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.

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.

Wolfram syndrome is a rare autosomal recessive disorder caused by pathogenic variants in the WFS1 gene, characterized by early-onset diabetes mellitus, optic atrophy, sensorineural hearing loss, arginine vasopressin deficiency, and progressive neurodegeneration. The condition selectively affects pancreatic {beta} cells and neurons via chronic endoplasmic reticulum (ER) stress, and no proven disease-modifying therapy currently exists. Diabetes mellitus is typically the first manifestation, presenting at a mean age of 6 years as an insulin-dependent phenotype with preserved C-peptide and negative diabetes-related autoantibodies. Glucagon-like peptide-1 receptor agonists (GLP-1 RAs) are well-established agents in the management of type 2 diabetes, augmenting glucose-dependent insulin secretion, suppressing glucagon, slowing gastric emptying, and promoting satiety. Preclinical evidence further suggests that GLP-1 RAs preserve {beta}-cell mass, attenuate ER stress, and confer neuroprotective effects, properties of particular therapeutic relevance to Wolfram syndrome. We conducted a retrospective cohort study of 84 participants with genetically confirmed Wolfram syndrome and insulin-dependent diabetes mellitus enrolled in the Washington University Wolfram Syndrome International Registry and Clinical Study. Clinical data were extracted from medical records; for participants concurrently enrolled in the Tracking Neurodegeneration in Early Wolfram Syndrome study, longitudinal data were obtained from that source as well. Thirty-five percent of eligible participants had received a GLP-1 RA at some point during follow-up. We characterize the prevalence of GLP-1 RA use, documented rationale for initiation, observed effects on glycemic control and visual outcomes, adverse effects, and reasons for discontinuation. No statistically significant changes in hemoglobin A1c (HbA1c) or body mass index (BMI) were observed. Visual acuity declined significantly at two years, consistent with expected disease progression. Gastrointestinal adverse effects were common and contributed to frequent discontinuation. These observational data provide important clinical context and a foundation for future prospective trials evaluating GLP-1 RAs as a potential disease-modifying strategy in Wolfram syndrome.

The detection of monogenic diabetes illustrates the potential of precision medicine, with treatments tailored to specific genes and diagnosis involving targeted genetic testing. Current detection criteria are derived from White populations. We investigated detection of monogenic diabetes in an unselected multiethnic cohort comprising 1,706 participants diagnosed with diabetes before the age of 30-years. Using broad biomarker criteria (triple pancreatic antibody negative and detectable C-peptide) to select for next generation sequencing of monogenic diabetes genes, we found a non-significantly different minimum cohort prevalence of monogenic diabetes of 2.1% in White, 2.0% in South Asian, 2.5% in African-Caribbean, and 3.6% in Mixed participants. The detection rate, however, varied significantly (17.7% in White, 5.3%in South Asian, 8.0% in African-Caribbean, and 15.2% in Mixed participants, p<0.001). Those without monogenic diabetes showed significant variations in BMI. No difference in phenotype of monogenic diabetes across ancestry groups was observed. Non-white ethnicity participants were significantly more likely to have undiagnosed monogenic diabetes than White with on average a 10-year duration before receiving a correct diagnosis. By applying ancestry-specific BMI cut-offs (White <30, South Asian <27, African-Caribbean and Mixed <35 kg/m{superscript 2}), the overall detection rate increased from 8.8 to 16%, reducing the number needed to test to identify one case from 11 to 6 and boosting detection rates to 39, 11, 9 and 26% in White, South Asian, African-Caribbean and Mixed-ethnicity participants, respectively. These findings were validated in an external real-world dataset. Applying broad biomarker criteria for initial selection, mitigates clinical biases leading to misclassification of monogenic diabetes in non-White ethnicities. However, further tailoring criteria with ethnic-specific BMI cut-offs doubled detection rates, improving cost-effectiveness by minimising unnecessary testing. Our study highlights the need to develop precision medicine approaches accounting for phenotypic variation across diverse populations, to ensure accurate diagnoses and cost-efficient healthcare provision.

