Pancreatic Beta Cell Autophagy is Impaired in Type 1 Diabetes

Aims/hypothesis: Pancreatic beta cells are highly metabolic secretory cells that are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species (ROS) during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce ROS to protect against apoptosis both in vitro and in vivo. While impaired islet autophagy has been demonstrated in human type 2 diabetes, it is unknown if islet autophagy is perturbed in the pathogenesis of type 1 diabetes. We hypothesized that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development. Methods: Mouse pancreata were collected from chloroquine injected and non-injected NOR, nondiabetic NOD, and diabetic NOD mice. Age and BMI-matched pancreas tissue sections from human organ donors (n=34) were obtained from the Network for Pancreatic Organ Donors with Diabetes (nPOD). To assess autophagic flux, we injected the mice with chloroquine to inhibit the final stages of autophagy. We analyzed tissues for markers of autophagy via immunofluorescence analysis. Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), LC3A/B (autophagosome marker), Lamp1 (lysosome marker), and p62 (autophagy adaptor protein and marker for autophagic flux). Images were collected on a scanning laser confocal microscope then analyzed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes (formerly called lipofuscin bodies, residual bodies or tertiary lysosomes) were analyzed in electron micrographs of pancreatic tissue sections from human organ donors (nPOD; n=12) deposited in www.nanotomy.org/OA/nPOD. Energy Dispersive X-ray (EDX) analysis was also performed on these tissues to analyze distribution of elements such as nitrogen, phosphorus, and osmium in secondary lysosomes and telolysosomes of nondiabetic and autoantibody positive donor tissues (n=5). Results: We observed increased autophagosome numbers in islets of diabetic NOD mice (p=0.0077) and increased p62 in islets of both nondiabetic and diabetic NOD mice (p<0.0001 in both cases) when compared to NOR mice. There was also a significant reduction in autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to both nondiabetic NOD mice (p=0.0004) and NOR mice (p=0.0003). Chloroquine infusions elicited accumulation of autophagosomes in the islets of NOR (p=0.0029) and nondiabetic NOD mice (p<0.0001), but not in the islets of diabetic NOD mice. Chloroquine also stimulated an accumulation of the autophagy adaptor protein p62 in the islets of NOR mice (p<0.001), however this was not observed in NOD mice (regardless of diabetes status). In the human pancreata, we observed significantly reduced autophagosome:lysosome colocalization (p=0.0002) in the residual beta cells of donors with type 1 diabetes compared to nondiabetic controls. We also observed reduced colocalization of proinsulin with lysosomes in the residual beta cells of donors with type 1 diabetes compared to both nondiabetic (p<0.0001) and autoantibody positive donors (p<0.0001). Electron microscopy based analysis of human pancreas sections also revealed accumulation of telolysosomes in beta cells of autoantibody positive donors (p=0.0084), the majority of which had an nitrogen dense ring outside a phospholipid core. Conclusions/interpretation: Collectively, we provide evidence of impairment in the final degradation stages of islet macroautophagy and crinophagy in human type 1 diabetes. We also document an accumulation of telolysosomes with nitrogen accumulation at their periphery in the beta cells of autoantibody positive donors. This demonstrates clear differences in the lysosome contents of autoantibody positive donors that may be associated with lysosome dysfunction prior to clinical hyperglycemia. We observe similar impairments in macroautophagy in the diabetic NOD mouse, a model of type 1 diabetes, suggesting that this mouse model can be appropriately used to study the pathogenesis of autophagy/crinophagy loss and how it relates to disease initiation and progression. Considering these data in the context of what is known regarding the cell-protective effects of islet autophagy, we suggest targeting beta cell autophagy pathways as an approach to reduce apoptosis in individuals at risk for type 1 diabetes development.