miR-146a-5p mediates inflammation-induced ß cell mitochondrial dysfunction and apoptosis.

We previously showed that miR-146a-5p is upregulated in pancreatic islets treated with pro-inflammatory cytokines. Others have reported that miR-146a-5p overexpression is associated with ß cell apoptosis and impaired insulin secretion. However, the molecular mechanisms mediating these effects remain elusive. To investigate the role of miR-146a-5p in ß cell function, we developed stable MIN6 cell lines to either overexpress or inhibit the expression of miR-146a-5p. Monoclonal cell populations were treated with pro-inflammatory cytokines (IL-1ß, IFNγ, and TNFα) to model T1D in vitro. We found that overexpression of miR-146a-5p increased cell death under conditions of inflammatory stress, whereas inhibition of miR-146a-5p reversed these effects. Additionally, inhibition of miR-146a-5p increased mitochondrial DNA copy number, respiration rate, and ATP production. Further, RNA sequencing data showed enrichment of pathways related to insulin secretion, apoptosis, and mitochondrial function when the expression levels of miR-146a-5p were altered. Finally, a temporal increase in miR-146a-5p expression levels and a decrease in mitochondria function markers was observed in islets derived from NOD mice. Collectively, these data suggest that miR-146a-5p may promote ß cell dysfunction and death during inflammatory stress by suppressing mitochondrial function.

Islet autoantibodies as precision diagnostic tools to characterize heterogeneity in type 1 diabetes: a systematic review.

BACKGROUND: Islet autoantibodies form the foundation for type 1 diabetes (T1D) diagnosis and staging, but heterogeneity exists in T1D development and presentation. We hypothesized that autoantibodies can identify heterogeneity before, at, and after T1D diagnosis, and in response to disease-modifying therapies. METHODS: We systematically reviewed PubMed and EMBASE databases (6/14/2022) assessing 10 years of original research examining relationships between autoantibodies and heterogeneity before, at, after diagnosis, and in response to disease-modifying therapies in individuals at-risk or within 1 year of T1D diagnosis. A critical appraisal checklist tool for cohort studies was modified and used for risk of bias assessment. RESULTS: Here we show that 152 studies that met extraction criteria most commonly characterized heterogeneity before diagnosis (91/152). Autoantibody type/target was most frequently examined, followed by autoantibody number. Recurring themes included correlations of autoantibody number, type, and titers with progression, differing phenotypes based on order of autoantibody seroconversion, and interactions with age and genetics. Only 44% specifically described autoantibody assay standardization program participation. CONCLUSIONS: Current evidence most strongly supports the application of autoantibody features to more precisely define T1D before diagnosis. Our findings support continued use of pre-clinical staging paradigms based on autoantibody number and suggest that additional autoantibody features, particularly in relation to age and genetic risk, could offer more precise stratification. To improve reproducibility and applicability of autoantibody-based precision medicine in T1D, we propose a methods checklist for islet autoantibody-based manuscripts which includes use of precision medicine MeSH terms and participation in autoantibody standardization workshops.

Islet autoantibodies are markers found in the blood when insulin-producing cells in the pancreas become damaged and can be used to predict future development of type 1 diabetes. We evaluated published literature to determine whether characteristics of islet antibodies (type, levels, numbers) could improve prediction and help understand differences in how individuals with type 1 diabetes respond to treatments. We found existing evidence shows that islet autoantibody type and number are most useful to predict disease progression before diagnosis. In addition, the age when islet autoantibodies first appear strongly influences rate of progression. These findings provide important information for patients and care providers on how islet autoantibodies can be used to understand future type 1 diabetes development and to identify individuals who have the potential to benefit from intervention or prevention therapy.

The Ailing ß-Cell in Diabetes: Insights From a Trip to the ER: The 2023 Outstanding Scientific Achievement Award Lecture.

The synthesis, processing, and secretion of insulin by the pancreatic ß-cell is key for the maintenance of systemic metabolic homeostasis, and loss or dysfunction of ß-cells underlies the development of both type 1 diabetes (T1D) and type 2 diabetes (T2D). Work in the Evans-Molina laboratory over the past 15 years has pioneered the idea that regulation of calcium dynamics is critical to ß-cell biology and diabetes pathophysiology. In this article, I will share three vignettes from the laboratory that demonstrate our bench-to-bedside approach to determining mechanisms of ß-cell stress that could improve therapeutic options and outcomes for individuals living with diabetes. The first of these vignettes will illustrate a role for the sarcoendoplasmic reticulum calcium ATPase (SERCA) pump in the regulation of endoplasmic reticulum (ER) calcium, protein trafficking, and proinsulin processing within the ß-cell. The second vignette will highlight how alterations in ß-cell calcium signaling intersect with T1D pathogenesis. The final vignette will demonstrate how activation of ß-cell stress pathways may serve as an anchor to inform biomarker strategies in T1D. Lastly, I will share my vision for the future of diabetes care, where multiple biomarkers of ß-cell stress may be combined with additional immune and metabolic biomarkers to better predict disease risk and improve therapies to prevent or delay T1D development.

