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The catabolic pathway of autophagy is critical for pancreatic beta-cell function and is defective in established type 1 diabetes (T1D). However, it is unclear when and how this critical cell process becomes defective during diabetes pathogenesis. To study the nature of autophagy dysfunction in the context of autoimmune diabetes, we utilized intravital microscopy to study autophagic fluxin vivoin real time. We generated a custom AAV8- packaged mCherry-eGFP-LC3B biosensor driven by the insulin promoter for beta-cell-selective expression. For real time autophagic flux evaluation, fluorescent signal from eGFP and mCherry fluorophores was correlated in space and time to follow the process of autophagosome-lysosome fusion. We observed autophagic flux defects in the beta-cells of non-obese diabetic (NOD) mouse model of T1D prior to hyperglycemia onset that were less apparent in mice without a functional immune system. We also evaluated autophagic flux in human donor islets that were transplanted under the kidney capsule of immune incompetent mice. Collectively, we provide the first evaluation of autophagic fluxin vivoin 4D and demonstrate that autophagy defects precede hyperglycemia in NOD mice suggesting a potential causative role for these defects in beta-cell demise during T1D pathogenesis.

Metabolic dysfunction of β-cells has been implicated as a contributor to diabetes pathogenesis, and efforts are ongoing to optimize analytical techniques that evaluate islet metabolism. High-resolution respirometry offers sensitive measurements of the respiratory effects of metabolic substrates and customizable manipulation of electron transport chain components, though the delicate nature of islets can pose challenges to conventional analyses. An affordable and reliable option for respirometry is the Oroboros Oxygraph-2 K system, which utilizes a stir bar to circulate reagents around cells. While this technique may be suitable for individual cells or mitochondria, the continual force exerted by the stir bar can have damaging effects on islet integrity. Herein, we demonstrate the protective benefits of a novel 3D-printed islet stabilization device and highlight the destructive effects of conventional Oxygraph analysis on islet integrity. Islet containment did not inhibit cellular responses to metabolic modulatory drugs, as indicated by robust fluctuations in oxygen consumption rates. The average size of wild-type mouse islets was significantly reduced following a standard Mito Stress Test within Oxygraph chambers, with a clear disruption in islet morphology and viability. Alternatively, containment of the islets within the interior chamber of the islet stabilization device yielded preservation of both islet morphology and increased cell viability/survival after respirometry analysis. Collectively, our study introduces a new and easily accessible tool to improve conventional Oxygraph respirometry of pancreatic islets by preserving natural islet structure and function throughout metabolic analysis.

Aims/hypothesis Pancreatic 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. Methods Pancreases 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). Results We 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 p p p = 0.003) and non-diabetic NOD mice (p p p p p = 0.002). Conclusions/interpretation We 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

Objective Adult polyglucosan body disease (APBD) is an adult‐onset neurological variant of glycogen storage disease type IV. APBD is caused by recessive mutations in the glycogen branching enzyme gene, and the consequent accumulation of poorly branched glycogen aggregates called polyglucosan bodies in the nervous system. There are presently no treatments for APBD. Here, we test whether downregulation of glycogen synthesis is therapeutic in a mouse model of the disease. Methods We characterized the effects of knocking out two pro‐glycogenic proteins in an APBD mouse model. APBD mice were crossed with mice deficient in glycogen synthase (GYS1), or mice deficient in protein phosphatase 1 regulatory subunit 3C (PPP1R3C), a protein involved in the activation of GYS1. Phenotypic and histological parameters were analyzed and glycogen was quantified. Results APBD mice deficient in GYS1 or PPP1R3C demonstrated improvements in life span, morphology, and behavioral assays of neuromuscular function. Histological analysis revealed a reduction in polyglucosan body accumulation and of astro‐ and micro‐gliosis in the brains of GYS1‐ and PPP1R3C‐deficient APBD mice. Brain glycogen quantification confirmed the reduction in abnormal glycogen accumulation. Analysis of skeletal muscle, heart, and liver found that GYS1 deficiency reduced polyglucosan body accumulation in all three tissues and PPP1R3C knockout reduced skeletal muscle polyglucosan bodies. Interpretation GYS1 and PPP1R3C are effective therapeutic targets in the APBD mouse model. These findings represent a critical step toward the development of a treatment for APBD and potentially other glycogen storage disease type IV patients.

