Diabetes

Aims/hypothesisType 1 diabetes is a complex autoimmune disorder in which autoreactive CD4 and CD8 T cells destroy pancreatic beta-cells, resulting in insulin deficiency and hyperglycemia. Although genetic susceptibility, particularly certain HLA alleles, contributes to disease risk, not all genetically predisposed individuals develop Type 1 diabetes. Screening first degree relatives (FDRs) for islet autoantibodies (GAD65, IAA, IA-2, ZnT8) helps detect autoimmune activity. However, these serum markers arise only after T-helper cell activation, limiting early intervention opportunities. Since protein antigen recognition by B cells requires T-helper cell assistance through linked recognition, T cell activation precedes B cell activation and autoantibody production. Activation of these T cells leads to shedding of the immune-regulatory (activation) surface protein LAG-3 (Lymphocyte Activation Gene-3 or CD223), generating its soluble form, sLAG-3, that is detectable in circulation. We hypothesized that sLAG-3 may serve as an early biomarker of autoimmune activity preceding islet autoantibody development in type 1 diabetes. MethodsPlasma sLAG-3 levels were measured longitudinally in female diabetes-prone NOD mice and analyzed in relation to islet antigen-specific CD4 T cell expansion and diabetes onset. To mechanistically link autoreactive T cell activation to sLAG-3 release. Naive autoreactive C6.6.9 TCR-transgenic (TCR-Tg) CD4 T cells were adoptively transferred into NOD.SCID mice and longitudinal assessment for plasma sLAG-3, beta-cell antigen specific CD4 T cell tetramer profiles, and circulating insulin (Ins2) mRNA to determine ongoing beta-cell stress. In parallel, sLAG-3 levels were analyzed from different human cohorts, including FDRs of individuals with type 1 diabetes, using cross-sectional and longitudinal approaches. ResultsIn murine models, elevated sLAG-3 correlated with expansion of islet-specific CD4 T cells that preceded hyperglycemia and diabetes onset. In the adoptive transfer model, early increases in sLAG-3 and circulating Ins2 mRNA marked immune activation and emerging beta-cell stress prior to overt diabetes. In our human cohorts, sLAG-3 was detectable in autoantibody-negative and single-autoantibody-positive FDRs, with higher levels observed in progressors compared to non-progressors, and associated with high-risk HLA genotypes. Conclusions/interpretationThese findings identify sLAG-3 as a candidate biomarker of early T cell activation in type 1 diabetes that may precede islet autoantibody development. Integration of sLAG-3 with antigen-specific T cell and beta-cell stress markers could improve early risk stratification and inform preventive strategies before substantial loss of beta-cell. Prospective longitudinal studies aligned to seroconversion are required to validate sLAG-3 as a surrogate marker of early disease activity. Research in contextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIBefore the clinical onset of hyperglycemia, type 1 diabetes is characterized by a prolonged preclinical phase in which autoreactive B and T cells mediate progressive beta-cell destruction. C_LIO_LICurrent risk stratification strategies rely mainly on genetic susceptibility (genomic DNA) and the detection of islet autoantibodies in plasma/serum. C_LIO_LIIslet autoantibodies arise only after CD4 T cell activation and therefore do not capture the earliest stages of immune dysregulation. C_LIO_LIConsequently, biomarkers that directly reflect early pathogenic T cell activity prior to, or independent of, seroconversion remain limited and insufficiently validated. C_LI What is the key question?Can plasma sLAG-3 levels, beta-cell antigen-specific CD4 T cell tetramer expression, and circulating Ins2 mRNA serve as very early biomarkers of autoimmune activity in type 1 diabetes and serve to better inform risk stratification, thereby informing preventive intervention strategies for the clinician? What are the new findings?O_LIsLAG-3 increases transiently during early antigen-specific CD4 T cell activation stage, precedes hyperglycemia in mouse models, and is elevated in autoantibody-negative and single-autoantibody-positive first-degree relatives who later progress to type 1 diabetes. C_LIO_LIsLAG-3 was associated with beta-cell antigen-specific CD4 T cell expansion, assessment of stress induced beta cell Ins2 mRNA release and high-risk HLA genotypes, indicating early autoimmune activation rather than established disease. C_LI How might this impact clinical practice in the foreseeable future?These findings support sLAG-3 as a candidate early biomarker of T cell activation, before or at the earliest stages of islet autoantibody development in some at-risk individuals. Integration of plasma sLAG-3 with beta-cell antigen specific CD4 T cell profiling and insulin mRNA measurements could complement current autoantibody-based screening, improve risk stratification, and enable earlier preventive interventions to preserve beta-cell function in patients at-risk for type 1 diabetes.