Regulation of ß-cell death by ADP-ribosylhydrolase ARH3 via lipid signaling in insulitis.

BACKGROUND: Lipids are regulators of insulitis and ß-cell death in type 1 diabetes development, but the underlying mechanisms are poorly understood. Here, we investigated how the islet lipid composition and downstream signaling regulate ß-cell death. METHODS: We performed lipidomics using three models of insulitis: human islets and EndoC-ßH1 ß cells treated with the pro-inflammatory cytokines interlukine-1ß and interferon-γ, and islets from pre-diabetic non-obese mice. We also performed mass spectrometry and fluorescence imaging to determine the localization of lipids and enzyme in islets. RNAi, apoptotic assay, and qPCR were performed to determine the role of a specific factor in lipid-mediated cytokine signaling. RESULTS: Across all three models, lipidomic analyses showed a consistent increase of lysophosphatidylcholine species and phosphatidylcholines with polyunsaturated fatty acids and a reduction of triacylglycerol species. Imaging assays showed that phosphatidylcholines with polyunsaturated fatty acids and their hydrolyzing enzyme phospholipase PLA2G6 are enriched in islets. In downstream signaling, omega-3 fatty acids reduce cytokine-induced ß-cell death by improving the expression of ADP-ribosylhydrolase ARH3. The mechanism involves omega-3 fatty acid-mediated reduction of the histone methylation polycomb complex PRC2 component Suz12, upregulating the expression of Arh3, which in turn decreases cell apoptosis. CONCLUSIONS: Our data provide insights into the change of lipidomics landscape in ß cells during insulitis and identify a protective mechanism by omega-3 fatty acids. Video Abstract.

Mapping the daily rhythmic transcriptome in the diabetic retina.

Retinal function changes dramatically from day to night, yet clinical diagnosis, treatments, and experimental sampling occur during the day. To begin to address this gap in our understanding of disease pathobiology, this study investigates whether diabetes affects the retina's daily rhythm of gene expression. Diabetic, Ins2Akita/J mice, and non-diabetic littermates were kept under a 12 h:12 h light/dark cycle until 4 months of age. mRNA sequencing was conducted in retinas collected every 4 h throughout the 24 hr light/dark cycle. Computational approaches were used to detect rhythmicity, predict acrophase, identify differential rhythmic patterns, analyze phase set enrichment, and predict upstream regulators. The retinal transcriptome exhibited a tightly regulated rhythmic expression with a clear 12-hr transcriptional axis. Day-peaking genes were enriched for DNA repair, RNA splicing, and ribosomal protein synthesis, night-peaking genes for metabolic processes and growth factor signaling. Although the 12-hr transcriptional axis is retained in the diabetic retina, it is phase advanced for some genes. Upstream regulator analysis for the phase-shifted genes identified oxygen-sensing mechanisms and HIF1alpha, but not the circadian clock, which remained in phase with the light/dark cycle. We propose a model in which, early in diabetes, the retina is subjected to an internal desynchrony with the circadian clock and its outputs are still light-entrained whereas metabolic pathways related to neuronal dysfunction and hypoxia are phase advanced. Further studies are now required to evaluate the chronic implications of such desynchronization on the development of diabetic retinopathy.

Protection of ß cells against pro-inflammatory cytokine stress by the GDF15-ERBB2 signaling.

