Abstract
Aims/hypothesisPancreatic beta cells are subjected to exogenous damaging factors such as proinflammatory cytokines or excess glucose that can cause accumulation of damage-inducing reactive oxygen species during the pathogenesis of diabetes. We and others have shown that beta cell autophagy can reduce reactive oxygen species to protect against apoptosis. 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 hypothesised that beta cell autophagy is dysfunctional in type 1 diabetes, and that there is a progressive loss during early diabetes development.MethodsPancreases were collected from chloroquine-injected and non-injected non-obese diabetes-resistant (NOR) and non-obese 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). Tissue sections were stained with antibodies against proinsulin or insulin (beta cell markers), microtubule-associated protein 1 light chain 3 A/B (LC3A/B; autophagosome marker), lysosomal-associated membrane protein 1 (LAMP1; lysosome marker) and p62 (autophagy adaptor). Images collected on a scanning laser confocal microscope were analysed with CellProfiler and ImageJ. Secondary lysosomes and telolysosomes were assessed in electron micrographs of human pancreatic tissue sections (n = 12), and energy dispersive x-ray analysis was performed to assess distribution of elements (n = 5).ResultsWe observed increased autophagosome numbers in islets of diabetic NOD mice (p = 0.008) and increased p62 in islets of both non-diabetic and diabetic NOD mice (p < 0.001) vs NOR mice. There was also a reduction in LC3–LAMP1 colocalisation in islets of diabetic NOD mice compared with both non-diabetic NOD (p < 0.001) and NOR mice (p < 0.001). Chloroquine elicited accumulation of autophagosomes in the islets of NOR (p = 0.003) and non-diabetic NOD mice (p < 0.001), but not in islets of diabetic NOD mice; and stimulated accumulation of p62 in NOR (p < 0.001), but not in NOD mice. We observed reduced LC3–LAMP1 colocalisation (p < 0.001) in residual beta cells of human donors with type 1 diabetes vs non-diabetic participants. We also observed reduced colocalisation of proinsulin with LAMP1 in donors with type 1 diabetes (p < 0.001). Electron microscopy also revealed accumulation of telolysosomes with nitrogen-dense rings in beta cells of autoantibody-positive donors (p = 0.002).Conclusions/interpretationWe provide evidence of islet macroautophagy/crinophagy impairment in human type 1 diabetes. We also document accumulation of telolysosomes with peripheral nitrogen in beta cells of autoantibody-positive donors, demonstrating altered lysosome content that may be associated with lysosome dysfunction before clinical hyperglycaemia. Similar macroautophagy impairments are present in the NOD mouse model of type 1 diabetes.Graphical abstract
Highlights
Beta cell reactive oxygen species (ROS) accumulation is implicated as a triggering event during the development of type 1 diabetes [1]
Evidence of impaired autophagy in diabetic nonobese diabetic (NOD) mice To determine if autophagy is impaired in a mouse model of spontaneous autoimmune diabetes, we analysed pancreases from diabetic NOD mice, comparing them with both non-diabetic NOD mice and non-obese diabetes-resistant (NOR) mice, which do not develop insulitis or diabetes and are MHC-matched to NOD mice [19]
We observed a significant increase in LC3 puncta in diabetic NOD mouse islets (p = 0.008) and a nonsignificant increase in autophagosomes in non-diabetic NOD islets (p = 0.083) when compared with islets of NOR mice (Fig. 1b)
Summary
Beta cell reactive oxygen species (ROS) accumulation is implicated as a triggering event during the development of type 1 diabetes [1]. Low levels of ROS can promote important signalling events in the beta cell such as insulin secretion [3] and proliferation [4], excess ROS can damage cellular proteins and organelles and overwhelm endogenous mechanisms that maintain homeostasis [5]. Autophagy is an endogenous mechanism to reduce ROS and promote beta cell survival [6]. Known types of autophagy, including macroautophagy, chaperone-mediated autophagy, microautophagy and crinophagy, function as important housekeeping catabolic processes to facilitate recycling of excess or damaged cellular components and promote cellular homeostasis [7]. Macroautophagy (autophagy) is a dynamic process involving a cascade of regulated events leading to engulfment of damaged proteins or organelles into doublemembraned autophagosomes containing microtubuleassociated protein 1 light chain 3 (MAP1LC3, or LC3), a widely used marker for mature autophagosomes. The mechanism of crinophagy is not well understood, this process plays a key role in the regulation of insulin granules [10]
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