Insulin resistance is a hallmark feature of Type 2 Diabetes (T2D), but the progression of the disease is closely linked to a deterioration in {beta}-cell mass and function. While the precise mechanisms of {beta}-cell failure are unclear, chronic hyperglycemia (glucotoxicity) and dyslipidemia (lipotoxicity) are considered contributing factors; however, the relative importance of these insults on {beta}-cell function remains controversial. To examine this, we dissociated glucotoxicity from lipotoxicity using a high-fat diet (HFD)-fed mouse model of T2D and the glucose-lowering SGLT2 inhibitor, canagliflozin (CANA). As expected, HFD-feeding impaired glucose tolerance and isolated islet function. However, despite improvements in glucose tolerance and indices of {beta}-cell insulin secretory function in vivo, CANA failed to restore isolated {beta}-cell function. Shotgun lipidomics analysis of isolated islets revealed that HFD-feeding induced glycerophospholipid remodeling with a persistent increase in arachidonic acid (20:4)-enriched molecular species. Further analysis revealed that lysophosphatidylcholine (LPC) was the predominant lipid class elevated in HFD islets following correction of glucotoxicity with CANA. In follow-up experiments, LPC stimulations acutely and dose-dependently impaired glucose-stimulated insulin secretion (GSIS) in isolated wild-type islets, mechanistically linking this lipid class to {beta}-cell dysfunction. Our findings indicate that persistent inflammatory lipotoxicity impedes {beta}-cell function in diet-induced obese (DIO) rodents even after normalization of hyperglycemia. If replicated in humans, these data suggest that interventions targeting lipotoxicity may be beneficial for the long-term protection of pancreatic {beta}-cell function in T2D.

Glucokinase (GK) catalyses the key regulatory step in glucose-stimulated insulin secretion. Correspondingly, hetero- and homozygous mutations in human GCK cause maturity-onset diabetes of the young (GCK-MODY) and permanent neonatal diabetes (PNDM), respectively. To explore the possible utility of glucokinase activators (GKA) and of glucagon-like receptor-1 (GLP-1) agonists in these diseases, we have developed a novel hypomorphic Gck allele in mice encoding an aberrantly spliced mRNA deleted for exons 2 and 3. In islets from homozygous knock-in (GckKI/KI) mice, GK immunoreactivity was reduced by >85%, and glucose-stimulated insulin secretion eliminated. Homozygous GckKI/KI mice were smaller than wildtype littermates and displayed frank diabetes (fasting blood glucose >18 mmol/L; HbA1c [~]12%), ketosis and nephropathy. Heterozygous GckKI/+ mice were glucose intolerant (HbA1c [~]5.5%). Abnormal glucose-stimulated Ca2+ dynamics and beta cell-beta cell connectivity in GckKI/+ islets were completely reversed by the recently-developed GKA, dorzagliatin, which was largely inactive in homozygous GckKI/KI mouse islets. The GLP-1 receptor agonist exendin-4 improved glucose tolerance in male GckKI/+ mice, an action potentiated by dorzagliatin, in male but not female mice. Sex-dependent additive effects of these agents were also observed on insulin secretion in vitro. Combined treatment with GKA and incretin may thus be useful in GCK-MODY or GCK-PNDM. Article Highlightsa. Glucokinase deficiency can drive maturity-onset diabetes of the young (GCK-MODY; heterozygotes) and permanent neonatal diabetes (GCK-PNDM; homozygotes) b. We describe a hypomorphic Gck allele where aberrant splicing in islets lowers GK activity to by [~]85%. We use these mice to explore the effects of the glucokinase activator, dorzagliatin, and incretin on insulin secretion c. Whereas heterozygous mutant mice are mildly hyperglycemic, homozygotes have frank diabetes but survive to adulthood. Dorzagliatin potentiates the effects of GLP-1 receptor activation sex-dependently in heterozygotes d. Combined use of these drugs may be useful in some forms of GCK diabetes