Aim/hypothesis: Growth/differentiation factor 15 (GDF15) is a therapeutic target for a variety of metabolic diseases, including type 1 diabetes (T1D). However, the nausea caused by GDF15 is a challenging point for therapeutic development. In addition, it is unknown why the endogenous GDF15 fails to protect from T1D development. Here, we investigate the GDF15 signaling in pancreatic islets towards opening possibilities for therapeutic targeting in ß cells and to understand why this protection fails to occur naturally. Methods: GDF15 signaling in islets was determined by proximity-ligation assay, untargeted proteomics, pathway analysis, and treatment of cells with specific inhibitors. To determine if GDF15 levels would increase prior to disease onset, plasma levels of GDF15 were measured in a longitudinal prospective study of children during T1D development (n=132 cases vs. n=40 controls) and in children with islet autoimmunity but normoglycemia (n=47 cases vs. n=40 controls) using targeted mass spectrometry. We also investigated the regulation of GDF15 production in islets by fluorescence microscopy and western blot analysis. Results: The proximity-ligation assay identified ERBB2 as the GDF15 receptor in islets, which was confirmed using its specific antagonist, tucatinib. The untargeted proteomics analysis and caspase assay showed that ERBB2 activation by GDF15 reduces ß cell apoptosis by downregulating caspase 8. In plasma, GDF15 levels were higher (p=0.0024) during T1D development compared to controls, but not in islet autoimmunity with normoglycemia. However, in the pancreatic islets GDF15 was depleted via sequestration of its mRNA into stress granules, resulting in translation halting. Conclusions/interpretation: GDF15 protects against T1D via ERBB2-mediated decrease of caspase 8 expression in pancreatic islets. Circulating levels of GDF15 increases pre-T1D onset, which is insufficient to promote protection due to its localized depletion in the islets. These findings open opportunities for targeting GDF15 downstream signaling for pancreatic ß cell protection in T1D and help to explain the lack of natural protection by the endogenous protein.

Gut microbial changes associated with obesity in youth with type 1 diabetes.

Obesity is increasingly prevalent in type 1 diabetes (T1D) and is associated with management problems and higher risk for diabetes complications. Gut microbiome changes have been described separately in each of T1D and obesity, however, it is unknown to what extent gut microbiome changes are seen when obesity and T1D concomitantly occur. OBJECTIVE: To describe the gut microbiome and microbial metabolite changes associated with obesity in T1D. We hypothesized significant gut microbial and metabolite differences between T1D youth who are lean (BMI: 5-<85%) vs. those with obesity (BMI: ≥95%). METHODS: We analyzed stool samples for gut microbial (using metagenomic shotgun sequencing) and short-chain fatty acid (SCFA) metabolite differences in lean (n=27) and obese (n=21) T1D youth. The mean±SD age was 15.3±2.2yrs, A1c 7.8±1.3%, diabetes duration 5.1±4.4yrs, 42.0% females, and 94.0% were White. Linear discriminant analysis (LDA) effect size (LEfSe) was used to identify taxa that best discriminated between the BMI groups. RESULTS: Bacterial community composition showed differences in species type (ß-diversity) by BMI group (p=0.013). At the genus level, there was a higher ratio of Prevotella to Bacteroides in the obese group (p=0.0058). LEfSe analysis showed a differential distribution of significantly abundant taxa in either the lean or obese groups, including increased relative abundance of Prevotella copri , among other taxa in the obese group. Functional profiling showed that pathways associated with decreased insulin sensitivity were upregulated in the obese group. Stool SCFAs (acetate, propionate and butyrate) were higher in the obese compared to the lean group (p<0.05 for all). CONCLUSIONS: Our findings identify gut microbiome, microbial metabolite and functional pathways differences associated with obesity in T1D. These findings could be helpful in identifying gut microbiome targeted therapies to manage obesity in T1D.

Histone Deacetylase Inhibitors Prevent Cytokine-Induced ß Cell Dysfunction Through Restoration of Stromal Interaction Molecule 1 Expression and Activation of Store-Operated Calcium Entry.

Histone deacetylase inhibitors (HDIs) modulate ß cell function in preclinical models of diabetes; however, the mechanisms underlying these beneficial effects have not been determined. In this study, we investigated the impact of the HDI sodium butyrate (NaB) on ß cell function and calcium (Ca2+) signaling using ex vivo and in vitro models of diabetes. Our results show that NaB significantly improved glucose-stimulated insulin secretion in islets from human organ donors with type 2 diabetes and in cytokine-treated INS-1 ß cells. Consistently, NaB partially rescued glucose-stimulated Ca2+ oscillations in mouse islets treated with proinflammatory cytokines. Because the oscillatory phenotype of Ca2+ in the ß cell is governed by changes in endoplasmic reticulum (ER) Ca2+ levels, next we explored the relationship between NaB and store-operated calcium entry (SOCE), a rescue mechanism that acts to refill ER Ca2+ levels through STIM1-mediated gating of plasmalemmal Orai channels. We found that NaB treatment preserved basal ER Ca2+ levels and restored SOCE in IL-1ß-treated INS-1 cells. Furthermore, we linked these changes with the restoration of STIM1 levels in cytokine-treated INS-1 cells and mouse islets, and we found that NaB treatment was sufficient to prevent ß cell death in response to IL-1ß treatment. Mechanistically, NaB counteracted cytokine-mediated reductions in phosphorylation levels of key signaling molecules, including AKT, ERK1/2, glycogen synthase kinase-3α (GSK-3α), and GSK-3ß. Taken together, these data support a model whereby HDI treatment promotes ß cell function and Ca2+ homeostasis under proinflammatory conditions through STIM1-mediated control of SOCE and AKT-mediated inhibition of GSK-3.

