), a transcriptional master-regulator of lysosomes and autophagy, contributes to β-cell failure and death under various ER stress-inducing conditions. Short-term β-cell autophagy-deficiency (inducible Atg5 knockout) reduced β-cell survival under hypoxic stress in vitro and impaired the ability of syngeneic marginal mass islet grafts to maintain glucose homeostasis in streptozotocin (STZ)-induced diabetic mice, with >80% of recipients of Atg5-deficient islets returning to diabetes within 10 days post-transplant (vs. 30% of controls, p=0.05). Hypoxic culture activated pro-apoptotic ER stress pathways, and confocal analysis of islet cells from LC3-RFP-EGFP autophagy reporter mice demonstrated that autophagic flux was interrupted. Hypoxia-induced impairment of autophagy was associated with reduced lysosomal cathepsin B activity and a significant loss of Tfeb, both of which could be partially rescued by Torin-1. Lipotoxicity and thapsigargin-induced ER-stress similarly reduced Tfeb protein and cell viability, suggesting ER stress-specific mechanisms may drive dysregulation of Tfeb and lysosomes. Conversely, overexpression of Tfeb:GFP reduced thapsigargin-induced death in MIN6 cells. To directly study the consequences of Tfeb loss we generated mice with Ins1Cre-driven, β-cell-specific, Tfeb deletion (TfebβKO). Notably, both male and female TfebβKO mice had normal glucose tolerance, but cultured Tfeb KO β-cells showed impaired lysosomal cathepsin B activity and TfebβKO mice had worsened glucose tolerance compared to Tfeb flox littermates following injection with multiple low-doses STZ. In summary, autophagy protects β-cells under hypoxia but is impaired under prolonged hypoxic stress, possibly through Tfeb dysregulation. Our data further suggest loss of Tfeb is a common feature of ER stress and that this loss exacerbates stress-induced β-cell failure and death. Disclosure Y. Zou: None. D. Pasula: None. B. Verchere: Advisory Panel; Self; Integrated Nanotherapeutics Inc., Sirona Biochem, Stock/Shareholder; Self; Integrated Nanotherapeutics Inc. D. S. Luciani: None. Funding JDRF (2-2013-50)

Proinsulin (PI) is processed to mature insulin by prohormone convertases 1/3 (Pcsk1) and 2 (Pcsk2). In individuals with T1D and T2D, there is an increase in the circulating PI:insulin ratio. We hypothesized that β-cell Pcsk1 and Pcsk2 deletion would result in β-cell dysfunction, loss of insulin production, and marked hyperglycemia. Single Pcsk1flox/flox Ins1cre/+ (Pcsk1βKO), Pcsk2flox/flox Ins1cre/+ (Pcsk2βKO), and double Pcsk1flox/flox Pcsk2flox/flox Ins1cre/+ (DPCβKO) β-cell prohormone convertase knockout mice were generated (with respective Pcsk1+/+ Pcsk2+/+ Ins1cre/+ controls) and monitored until 30 weeks old. Pcsk2βKO mice displayed no glycemic phenotype, while Pcsk1βKO mice displayed mild hyperglycemia and impaired glucose tolerance by 26 weeks of age. DPCβKO mice displayed mild hyperglycemia (10.1±1.6 vs. 12.3±2.1 mM; p=0.006) and marked glucose intolerance by 10 weeks of age (AUC: 418±135 vs. 1485±360; p<0.0001). No animals displayed obesity relative to controls. Insulin+ pancreas area was doubled in DPCβKO male mice (p=0.0004) and DPCβKO islets showed early glucose-induced Ca2+ influx and loss of oscillatory Ca2+ flux. Pcsk2βKO islets had unaltered mature insulin, while Pcsk1βKO islets had reduced mature insulin. DPCβKO islets contained no detectable mature insulin by western blot. In contrast, Pcsk2βKO islets had markedly reduced mature islet amyloid polypeptide (IAPP), while Pcsk1βKO islets contained normal mature IAPP levels. Our results suggest that complete loss of β-cell prohormone processing, including PI processing, can drive β-cell dysfunction and expansion, but only mild diabetes and glucose intolerance. Although Pcsk2 deletion in β cells does not impact glycemia or PI processing, it is required for normal proIAPP processing. In total, our data support the idea that altered β-cell prohormone processing does not impact obesity but can contribute to the pathogenesis of diabetes via β-cell dysfunction. Disclosure A. Taylor: None. Y. Chen: None. D. Pasula: None. D. S. Luciani: None. B. Verchere: Advisory Panel; Self; Integrated Nanotherapeutics Inc., Sirona Biochem, Stock/Shareholder; Self; Integrated Nanotherapeutics Inc. Funding Canadian Institutes of Health Research

Nutrient stress contributes to β-cell failure in type 2 diabetes. We previously reported that antiapoptotic Bcl-xL dampens β-cell glucose signalling. Here, we examined if Bcl-xL is important for β-cell mitochondrial homeostasis during prolonged exposure to excess glucose.

INVESTIGATING THE ROLE OF AXIN2 IN THE REGULATION OF THE WNT SIGNALING PATHWAY IN EARLY ZEBRAFISH DEVELOPMENT Daniel Pasula Advisor: University of Guelph, 2015 Professor T. Van Raay The negative feedback inhibitor Axin2 is known to inhibit Wnt signaling by re-enabling the destruction complex’s role in the degradation of cytoplasmic β-catenin. To better understand how Axin2 regulates Wnt signaling, I tested the hypothesis that the role of Axin2 is to mediate temporal control of Wnt signaling and that loss of Axin2 in zebrafish embryos will prolong Wnt signaling, especially in embryos challenged with excess Wnt ligand. The CRISPR/Cas9 genome editing system was used to knock out axin2 in zebrafish embryos. Loss of Axin2 had no obvious effect on the developing zebrafish embryo and there was no evidence of hyperactivated Wnt signaling. Instead, loss of Axin2 appeared to hyper-sensitize the embryos to excess Wnt signaling, resulting in significant death. Contrary to my hypothesis, loss of Axin2 did not affect the duration of Wnt signaling, but instead appeared to control the intensity of the Wnt signal.