Carboxypeptidase E (CPE) facilitates the conversion of prohormones into mature hormones and is highly expressed in multiple neuroendocrine tissues. Carriers of CPE mutations have elevated plasma proinsulin and develop severe obesity and hyperglycemia. We aimed to determine whether loss of Cpe in pancreatic beta cells disrupts proinsulin processing and accelerates development of diabetes and obesity in mice. Pancreatic beta cell-specific Cpe knockout mice ({beta}CpeKO; Cpefl/fl x Ins1Cre/+) lack mature insulin granules and have elevated proinsulin in plasma; however, glucose-and KCl-stimulated insulin secretion in {beta}CpeKO islets remained intact. High fat diet-fed {beta}CpeKO mice showed comparable weight gain and glucose tolerance compared to Wt littermates. Notably, beta-cell area was increased in chow-fed {beta}CpeKO mice and beta-cell replication was elevated in {beta}CpeKO islets. Transcriptomic analysis of {beta}CpeKO beta cells revealed elevated glycolysis and Hif1-target gene expression. Upon high glucose challenge, beta cells from {beta}CpeKO mice showed reduced mitochondrial membrane potential, increased reactive oxygen species, reduced MafA, and elevated Aldh1a3 transcript levels. Following multiple low-dose streptozotocin treatment, {beta}CpeKO mice had accelerated hyperglycemia with reduced beta-cell insulin and Glut2 expression. These findings suggest that Cpe and proper proinsulin processing are critical in maintaining beta cell function during the development of diabetes.

In type 1 diabetes (T1D), the innate and adaptive immune systems attack and eventually destroy the insulin-secreting pancreatic {beta} cells. During this process, {beta} cells activate inflammatory signaling pathways that augment the dysfunction and destruction imposed by cellular autoimmunity. The 12-lipoxygenase (12-LOX) pathway produces the pro-inflammatory eicosanoid 12-HETE, which induces oxidative and endoplasmic reticulum stress and results in diminished insulin secretion and apoptosis. The G protein-coupled receptor GPR31 has been identified as a putative receptor for 12-HETE. In this study, we generated conventional GPR31 knockout (KO) mice on the C57BL/6J background. To interrogate the role of GPR31 in {beta} cells, we treated islets from wildtype and Gpr31b KO mice with pro-inflammatory cytokines and subjected the islets to RNA sequencing. Differentially expressed pathways in Gpr31b KO islets included those pertaining to inflammation and oxidative stress, consistent with functional studies that demonstrated reduced cytokine-induced oxidative stress in Gpr31b KO islets compared to wildtype controls. Bone marrow-derived macrophages from Gpr31b KO mice showed reduced macrophage migration and decreased inflammatory IFN- and IFN-{gamma} signaling by RNA sequencing. To mimic islet and macrophage inflammation as seen in T1D, wildtype and Gpr31b KO mice were treated with the diabetogenic toxin streptozotocin. Compared to wildtype, Gpr31b KO mice had improved glucose tolerance and preserved {beta}-cell mass. siRNA knockdown of Gpr31b in non-obese diabetic (NOD) mice reduced insulitis, macrophage infiltration, and oxidative stress. Collectively, these findings are consistent with previously published data using 12/15-LOX KO mice and suggest that GPR31 mediates the pro-inflammatory responses of 12-HETE in both {beta} cells and macrophages.

When homozygous for the LeptinOb mutation (Ob), Black-and-Tan Brachyury (BTBR) mice become morbidly obese and severely insulin resistant, and by 10 weeks of age, frankly diabetic. Previous work has shown Prostaglandin EP3 Receptor (EP3) expression and activity is up-regulated in islets from BTBR-Ob mice as compared to lean controls, actively contributing to their beta-cell dysfunction. In this work, we aimed to test the impact of beta-cell-specific EP3 loss on the BTBR-Ob phenotype by crossing Ptger3 floxed mice with the Rat insulin promoter (RIP)-CreHerr driver strain. Instead, germline recombination of the floxed allele in the founder mouse - an event whose prevalence we identified as directly associated with underlying insulin resistance of the background strain - generated a full-body knockout. Full-body EP3 loss provided no diabetes protection to BTBR-Ob mice, but, unexpectedly, significantly worsened BTBR-lean insulin resistance and glucose tolerance. This in vivo phenotype was not associated with changes in beta-cell fractional area or markers of beta-cell replication ex vivo. Instead, EP3-null BTBR-lean islets had essentially uncontrolled insulin hypersecretion. The selective up-regulation of constitutively-active EP3 splice variants in islets from young, lean BTBR mice as compared to C57BL/6J, where no phenotype of EP3 loss has been observed, provides a potential explanation for the hypersecretion phenotype. In support of this, high islet EP3 expression in Balb/c females vs. Balb/c males was fully consistent with their sexually-dimorphic metabolic phenotype after loss of EP3-coupled Gz protein. Taken together, our findings provide a new dimension to the understanding of EP3 as a critical brake on insulin secretion. New and NoteworthyIslet Prostaglandin EP3 receptor (EP3) signaling is well-known as up-regulated in the pathophysiological conditions of type 2 diabetes, contributing to beta-cell dysfunction. Unexpected findings in mouse models of non-obese insulin sensitivity and resistance provide a new dimension to our understanding of EP3 as a key modulator of insulin secretion. A previously-unknown relationship between mouse insulin resistance and the penetrance of Rat insulin promoter-driven germline floxed allele recombination is critical to consider when creating beta-cell-specific knockouts. For Table of Contents Use Only O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=121 SRC="FIGDIR/small/671289v3_ufig1.gif" ALT="Figure 1"> View larger version (37K): org.highwire.dtl.DTLVardef@15cf1d5org.highwire.dtl.DTLVardef@108f565org.highwire.dtl.DTLVardef@126e479org.highwire.dtl.DTLVardef@61fdf8_HPS_FORMAT_FIGEXP M_FIG C_FIG