Advanced Imaging Techniques for the Characterization of Subcellular Organelle Structure in Pancreatic Islet ß Cells.

Type 2 diabetes (T2D) affects more than 32.3 million individuals in the United States, creating an economic burden of nearly $966 billion in 2021. T2D results from a combination of insulin resistance and inadequate insulin secretion from the pancreatic ß cell. However, genetic and physiologic data indicate that defects in ß cell function are the chief determinant of whether an individual with insulin resistance will progress to a diagnosis of T2D. The subcellular organelles of the insulin secretory pathway, including the endoplasmic reticulum, Golgi apparatus, and secretory granules, play a critical role in maintaining the heavy biosynthetic burden of insulin production, processing, and secretion. In addition, the mitochondria enable the process of insulin release by integrating the metabolism of nutrients into energy output. Advanced imaging techniques are needed to determine how changes in the structure and composition of these organelles contribute to the loss of insulin secretory capacity in the ß cell during T2D. Several microscopy techniques, including electron microscopy, fluorescence microscopy, and soft X-ray tomography, have been utilized to investigate the structure-function relationship within the ß cell. In this overview article, we will detail the methodology, strengths, and weaknesses of each approach. © 2024 American Physiological Society. Compr Physiol 14:5243-5267, 2024.

Inhibition of polyamine biosynthesis preserves ß cell function in type 1 diabetes.

In preclinical models, α-difluoromethylornithine (DFMO), an ornithine decarboxylase (ODC) inhibitor, delays the onset of type 1 diabetes (T1D) by reducing ß cell stress. However, the mechanism of DFMO action and its human tolerability remain unclear. In this study, we show that mice with ß cell ODC deletion are protected against toxin-induced diabetes, suggesting a cell-autonomous role of ODC during ß cell stress. In a randomized controlled trial (ClinicalTrials.gov: NCT02384889) involving 41 recent-onset T1D subjects (3:1 drug:placebo) over a 3-month treatment period with a 3-month follow-up, DFMO (125-1,000 mg/m2) is shown to meet its primary outcome of safety and tolerability. DFMO dose-dependently reduces urinary putrescine levels and, at higher doses, preserves C-peptide area under the curve without apparent immunomodulation. Transcriptomics and proteomics of DFMO-treated human islets exposed to cytokine stress reveal alterations in mRNA translation, nascent protein transport, and protein secretion. These findings suggest that DFMO may preserve ß cell function in T1D through islet cell-autonomous effects.

Hailey-Hailey Disease is Associated with Diabetes: A Population-based Cohort Study, Clinical Cohort Study, and Pedigree Analysis.

Hailey-Hailey disease is a rare hereditary skin disease caused by mutations in the ATP2C1 gene encoding the secretory pathway Ca2+/Mn2+-ATPase 1 (SPCA1) protein. Extracutaneous manifestations of Hailey-Hailey disease are plausible but still largely unknown. The aim of this study was to explore the association between Hailey-Hailey disease and diabetes. A population-based cohort study of 347 individuals with Hailey-Hailey  disease was performed to assess the risks of type 1  diabetes and type 2 diabetes, using Swedish nationwide registries. Pedigrees from 2 Swedish families with Hailey-Hailey disease were also investigated: 1 with concurrent type 1 diabetes and HLA-DQ3, the other with type 2 diabetes. Lastly, a clinical cohort with 23 individuals with Hailey-Hailey disease and matched healthy controls was evaluated regarding diabetes. In the register data males with Hailey-Hailey disease had a 70% elevated risk of type 2 diabetes, whereas no  excess risk among women could be confirmed. In both pedigrees an unusually high inheritance for diabetes was observed. In the clinical cohort, individuals with Hailey-Hailey disease displayed a metabolic phenotype indicative of type 2 diabetes. Hailey-Hailey disease seems to act as a synergistic risk factor for diabetes. This study indicates, for the first time, an association between Hailey-Hailey disease and diabetes and represents human evidence that SPCA1 and the Golgi apparatus may be implicated in diabetes pathophysiology.