Autophagic degradation is a dynamic recycling process that contributes to cellular homeostasis and mitigation of cellular stress. While there are multiple forms of -phagic degradation processes, a common endpoint is the final degradation step in the lumen of the acidic lysosomes. Post-mitotic cells, including beta cells, can accumulate tertiary lysosomes called telolysosomes which contain oxidized proteins and lipids that are not completely digested. We recently found that autophagy is impaired in the beta cells of individuals with T1D and also in islets of diabetic NOD mice. We also observed a significant increase in the number of autophagosomes, and telolysosomes in the beta cells of autoantibody positive organ donors when compared to nondiabetic organ donors. We now also find a 1.5-fold increase in the number of telolysosomes in the alpha cells of autoantibody positive donors when compared to nondiabetic organ donors (p=0.03). This was mirrored by our previous observation of globally increased lysosome numbers across the islets of pre-diabetic NOD mice, suggesting that these lysosomes may be dysfunctional. To test this, we performed a lysosomal enzyme activity assay to analyze the lysosomal cysteine protease Cathepsin H (a T1D susceptibility locus). Interestingly, we found that the activity of Cathepsin H was increased by ~2-fold in islets of pre-diabetic NOD mice when compared to NOR controls (p=0.02). Taken together, these data suggest a compensatory hyperactivation of the lysosomal enzyme Cathepsin H in the islets of pre-diabetic NOD mice, potentially as a mechanism to rid the cell of degradation-resistant oxidized proteins and lipids whose accumulation in lysosomes might lead to the presence of telolysosomes. Disclosure C. Muralidharan: None. J. Crowder: None. A. K. Linnemann: None. Funding Network for Pancreatic Organ Donors with Diabetes (nPOD; RRID:SCR_014641); JDRF (nPOD: 5-SRA-2018-557-Q-R); The Leona M. and Harry B. Helmsley Charitable Trust (2018PG-T1D053)

β-cells are highly metabolic and vulnerable to a variety of exogenous stressors, many of which elicit the generation of reactive oxygen species (ROS). Endogenous adaptive responses can restore homeostasis after excessive ROS elevation. If the endogenous adaptive response fails, ROS accumulation can cause damage to DNA, proteins, and organelles, ultimately leading to apoptosis. A wealth of evidence supports that ROS accumulation contributes to β-cell dysfunction and destruction during diabetes development. These findings suggest that stimulating the antioxidant response could prevent or treat diabetes. However, limitations exist in that persistent systemic activation of the antioxidant response can perturb the normal second messenger function of ROS, and that exogenous antioxidants have poor bioavailability and lack specificity. To overcome these obstacles, we developed β-cell targeted nanoparticles to selectively deliver antioxidants to β-cells. We conjugated modified Exendin-4 (Ex4) to an amphiphilic polymer, allowing us to generate lipid nanoparticles to deliver small hydrophobic compounds to GLP-1R expressing cells. We assessed β-cell targeting of our nanoparticles using isolated islets from mice expressing tdTomato selectively in the β-cell. Isolated islets were treated with nanoparticles containing a lipid intercalating dye, DiO, and we observed selective DiO accumulation in tdTomato positive cells. We also performed in vivo imaging of live mice injected with targeted or non-targeted nanoparticles containing a near-IR lipid intercalating dye. These experiments demonstrated selective retention of targeted nanoparticles both systemically and in the pancreata of animals 1 hour after tail vein injection. Collectively, we provide evidence that Ex4 coated micelles are selective to β-cells. Future experiments will explore this targeted approach to deliver drugs to stimulate the antioxidant response and reduce β-cell apoptosis under diabetogenic conditions. Disclosure A. Novak: None. T. Nallan chakravarthula: None. J. Crowder: None. N. J. Alves: None. J. Broichhagen: None. D. Hodson: None. A. Bedwell: None. A. K. Linnemann: None.

Aims/hypothesisPancreatic 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.MethodsMouse 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).ResultsWe 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 (pConclusions/interpretationCollectively, 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.Research in contextWhat is already known about this subject?Autophagy confers a cytoprotective role in the beta cell.Autophagy is reduced in type 2 diabetes.Autophagy in the context of type 1 diabetes is unexplored.What is the key question?Is autophagy reduced during the pathogenesis of human type 1 diabetes?What are the new findings?We provide evidence of reduced autophagy and crinophagy in human type 1 diabetes.These data are supported by observations of reduced autophagy in a mouse model of autoimmune diabetes.How might this impact on clinical practice in the foreseeable future?This study provides evidence that autophagy is impaired in human type 1 diabetes. Prior studies have shown that loss of autophagy in the islet is associated with increased beta cell apoptosis, therefore we propose that therapeutic targeting of autophagy pathways may reduce beta cell death in type 1 diabetes development.