Type 2 diabetes (T2D) arises in the context of obesity and overnutrition; however, additional demographic features including age and biological sex contribute to T2D risk. Estradiol (E2) is thought to play a protective metabolic role that may govern sex differences in the development of T2D. The mechanisms by which E2 exerts these effects and the impact of reduced E2 signaling in {beta} cells during menopause remain incompletely understood. We analyzed publicly available whole islet transcriptome datasets from female and male cadaveric donors and showed significant age-related modulation of gene expression, including changes in pathways related to {beta} cell function, in islets from female donors. Importantly, these patterns were not observed in islets from male donors. To test the in vivo relationship between E2 signaling and {beta} cell function, 10-week- old female C57BL6/J mice underwent an ovariectomy (OVX) or sham (CTR) surgery, followed by 4 weeks of high-fat diet (HFD) treatment. HFD-OVX mice exhibited obesity-induced glucose intolerance, increased cell mass, and reduced expression of {beta} cell identity markers. Furthermore, ex vivo treatment of islets with the G protein coupled estrogen receptor (GPER)- specific agonist G-1 restored {beta} cell identity gene expression. Together, these data identify a novel connection between GPER signaling and {beta} cell identity and suggest that menopausal loss of E2 signaling through GPER may be linked with loss of {beta} cell identity.

The mechanisms that underlie the {beta}-cell pathophysiology of Type 1 Diabetes (T1D) are not fully understood. Our group has defined the unique heterotrimeric G protein alpha-subunit, Gz, as a key negative regulator of {beta}-cell signal transduction pathways. Non-obese diabetic (NOD) mice lacking Gz throughout the body are protected from developing T1D-like hyperglycemia. To determine whether this phenotype is {beta}-cell autonomous, we generated and validated a {beta}-cell-specific Gz knockout ({beta}KO) on the NOD background and characterized the phenotype of female and male cohorts. Long-term hyperglycemia incidence was lower in Gz {beta}KO mice as compared to wild-type (WT) controls, but, unlike global Gz knockout mice, this protection was incomplete. While young male and female Gz {beta}KO NOD mice had improved glucose tolerance, WT NOD males were significantly less glucose tolerant than females, and only female Gz {beta}KO mice retained improved glucose tolerance at 28-29 weeks of age. Conversely, {beta}-cell-specific Gz loss only influenced insulitis in 28-29-week old male NOD mice, a phenotype correlating directly with body burden of glucose during oral glucose challenge. Using surrogates for {beta}-cell function and apoptosis, the partial penetrance of euglycemia in Gz {beta}KO NOD was best explained by an early failure to up-regulate {beta}-cell proliferation. We conclude {beta}-cell Gz is an important regulator of the sexually-dimorphic T1D-like phenotype of NOD mice. Yet, other factors must be important in imparting full protection from the disease.

Seroconversion (SV) marks the initiation of islet autoimmunity (IA) and pre-clinical phase of type 1 diabetes, yet the contributions of immune cells beyond cytotoxic T cells remain unclear. We applied high-resolution immune cell-type deconvolution using peripheral blood DNA methylation data from nested case-control samples of the Diabetes Autoimmunity Study in the Young (DAISY; n=151) and The Environmental Determinants of Diabetes in the Young (TEDDY; n=166) to estimate immune cell proportions across pre-SV and SV timepoints and construct functional ratios, such as the neutrophil-to-lymphocyte ratio (NLR). Using linear models, we evaluated differences between type 1 diabetes cases and controls at pre-SV, SV, and the change across timepoints. Pre-SV, cases had higher NLR and lower CD4T/CD8T cell ratios. At SV, the combined B-CD4T-CD8T memory/naive ratio was reduced in cases. From pre-SV to SV, cases showed attenuations in NLR, B-memory/naive, and B-CD4T-CD8T memory/naive ratios. These patterns may reflect delayed or disrupted immune maturation with the persistence or expansion of naive cells or impaired transition to memory subsets following antigen exposure. Our findings highlight early shifts in innate and adaptive immune cell dynamics during type 1 diabetes pathogenesis and support immune cell ratios as potential biomarkers for risk stratification and mechanistic insight. Article HighlightsO_LIWe sought to examine immune cells around the time of IA seroconversion in children at higher risk for type 1 diabetes. C_LIO_LIWe wanted to answer whether immune cell ratio differences exist between type 1 diabetes cases and controls around IA at pre-SV, SV, and the change pre-SV to SV. C_LIO_LIWe found immune cell ratio differences between type 1 diabetes cases and controls before, during, and across SV timepoints, suggesting potential etiological and pathophysiological roles. C_LIO_LIOur findings highlight the complexity of immunodynamics around IA seroconversion and potential role for immune cell ratios in type 1 diabetes risk stratification and intervention. C_LI