Disease-modifying therapies and features linked to treatment response in type 1 diabetes prevention: a systematic review.

BACKGROUND: Type 1 diabetes (T1D) results from immune-mediated destruction of insulin-producing beta cells. Prevention efforts have focused on immune modulation and supporting beta cell health before or around diagnosis; however, heterogeneity in disease progression and therapy response has limited translation to clinical practice, highlighting the need for precision medicine approaches to T1D disease modification. METHODS: To understand the state of knowledge in this area, we performed a systematic review of randomized-controlled trials with ≥50 participants cataloged in PubMed or Embase from the past 25 years testing T1D disease-modifying therapies and/or identifying features linked to treatment response, analyzing bias using a Cochrane-risk-of-bias instrument. RESULTS: We identify and summarize 75 manuscripts, 15 describing 11 prevention trials for individuals with increased risk for T1D, and 60 describing treatments aimed at preventing beta cell loss at disease onset. Seventeen interventions, mostly immunotherapies, show benefit compared to placebo (only two prior to T1D onset). Fifty-seven studies employ precision analyses to assess features linked to treatment response. Age, beta cell function measures, and immune phenotypes are most frequently tested. However, analyses are typically not prespecified, with inconsistent methods of reporting, and tend to report positive findings. CONCLUSIONS: While the quality of prevention and intervention trials is overall high, the low quality of precision analyses makes it difficult to draw meaningful conclusions that inform clinical practice. To facilitate precision medicine approaches to T1D prevention, considerations for future precision studies include the incorporation of uniform outcome measures, reproducible biomarkers, and prespecified, fully powered precision analyses into future trial design.

Type 1 diabetes (T1D) is a condition that results from the destruction of a type of cell in the pancreas that produces the hormone insulin, leading to lifelong dependence on insulin injections. T1D prevention remains a challenging goal, largely due to the immense variability in disease processes and progression. Therapies tested to date in medical research settings (clinical trials) work only in a subset of individuals, highlighting the need for more tailored prevention approaches. We reviewed clinical trials of therapies targeting the disease process in T1D. While the overall quality of trials was high, studies testing individual features affecting responses to treatments were low. This review reveals an important need to carefully plan high-quality analyses of features that affect treatment response in T1D, to ensure that tailored approaches may one day be applied to clinical practice.

Islet-on-chip: promotion of islet health and function via encapsulation within a polymerizable fibrillar collagen scaffold.

The protection and interrogation of pancreatic ß-cell health and function ex vivo is a fundamental aspect of diabetes research, including mechanistic studies, evaluation of ß-cell health modulators, and development and quality control of replacement ß-cell populations. However, present-day islet culture formats, including traditional suspension culture as well as many recently developed microfluidic devices, suspend islets in a liquid microenvironment, disrupting mechanochemical signaling normally found in vivo and limiting ß-cell viability and function in vitro. Herein, we present a novel three-dimensional (3D) microphysiological system (MPS) to extend islet health and function ex vivo by incorporating a polymerizable collagen scaffold to restore biophysical support and islet-collagen mechanobiological cues. Informed by computational models of gas and molecular transport relevant to ß-cell physiology, a MPS configuration was down-selected based on simulated oxygen and nutrient delivery to collagen-encapsulated islets, and 3D-printing was applied as a readily accessible, low-cost rapid prototyping method. Recreating critical aspects of the in vivo microenvironment within the MPS via perfusion and islet-collagen interactions mitigated post-isolation ischemia and apoptosis in mouse islets over a 5-day period. In contrast, islets maintained in traditional suspension formats exhibited progressive hypoxic and apoptotic cores. Finally, dynamic glucose-stimulated insulin secretion measurements were performed on collagen-encapsulated mouse islets in the absence and presence of well-known chemical stressor thapsigargin using the MPS platform and compared to conventional protocols involving commercial perifusion machines. Overall, the MPS described here provides a user-friendly islet culture platform that not only supports long-term ß-cell health and function but also enables multiparametric evaluations.

Evaluating the effect of prebiotics on the gut microbiome profile and ß cell function in youth with newly diagnosed type 1 diabetes: protocol of a pilot randomized controlled trial.