Autophagy is a dynamic recycling mechanism that maintains beta cell homeostasis, whereas the related process of crinophagy also plays a specific role in insulin granule regulation. Impairment of autophagy and crinophagy have been implicated in beta cell death and dysfunction in the context of type 2 diabetes; however, there is currently no literature describing beta cell autophagy in type 1 diabetes pathogenesis. Therefore, we set out to determine if beta cell autophagy and crinophagy are impaired in the context of type 1 diabetes. We utilized immunofluorescent staining of LC3 (autophagosome marker), Lamp1 (lysosome marker), and proinsulin to analyze pancreatic islet autophagy and crinophagy in the NOD (non-obese diabetic) mouse model of type 1 diabetes as well as in human pancreas tissue obtained from the network for Pancreatic Organ donors with Diabetes (nPOD). We observed evidence of reduced macroautophagy in the residual proinsulin-positive islet cells of human donors with type 1 diabetescompared to controls, as measured by significantly reduced autophagosome:lysosome colocalization (p=0.034). Similarly, there was a trend towards reduced autophagosome:lysosome colocalization in islets of diabetic NOD mice compared to nondiabetic NOR mice (p=0.08). We also observed significantly reduced colocalization of proinsulin with lysosomes in the proinsulin positive cells from human donors with type 1 diabetes in comparison to both controls without diabetes (p=0.0014) and autoantibody positive donors (p= 0.0278), as well as in diabetic NOD vs. nondiabetic NOR mice (p=0.0121), suggesting that crinophagy is also reduced as a function of type 1 diabetes. Collectively, we provide evidence that macroautophagy and crinophagy are reduced in the context of type 1 diabetes. 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. Disclosure C. Muralidharan: None. M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. J. Crowder: Employee; Spouse/Partner; Eli Lilly and Company. A.M. Conteh: None. A.K. Linnemann: None.

Pathways that mitigate cellular stress are critical for pancreatic beta cell function and survival. We have reported that the cytokine interleukin-6 (IL-6) coordinately activates two such pathways, autophagy and the antioxidant response, resulting in decreased oxidative stress and increased beta cell survival under diabetogenic conditions. Our investigation of this protective effect of IL-6 on the beta cells subsequently revealed that members of the IL-6 family of cytokines stimulated the mitochondrial translocation of NRF2, the central antioxidant regulator. This was surprising since NRF2 is well-characterized as a transcription factor. Under these conditions, we also observe translocation of another transcription factor, STAT3, to the mitochondria, consistent with previously published observations. Here, we present data to validate these findings in the islet in vivo and investigate the downstream events associated with NRF2 mitochondrial translocation. We used a mouse model where animals were intraperitoneally injected with either IL-6 or another IL-6 family member, leukemia inhibitory factor (LIF), and pancreata were collected at 30 minutes post-injection. We then used immunofluorescence analysis of paraffin tissue sections. We observed that both IL-6 and LIF rapidly elicit a reduction in the autophagy receptor p62 both in the cytosol and at the mitochondria when compared to saline-injected mice. This suggests that both global autophagy and selective mitophagy are stimulated in the beta cell by both IL-6 and LIF. Furthermore, as both IL-6 and LIF selectively stimulate mitochondrial but not nuclear translocation of NRF2, our data suggests that NRF2 may play a role in the stimulation of mitophagy by IL-6 family cytokines. Further studies investigating the requirement of NRF2 for beta cell mitophagy under conditions of beta cell stress response will shed light on this novel pathway of the cellular antioxidant response. Disclosure M. Marasco: Employee; Spouse/Partner; Eli Lilly and Company. Z. Akbar: None. J. Crowder: Employee; Spouse/Partner; Eli Lilly and Company. A. Novak: None. C. Muralidharan: None. M. Arvin: None. A.K. Linnemann: None. Funding National Institutes of Health (T32DK064466-16, R03DK115990)

Background and Hypothesis: Glycogen is a highly branched polymer of glucose and is an important form of energy storage in mammals. The brain is able to form glycogen in astrocytes and neurons via glycogen synthase and branching enzyme. Once formed, brain glycogen functions as the only stored energy source for these cells. Various physiological roles for brain glycogen have been hypothesized, including memory consolidation and sleep regulation, as well as a protective role during various physiological stressors, such as hypoglycemia and hypoxia. For instance, rat brain glycogen levels were decreased 10 minutes after vaginal birth, but not after a C-section. This suggested that cerebral hypoxia experienced during vaginal birth induced the utilization of brain glycogen to minimize neurodegeneration during the hypoxic event. Symptoms of hypoxia can range from tachycardia, tachypnea, shortness of breath, and diaphoresis to confusion, loss of motor coordination and cognitive function, neurodegeneration, and brain death. The many causes of hypoxia include lungs diseases (COPD, pneumonia, pulmonary edema), CNS depressants (opiates), heart problems (CHF), anemia, and obstructive sleep apnea (OSA). We hypothesized that the lack of brain glycogen would cause a noticeable detrimental effect to the survival time and physiologic functions of mice exposed to acute hypoxia. Experimental Design or Project Methods: We subjected mice, with or without glycogen synthase disrupted in the brain, to carbon dioxide- or nitrogen-induced hypoxia and monitored effects on brain glycogen levels, behavior, and survival time. Results: We found that mice lacking brain glycogen exhibited the characteristic physiologic responses to hypoxia, but expired ~50% sooner than mice with brain glycogen. Conclusion and Potential Impact: These results provide evidence that brain glycogen is imperative in responding to hypoxic events. Further, these findings suggest that brain glycogen may protect patients with OSA against other comorbidities, especially neurodegeneration and cognitive impairment.