BackgroundEarly-life nutritional exposures are increasingly recognized as critical determinants of long-term metabolic health, yet the molecular mechanisms linking maternal diet to offspring type 2 diabetes susceptibility remain incompletely understood. Experimental models are essential to disentangle maternal dietary effects from later-life metabolic influences. MethodsUsing the Nile rat, a genetically heterogeneous model of diet-induced diabetes, we quantified the impact of maternal high-fiber diet on offspring diabetes incidence using sex-stratified time-to-event analyses in 762 offspring. To identify molecular mediators, we performed transcriptomic profiling across 13 offspring tissues, independently contrasting maternal diet exposure and early-onset diabetes status. Overlapping differentially expressed genes were prioritized and evaluated for cardiometabolic associations in human whole-blood transcriptomic data from the GENE-FORECAST cohort. Untargeted plasma metabolomics was integrated to identify circulating metabolites associated with candidate genes. ResultsOffspring born to dams maintained on a high-fiber diet exhibited a markedly reduced risk of developing type 2 diabetes, with approximately 70% lower hazard of diabetes onset in both males and females compared with offspring from regular chow-fed dams. Multi-tissue transcriptomic analyses identified 147 genes differentially expressed in association with both maternal diet and early-onset diabetes, with most effects being tissue-specific. Asnsd1 uniquely showed consistent regulation across the aorta, brown adipose tissue, and skeletal muscle, with higher expression in offspring exposed to a high-fiber maternal diet and lower expression in offspring with early-onset diabetes. In human whole-blood transcriptomic data, ASNSD1 expression was significantly associated with blood pressure-related cardiometabolic traits, including hypertension, systolic blood pressure, and mean arterial pressure. In the animal model, circulating succinic acid was positively correlated with Asnsd1 expression in the aorta but not in other tissues. ConclusionsThis study provides a multi-tissue transcriptomic-metabolomic framework linking maternal high-fiber diet to reduced offspring type 2 diabetes risk. The findings identify ASNSD1 as a maternal diet-sensitive gene associated with diabetes susceptibility across multiple tissues and with cardiometabolic traits in humans, while highlighting tissue-specific relationships between gene expression and circulating metabolites. Together, these results offer mechanistic insight into how early-life nutrition can durably influence diabetes risk across the life course.

Abstract: Type 1 diabetes (T1D) affects millions worldwide, yet few non-invasive biomarkers detect immune-mediated {beta}-cell dysfunction during the presymptomatic phase, a critical window for therapeutic intervention. Previously, we identified reduced double C2-like domain containing beta protein (DOC2B) levels in circulating platelets as a marker of reduced {beta}-cell function in early-onset T1D cohorts and nonobese diabetic (NOD) mice. Here, we assessed whether plasma DOC2B could serve as a sensitive early biomarker of T1D progression risk in the autoantibody-positive pediatric cohort (progressors vs non-progressors) from the longitudinal Diabetes Evaluation in Washington (DEW-IT) study; T1D patients and non-diabetic cohorts from the DEW-IT study served as controls. At pre-onset, progressors showed a decline in DOC2B that preceded measurable changes in random C-peptide and HbA1c levels, while non-progressors maintained stable levels. These observations were further supported by our analysis in prediabetic NOD mice. Comparisons of plasma levels pre- and post-clinical islet transplantation in long-standing T1D patients highlights its potential utility as a reporter of {beta}-cell functional mass. Together, these findings suggest that DOC2B decline may precede C-peptide decline in early presymptomatic T1D progression. This work could have significant future implications for clinical trial stratification and assessing response outcomes to disease-modifying or cell replacement therapies.