INTRODUCTION: Data show that disturbances in the gut microbiota play a role in glucose homeostasis, type 1 diabetes (T1D) risk and progression. The prebiotic high amylose maize starch (HAMS) alters the gut microbiome profile and metabolites favorably with an increase in bacteria producing short chain fatty acids (SCFAs) that have significant anti-inflammatory effects. HAMS also improves glycemia, insulin sensitivity, and secretion in healthy non-diabetic adults. Additionally, a recent study testing an acetylated and butyrylated form of HAMS (HAMS-AB) that further increases SCFA production prevented T1D in a rodent model without adverse safety effects. The overall objective of this human study will be to assess how daily HAMS-AB consumption impacts the gut microbiome profile, SCFA production, ß cell heath, function, and glycemia as well as immune responses in newly diagnosed T1D youth. METHODS AND ANALYSIS: We hypothesize that HAMS-AB intake will improve the gut microbiome profile, increase SCFA production, improve ß cell health, function and glycemia as well as modulate the immune system. We describe here a pilot, randomized crossover trial of HAMS-AB in 12 newly diagnosed T1D youth, ages 11-17 years old, with residual ß cell function. In Aim 1, we will determine the effect of HAMS-AB on the gut microbiome profile and SCFA production; in Aim 2, we will determine the effect of HAMS-AB on ß cell health, function and glycemia; and in Aim 3, we will determine the peripheral blood effect of HAMS-AB on frequency, phenotype and function of specific T cell markers. Results will be used to determine the effect-size estimate of using HAMS-AB. We anticipate beneficial effects from a simple, inexpensive, and safe dietary approach. ETHICS AND DISSEMINATION: The Institutional Review Board at Indiana University approved the study protocol. The findings of this trial will be submitted to a peer-reviewed pediatric journal. Abstracts will be submitted to relevant national and international conferences. TRIAL REGISTRATION: NCT04114357; Pre-results.

SERCA2 regulates proinsulin processing and processing enzyme maturation in pancreatic beta cells.

AIMS/HYPOTHESIS: Increased circulating levels of incompletely processed insulin (i.e. proinsulin) are observed clinically in type 1 and type 2 diabetes. Previous studies have suggested that Ca2+ signalling within beta cells regulates insulin processing and secretion; however, the mechanisms that link impaired Ca2+ signalling with defective insulin maturation remain incompletely understood. METHODS: We generated mice with beta cell-specific sarcoendoplasmic reticulum Ca2+ ATPase-2 (SERCA2) deletion (ßS2KO mice) and used an INS-1 cell line model of SERCA2 deficiency. Whole-body metabolic phenotyping, Ca2+ imaging, RNA-seq and protein processing assays were used to determine how loss of SERCA2 impacts beta cell function. To test key findings in human model systems, cadaveric islets were treated with diabetogenic stressors and prohormone convertase expression patterns were characterised. RESULTS: ßS2KO mice exhibited age-dependent glucose intolerance and increased plasma and pancreatic levels of proinsulin, while endoplasmic reticulum (ER) Ca2+ levels and glucose-stimulated Ca2+ synchronicity were reduced in ßS2KO islets. Islets isolated from ßS2KO mice and SERCA2-deficient INS-1 cells showed decreased expression of the active forms of the proinsulin processing enzymes PC1/3 and PC2. Additionally, immunofluorescence staining revealed mis-location and abnormal accumulation of proinsulin and proPC2 in the intermediate region between the ER and the Golgi (i.e. the ERGIC) and in the cis-Golgi in beta cells of ßS2KO mice. Treatment of islets from human donors without diabetes with high glucose and palmitate concentrations led to reduced expression of the active forms of the proinsulin processing enzymes, thus phenocopying the findings observed in ßS2KO islets and SERCA2-deficient INS-1 cells. Similar findings were observed in wild-type mouse islets treated with brefeldin A, a compound that perturbs ER-to-Golgi trafficking. CONCLUSIONS/INTERPRETATION: Taken together, these data highlight an important link between ER Ca2+ homeostasis and proinsulin processing in beta cells. Our findings suggest a model whereby chronic ER Ca2+ depletion due to SERCA2 deficiency impairs the spatial regulation of prohormone trafficking, processing and maturation within the secretory pathway. DATA AVAILABILITY: RNA-seq data have been deposited in the Gene Expression Omnibus (GEO; accession no.: GSE207498).

ß Cell microRNAs Function as Molecular Hubs of Type 1 Diabetes Pathogenesis and as Biomarkers of Diabetes Risk.