Conserved homologues of the Hrd1 ubiquitin ligase target for degradation proteins that persistently or aberrantly engage the endoplasmic reticulum translocon, including mammalian apolipoprotein B (apoB; the major protein component of low-density lipoproteins) and the artificial yeast proteinDeg1-Sec62. A complete understanding of the molecular mechanism by which translocon-associated proteins are recognized and degraded may inform the development of therapeutic strategies for cholesterol-related pathologies. Both apoB andDeg1-Sec62 are extensively post-translationally modified. Mass spectrometry of a variant ofDeg1-Sec62 revealed that the protein is acetylated at the N-terminal methionine and two internal lysine residues. N-terminal and internal acetylation regulates the degradation of a variety of unstable proteins. However, preventing N-terminal and internal acetylation had no detectable consequence for Hrd1-mediated proteolysis ofDeg1-Sec62. Our data highlight the importance of empirically validating the role of post-translational modifications and sequence motifs on protein degradation, even when such elements have previously been demonstrated sufficient to destine other proteins for destruction.

Aberrant nonstop proteins arise from translation of mRNA molecules beyond the coding sequence into the 3′-untranslated region. If a stop codon is not encountered, translation continues into the poly(A) tail, resulting in C-terminal appendage of a polylysine tract and a terminally stalled ribosome. In Saccharomyces cerevisiae, the ubiquitin ligase Rkr1/Ltn1 has been implicated in the proteasomal degradation of soluble cytosolic nonstop and translationally stalled proteins. Rkr1 is essential for cellular fitness under conditions associated with increased prevalence of nonstop proteins. Mutation of the mammalian homolog causes significant neurological pathology, suggesting broad physiological significance of ribosome-associated quality control. It is not known whether and how soluble or transmembrane nonstop and translationally stalled proteins targeted to the endoplasmic reticulum (ER) are detected and degraded. We generated and characterized model soluble and transmembrane ER-targeted nonstop and translationally stalled proteins. We found that these proteins are indeed subject to proteasomal degradation. We tested three candidate ubiquitin ligases (Rkr1 and ER-associated Doa10 and Hrd1) for roles in regulating abundance of these proteins. Our results indicate that Rkr1 plays the primary role in targeting the tested model ER-targeted nonstop and translationally stalled proteins for degradation. These data expand the catalog of Rkr1 substrates and highlight a previously unappreciated role for this ubiquitin ligase at the ER membrane.

Regulated protein degradation is crucial for virtually every cellular function. Much of what is known about the molecular mechanisms and genetic requirements for eukaryotic protein degradation was initially established in Saccharomyces cerevisiae. Classical analyses of protein degradation have relied on biochemical pulse-chase and cycloheximide-chase methodologies. While these techniques provide sensitive means for observing protein degradation, they are laborious, time-consuming, and low-throughput. These approaches are not amenable to rapid or large-scale screening for mutations that prevent protein degradation. Here, a yeast growth-based assay for the facile identification of genetic requirements for protein degradation is described. In this assay, a reporter enzyme required for growth under specific selective conditions is fused to an unstable protein. Cells lacking the endogenous reporter enzyme but expressing the fusion protein can grow under selective conditions only when the fusion protein is stabilized (i.e. when protein degradation is compromised). In the growth assay described here, serial dilutions of wild-type and mutant yeast cells harboring a plasmid encoding a fusion protein are spotted onto selective and non-selective medium. Growth under selective conditions is consistent with degradation impairment by a given mutation. Increased protein abundance should be biochemically confirmed. A method for the rapid extraction of yeast proteins in a form suitable for electrophoresis and western blotting is also demonstrated. A growth-based readout for protein stability, combined with a simple protocol for protein extraction for biochemical analysis, facilitates rapid identification of genetic requirements for protein degradation. These techniques can be adapted to monitor degradation of a variety of short-lived proteins. In the example presented, the His3 enzyme, which is required for histidine biosynthesis, was fused to Deg1-Sec62. Deg1-Sec62 is targeted for degradation after it aberrantly engages the endoplasmic reticulum translocon. Cells harboring Deg1-Sec62-His3 were able to grow under selective conditions when the protein was stabilized.