ObjectivePancreatic islet {beta}-cells are factories for insulin production; however ectopic expression of insulin is also well recognized. The gallbladder is a next-door neighbour to the developing pancreas. Here, we wanted to understand if gallbladders contain functional insulin-producing cells. DesignWe compared developing and adult mouse as well as human gallbladder epithelial cells and islets using immunohistochemistry, flow cytometry, ELISAs, RNA-sequencing, real-time PCR, chromatin immunoprecipitation and functional studies. ResultsWe demonstrate that the epithelial lining of developing, as well as adult mouse and human gallbladders naturally contain interspersed cells that retain the capacity to actively transcribe, translate, package, and release insulin. We show for the first time that human gallbladders also contain functional insulin-secreting cells with the potential to naturally respond to glucose in vitro and in situ. Notably, in a NOD mouse model of type 1 diabetes, we observed that insulin-producing cells in the gallbladder are not targeted by autoimmune cells. Conclusion: In summary, our biochemical, transcriptomic, and functional data in human gallbladder epithelial cells collectively demonstrate their potential for insulin-production under pathophysiological conditions, and open newer areas for type 1 diabetes research and therapy. Significance of the study What is already known about this subject?O_LIDeveloping pancreas and gallbladder are next-door neighbours and share similar developmental pathways. C_LIO_LIHuman Gallbladder-derived progenitor cells were shown to differentiate into insulin-producing cells. C_LI What are the new findings?O_LIGallbladder epithelium contains interspersed cells that can transcribe, translate, package and secrete insulin. C_LIO_LIInsulin-producing cells in the gallbladder are not destroyed by immune cells in an animal model of type 1 diabetes (T1D). C_LIO_LIOur studies demonstrating the absence of insulin splice variants in human gallbladder cells, and higher splice forms in human islets, suggest a potential mechanism (via defective ribosomal products) in escaping islet autoimmunity. C_LI How might it impact clinical practice?O_LIDeciphering mechanisms of protection of insulin-producing cells from immune cells in the gallbladder could help in developing strategies to prevent islet autoimmunity in T1D. C_LI

Aims/hypothesisImmunotherapeutics targeting T cells are crucial for inhibiting autoimmune disease progression proximal to disease onset in type 1 diabetes. A growing number of T cell-directed therapeutics have demonstrated partial therapeutic efficacy, with anti-CD3 (-CD3) representing the only regulatory agency-approved drug capable of slowing disease progression through a mechanism involving the induction of partial T cell exhaustion. There is an outstanding need to augment the durability and effectiveness of T cell targeting by directly restraining proinflammatory T helper type 1 (Th1) and type 1 cytotoxic CD8+ T cell (Tc1) subsets, while simultaneously augmenting regulatory T cell (Treg) activity. Here, we present a novel strategy for reducing diabetes incidence in the NOD mouse model using a blocking monoclonal antibody targeting the type 1 diabetes-risk associated T cell co-stimulatory receptor, CD226. MethodsFemale NOD mice were treated with anti-CD226 between 7-8 weeks of age and then monitored for diabetes incidence and therapeutic mechanism of action. ResultsCompared to isotype-treated controls, anti-CD226 treated NOD mice showed reduced insulitis severity at 12 weeks and decreased disease incidence at 30 weeks. Flow cytometric analysis performed five weeks post-treatment demonstrated reduced proliferation of CD4+ and CD8+ effector memory T cells in spleens of anti-CD226 treated mice. Phenotyping of pancreatic Tregs revealed increased CD25 expression and STAT5 phosphorylation following anti-CD226, with splenic Tregs displaying augmented suppression of CD4+ T cell responders in vitro. Anti-CD226 treated mice exhibited reduced frequencies of islet-specific glucose-6-phosphatase catalytic subunit related protein (IGRP)-reactive CD8+ T cells in the pancreas, using both ex vivo tetramer staining and single-cell T cell receptor sequencing (scTCR-seq) approaches. 51Cr-release assays demonstrated reduced cell-mediated lysis of beta-cells by anti-CD226-treated autoreactive cytotoxic T lymphocytes. Conclusions/interpretationCD226 blockade reduces T cell cytotoxicity and improves Treg function, representing a targeted and rational approach for restoring immune regulation in type 1 diabetes. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIThe co-stimulatory receptor CD226 is upregulated upon activation and is highly expressed on NK cell subsets, myeloid cells, and effector T cells. C_LIO_LIA single nucleotide polymorphism in CD226 (rs763361; C>T) results in a Gly307Ser missense mutation linked to genetic susceptibility for type 1 diabetes. C_LIO_LIGlobal knockout of Cd226 and conditional Cd226 knockout in FoxP3+ Tregs reduced insulitis severity and diabetes incidence in NOD mice, indicating a crucial role for CD226 in disease pathogenesis. C_LI What is the key question?O_LICan CD226 blockade reduce T cell cytotoxicity and improve Treg function to diminish diabetes incidence in NOD mice? C_LI What are the new findings?O_LIAnti-CD226 treatment reduced insulitis, decreased disease incidence, and inhibited splenic CD4+ and CD8+ effector memory T cell proliferation. C_LIO_LIPancreatic Tregs from anti-CD226 treated mice exhibited increased CD25 expression; splenic Tregs displayed augmented STAT5 phosphorylation and suppressive capacity in vitro. C_LIO_LIAnti-CD226 treatment reduced IGRP-specific pancreatic CD8+ T cell frequencies, and reduced autoreactive CD8+ T cell-mediated lysis of beta-cells in vitro. C_LI How might this impact on clinical practice in the foreseeable future?O_LICD226 blockade could reduce autoreactive T cell cytotoxicity, enhance Treg function, and slow disease progression in high-risk or recent-onset type 1 diabetes cases. C_LI