MicroRNAs (miRNAs) are small non-coding RNAs that play a crucial role in modulating gene expression and are enriched in cell-derived extracellular vesicles (EVs). We investigated whether miRNAs from human islets and islet-derived EVs could provide insight into ß cell stress pathways activated during type 1 diabetes (T1D) evolution, therefore serving as potential disease biomarkers. We treated human islets from 10 cadaveric donors with IL-1ß and IFN-γ to model T1D ex vivo. MicroRNAs were isolated from islets and islet-derived EVs, and small RNA sequencing was performed. We found 20 and 14 differentially expressed (DE) miRNAs in cytokine- versus control-treated islets and EVs, respectively. Interestingly, the miRNAs found in EVs were mostly different from those found in islets. Only two miRNAs, miR-155-5p and miR-146a-5p, were upregulated in both islets and EVs, suggesting selective sorting of miRNAs into EVs. We used machine learning algorithms to rank DE EV-associated miRNAs, and developed custom label-free Localized Surface Plasmon Resonance-based biosensors to measure top ranked EVs in human plasma. Results from this analysis revealed that miR-155, miR-146, miR-30c, and miR-802 were upregulated and miR-124-3p was downregulated in plasma-derived EVs from children with recent-onset T1D. In addition, miR-146 and miR-30c were upregulated in plasma-derived EVs of autoantibody positive (AAb+) children compared to matched non-diabetic controls, while miR-124 was downregulated in both T1D and AAb+ groups. Furthermore, single-molecule fluorescence in situ hybridization confirmed increased expression of the most highly upregulated islet miRNA, miR-155, in pancreatic sections from organ donors with AAb+ and T1D.

Stromal Interaction Molecule 1 Maintains ß-Cell Identity and Function in Female Mice Through Preservation of G-Protein-Coupled Estrogen Receptor 1 Signaling.

Altered endoplasmic reticulum (ER) Ca2+ signaling has been linked with ß-cell dysfunction and diabetes development. Store-operated Ca2+ entry replenishes ER Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). For characterization of the in vivo impact of STIM1 loss, mice with ß-cell-specific STIM1 deletion (STIM1Δß mice) were generated and challenged with high-fat diet. Interestingly, ß-cell dysfunction was observed in female, but not male, mice. Female STIM1Δß mice displayed reductions in ß-cell mass, a concomitant increase in α-cell mass, and reduced expression of markers of ß-cell maturity, including MafA and UCN3. Consistent with these findings, STIM1 expression was inversely correlated with HbA1c levels in islets from female, but not male, human organ donors. Mechanistic assays demonstrated that the sexually dimorphic phenotype observed in STIM1Δß mice was due, in part, to loss of signaling through the noncanonical 17-ß estradiol receptor (GPER1), as GPER1 knockdown and inhibition led to a similar loss of expression of ß-cell maturity genes in INS-1 cells. Together, these data suggest that STIM1 orchestrates pancreatic ß-cell function and identity through GPER1-mediated estradiol signaling. ARTICLE HIGHLIGHTS: Store-operated Ca2+ entry replenishes endoplasmic reticulum (ER) Ca2+ through reversible gating of plasma membrane Ca2+ channels by the ER Ca2+ sensor, stromal interaction molecule 1 (STIM1). ß-Cell-specific deletion of STIM1 results in a sexually dimorphic phenotype, with ß-cell dysfunction and loss of identity in female but not male mice. Expression of the noncanonical 17-ß estradiol receptor (GPER1) is decreased in islets of female STIM1Δß mice, and modulation of GPER1 levels leads to alterations in expression of ß-cell maturity genes in INS-1 cells.

ß-Cell Glucose Sensitivity to Assess Changes in ß-Cell Function in Recent-Onset Stage 3 Type 1 Diabetes.