Co-stimulation serves as a critical checkpoint for T cell development and activation, and several genetic variants affecting co-stimulatory pathways confer risk for autoimmune diseases. A single nucleotide polymorphism in CD226 (rs763361; G307S) has been shown to increase susceptibility to type 1 diabetes, multiple sclerosis, and rheumatoid arthritis. CD226 competes with the co-inhibitory receptor TIGIT (T cell immunoreceptor with Ig and ITIM domains) to bind CD155 to amplify TCR signaling. We previously found that Cd226 knockout protected non-obese diabetic (NOD) mice from disease, but the impact of CD226 signaling on individual immune subsets remained unclear. We focused on regulatory T cells (Tregs) as a population of interest, as prior reports demonstrated that human CD226+ Tregs exhibit reduced FOXP3+Helios+ purity and suppressive function following expansion. Hence, we hypothesized that global deletion of Cd226 would increase Treg stability and accordingly, Treg-specific Cd226 deletion would inhibit diabetes in NOD mice. Indeed, crossing the NOD.Cd226-/- and NOD.Foxp3-GFP-Cre.R26-loxP-STOP-loxP-YFP Treg-fate tracking strains resulted in increased Treg induction and decreased FoxP3-deficient "ex-Tregs" in the pancreatic lymph nodes. We generated a Treg-conditional knockout (Treg{Delta}Cd226) strain and found that female Treg{Delta}Cd226 mice had decreased insulitis and diabetes incidence compared to TregWT mice. Additionally, we observed increased TIGIT expression on Tregs and conventional CD4+ T cells within the pancreas of Treg{Delta}Cd226 versus TregWT mice. These findings demonstrate that an imbalance of CD226/TIGIT signaling may contribute to Treg destabilization in the NOD mouse and highlight the potential for therapeutic targeting of this pathway to prevent or reverse autoimmunity.

The capacity of the brain to elicit sustained remission of hyperglycemia in rodent models of type 2 diabetes following intracerebroventricular (icv) injection of fibroblast growth factor 1 (FGF1) is well established. Here, we show that following icv FGF1 injection, hypothalamic signaling by extracellular signal-regulated kinases 1 and 2 (ERK1/2), members of the mitogen-activated protein kinase (MAPK) family is induced for at least 24h. Further, we show that in diabetic Lepob/ob mice, this prolonged response is required for the sustained antidiabetic action of FGF1, since it is abolished by sustained (but not acute) pharmacologic blockade of hypothalamic MAPK/ERK signaling. We also demonstrate that FGF1 R50E, a FGF1 mutant that activates FGF receptors but induces only transient hypothalamic MAPK/ERK signaling, fails to mimic the sustained glucose lowering induced by FGF1. These data identify sustained activation of hypothalamic MAPK/ERK signaling as playing an essential role in the mechanism underlying diabetes remission induced by icv FGF1 administration.

There are multiple independent genetic signals at the Ras-responsive element binding protein 1 (RREB1) locus associated with type 2 diabetes risk, fasting glucose, ectopic fat, height, and bone mineral density. We have previously shown that loss of RREB1 in pancreatic beta cells reduces insulin content and impairs islet cell development and function. However, RREB1 is a widely expressed transcription factor and the metabolic impact of RREB1 loss in vivo remains unknown. Here, we show that male and female global heterozygous knockout (Rreb1+/-) mice have reduced body length, weight, and fat mass on high-fat diet. Rreb1+/- mice have sex- and diet-specific decreases in adipose tissue and adipocyte size; male mice on high-fat diet had larger gonadal adipocytes, while males on standard chow and females on high-fat diet had smaller, more insulin sensitive subcutaneous adipocytes. Mouse and human precursor cells lacking RREB1 have decreased adipogenic gene expression and activated transcription of genes associated with osteoblast differentiation, which was associated with Rreb1+/- mice having increased bone mineral density in vivo. Finally, human carriers of RREB1 T2D protective alleles have smaller adipocytes, consistent with RREB1 loss-of-function reducing diabetes risk.

Natural killer (NK) cells contribute to the development of obesity-associated insulin resistance and have previously been shown to up-regulate the expression of the P2Y purinoreceptor 6 (P2Y6R) upon high fat diet (HFD)-induced obesity. Here, we reveal that NK cell-specific inactivation of the P2Y6R gene improves insulin sensitivity in obese mice and reduces the expression of chemokines in adipose tissue infiltrating NK-cells. Obese mice lacking P2Y6R specifically in NK cells exhibited a reduction in adipose tissue inflammation, exhibited improved insulin-stimulated suppression of lipolysis in adipose tissue and a reduction in hepatic glucose production, leading to an overall improvement of systemic insulin sensitivity. In contrast, myeloid lineage specific P2Y6R inactivation does not affect energy or glucose homeostasis in obesity. Collectively, we show that P2Y6R signaling in NK cells contributes to the development of obesity-associated insulin resistance and thus might be a future target for the treatment of obesity-associated insulin resistance and type 2 diabetes.

ObjectiveSORCS2 is an intracellular sorting receptor genetically associated with body mass index (BMI) in humans, yet its mode of action remains unknown. Elucidating the receptor function that defines its role in metabolic health is the objective of this work. MethodsCombining in vivo metabolic studies in SORCS2-deficient mouse models with ex vivo structural and functional analyses as well as single-cell transcriptomics of murine pancreatic tissues, we studied the pathophysiological consequences of receptor dysfunction for metabolism. ResultsOur studies identified an important role for SORCS2 in islet stress response essential to sustain glucose-stimulated insulin release. In detail, we show that SORCS2 is predominantly expressed in islet alpha cells. Loss of receptor expression coincides with the inability of these cells to produce osteopontin, a secreted factor that facilitates insulin release from beta cells under stress. In line with diminished osteopontin levels, beta cells in SORCS2- deficient islets show changes in gene expression patterns related to aggravated ER stress, protein misfolding, as well as mitochondrial dysfunction; and they exhibit defects in insulin granule maturation and a blunted response to glucose stimulation in vivo and ex vivo. Impaired glucose tolerance in receptor mutant mice coincides with alterations in body weight and composition. ConclusionOur data identified a novel concept in protective islet stress response involving the alpha cell receptor SORCS2 and provide experimental support for association of SORCS2 with metabolic control in humans.

The transcription factor FOXM1 regulates {beta}-cell proliferation and insulin secretion. Our previous work demonstrates that expressing an activated form of FOXM1 (FOXM1*) in {beta} cells increases {beta}-cell proliferation and mass in aged male mice. Additionally, FOXM1* enhances {beta}-cell function even in young mice, in which no {beta}-cell mass elevation occurs. Here, we demonstrate that FOXM1 acts in a sexually dimorphic manner in the {beta} cell. Expression of FOXM1* in female mouse {beta} cells does not affect {beta}-cell proliferation or glucose tolerance. Transduction of male but not female human islets with FOXM1* enhances insulin secretion in response to elevated glucose. Estrogen contributes to diabetes susceptibility differences between males and females, and the estrogen receptor (ER) is the primary mediator of {beta}-cell estrogen signaling. We show that FOXM1* can rescue impaired glucose tolerance in female mice with a pancreas-wide ER deletion. Further, FOXM1 and ER binding sites overlap with each other and with other {beta}-cell-enriched transcription factors, including ISL1, PAX6, MAF, and GATA. These data indicate that FOMX1 and ER cooperate to regulate {beta}-cell function and suggest a general mechanism contributing to the lower incidence of diabetes observed in women.

{beta} cells may participate and contribute to their own demise during Type 1 diabetes (T1D). We identified a novel role of Tet2 in regulating immune killing of {beta} cells. Tet2 is induced in murine and human {beta} cells with inflammation but its expression is reduced in surviving {beta} cells. Tet2-KO mice that receive WT bone marrow transplants develop insulitis but not diabetes and islet infiltrates do not eliminate {beta} cells even though immune cells from the mice can transfer diabetes to NOD/scid recipients. Tet2-KO {beta} cells show reduced expression of inflammatory genes, associated with closed transcription factor binding sites. Tet2-KO recipients are protected from transfer of disease by diabetogenic immune cells. We conclude that Tet2 regulates pathologic interactions between {beta} cells and immune cells and controls intrinsic protective pathways. Modulating TET2 may enable survival of {beta} cells or their replacements in the setting of pathologic immune cells.