Following a diagnosis of type 1 diabetes (T1D), persisting C-peptide secretion leads to improved glycemic control and outcomes. Residual ß-cell function is often assessed with serial mixed-meal tolerance tests, but these tests do not correlate well with clinical outcomes. Herein, we instead use ß-cell glucose sensitivity (ßGS) to assess changes in ß-cell function, incorporating insulin secretion for a given serum glucose into the assessment of ß-cell function. We evaluated changes in ßGS in individuals enrolled in the placebo arm of 10 T1D trials performed at diabetes onset. We found that ßGS showed a more rapid decline in children, as compared with adolescents and adults. Individuals in the top quartile of ßGS baseline distribution had a slower rate in loss of glycemic control time over time. Notably, half of this group were children and adolescents. Finally, to identify predictors of glycemic control throughout follow-up, we ran multivariate Cox models and found that incorporating ßGS significantly improved the overall model. Taken together, these data suggest that ßGS may be of great utility in predicting those more likely to have a more robust clinical remission and may be of use in design of new-onset diabetes clinical trials and in evaluating response to therapies. ARTICLE HIGHLIGHTS: We undertook this study to better predict ß-cell loss following type 1 diabetes diagnosis. We set out to answer whether ß-cell glucose sensitivity (ßGS) improves means to evaluate ß-cell function postdiagnosis and whether ßGS correlates with clinical outcomes. We found that ßGS declines faster in children, subjects in the top baseline quartile of ßGS exhibit slower ß-cell decline (half are children), and incorporating ßGS into multivariate Cox models for glycemic improves the model. The implications of our findings are that ßGS predicts those likely to have robust clinical remissions and may help with clinical trials design.

Type 1 Diabetes Prevention: a systematic review of studies testing disease-modifying therapies and features linked to treatment response.

Background: Type 1 diabetes (T1D) results from immune-mediated destruction of insulin-producing beta cells. Efforts to prevent T1D have focused on modulating immune responses and supporting beta cell health; however, heterogeneity in disease progression and responses to therapies have made these efforts difficult to translate to clinical practice, highlighting the need for precision medicine approaches to T1D prevention. Methods: To understand the current state of knowledge regarding precision approaches to T1D prevention, we performed a systematic review of randomized-controlled trials from the past 25 years testing disease-modifying therapies in T1D and/or identifying features linked to treatment response, analyzing bias using a Cochrane-risk-of-bias instrument. Results: We identified 75 manuscripts, 15 describing 11 prevention trials for individuals with increased risk for T1D, and 60 describing treatments aimed at preventing beta cell loss in individuals at disease onset. Seventeen agents tested, mostly immunotherapies, showed benefit compared to placebo (only two prior to T1D onset). Fifty-seven studies employed precision analyses to assess features linked to treatment response. Age, measures of beta cell function and immune phenotypes were most frequently tested. However, analyses were typically not prespecified, with inconsistent methods reporting, and tended to report positive findings. Conclusions: While the quality of prevention and intervention trials was overall high, low quality of precision analyses made it difficult to draw meaningful conclusions that inform clinical practice. Thus, prespecified precision analyses should be incorporated into the design of future studies and reported in full to facilitate precision medicine approaches to T1D prevention. Plain Language Summary: Type 1 diabetes (T1D) results from the destruction of insulin-producing cells in the pancreas, necessitating lifelong insulin dependence. T1D prevention remains an elusive goal, largely due to immense variability in disease progression. Agents tested to date in clinical trials work in a subset of individuals, highlighting the need for precision medicine approaches to prevention. We systematically reviewed clinical trials of disease-modifying therapy in T1D. While age, measures of beta cell function, and immune phenotypes were most commonly identified as factors that influenced treatment response, the overall quality of these studies was low. This review reveals an important need to proactively design clinical trials with well-defined analyses to ensure that results can be interpreted and applied to clinical practice.

NMP4, an Arbiter of Bone Cell Secretory Capacity and Regulator of Skeletal Response to PTH Therapy.

The skeleton is a secretory organ, and the goal of some osteoporosis therapies is to maximize bone matrix output. Nmp4 encodes a novel transcription factor that regulates bone cell secretion as part of its functional repertoire. Loss of Nmp4 enhances bone response to osteoanabolic therapy, in part, by increasing the production and delivery of bone matrix. Nmp4 shares traits with scaling factors, which are transcription factors that influence the expression of hundreds of genes to govern proteome allocation for establishing secretory cell infrastructure and capacity. Nmp4 is expressed in all tissues and while global loss of this gene leads to no overt baseline phenotype, deletion of Nmp4 has broad tissue effects in mice challenged with certain stressors. In addition to an enhanced response to osteoporosis therapies, Nmp4-deficient mice are less sensitive to high fat diet-induced weight gain and insulin resistance, exhibit a reduced disease severity in response to influenza A virus (IAV) infection, and resist the development of some forms of rheumatoid arthritis. In this review, we present the current understanding of the mechanisms underlying Nmp4 regulation of the skeletal response to osteoanabolics, and we discuss how this unique gene contributes to the diverse phenotypes among different tissues and stresses. An emerging theme is that Nmp4 is important for the infrastructure and capacity of secretory cells that are critical for health and disease.