Individuals with Down syndrome (trisomy of human chromosome 21) are at a significantly higher risk of developing type 2 diabetes (T2D) than the general population. Systemic metabolic defects in Down syndrome have been linked to gene expression dysregulation in peripheral tissues like the liver, muscle, brain, and adipose. However, the contribution of gene expression dysregulation in the islets of Langerhans to the increased risk of T2D in Down syndrome has not been explored. Here we show that trisomic Ts65Dn mice, a common Down syndrome mouse model, are glucose intolerant and display reduced {beta}-to- cell ratio compared to disomic controls. Using single cell RNA sequencing on islets from Ts65Dn mice we found genome-wide, cell type-specific, and sex-specific transcriptional dysregulation in trisomic islets compared to controls. The Down syndrome-associated transcriptional signature revealed important islet defects, both at the cell autonomous level and at the whole-islet level, increasing T2D susceptibility. Our results put forth innate islet defects as a central underlying cause of Down syndrome-related T2D, warranting additional studies.

The clinical characteristics of type 2 diabetes (T2D) differ between the sexes. For example, the risk of T2D is higher in males than in premenopausal females, whereas the risk of T2D-associated cardiovascular disease is higher in females. However, the sex-dependent mechanisms of T2D pathogenesis remain incompletely understood. Publicly available human islet datasets, such as HPAP and Humanislets.com, offer a valuable tool for uncovering the impact of biological sex on islet structure, gene expression, and function at a scale that was not previously possible. We performed an integrated analysis of data from publicly available sources to identify sex differences in baseline islet characteristics in donors without diabetes and subsequently examined these features in donors who lived with T2D. Among donors without diabetes, female islets had a greater proportion of alpha-cells compared with male islets and showed enriched expression of ribosomal and mitochondrial pathways in both beta- and alpha-cells. Measurements of mitochondrial function in female islets revealed lower spare respiratory capacity compared to male islets. Male and female islets had distinct changes in gene and protein expression in the context of T2D with female islets having greater preservation of insulin content and fewer defects in islet function. Together, these data show female islets have fewer islet impairments in T2D. This highlights the need for detailed mechanistic studies in both sexes to support effective and sex-informed interventions for T2D.

Many genetic variants associated with increased type 1 diabetes (T1D) risk are located within the SKAP2 gene; however, the mechanisms by which these variants confer disease risk remain unclear. SKAP2 encodes an adapter protein that functions within the integrin signaling pathway and is found at the highest levels in myeloid leukocytes. We recently identified a de novo gain-of-function SKAP2 mutation in an individual with T1D, leading to hyperactive integrin signaling in myeloid cells. To dissect the mechanisms by which this mutation may lead to T1D, we generated a knock-in mouse line containing the orthologous p.G153R substitution in mouse SKAP2 on the diabetes-prone nonobese diabetic (NOD) genetic background. Both female and male SKAP2G153R/G153R mice developed accelerated T1D. The SKAP2G153R/G153R mice also exhibited a unique spectrum of autoantibodies, leading to immune-complex nephritis. Accelerated infiltration of pancreatic islets by myeloid cells, B lymphocytes, and activated T cells was observed in SKAP2G153R/G153R mice. Single-cell RNA sequencing demonstrated a type 1 IFN{gamma}-driven inflammatory program within the pancreatic islets of SKAP2G153R/G153R mice. Dendritic cells from SKAP2G153R/G153R mice demonstrated increased antigen-presenting capacity, characterized by enhanced adhesion to T cells during immune synapse formation. Macrophages and neutrophils from SKAP2G153R/G153R mice also showed increased integrin signaling responses, with neutrophils expressing high levels of activated {beta}2 integrins on the cell surface. When backcrossed onto the C57BL/6J genetic background, the SKAP2G153R/G153R mice developed spontaneous autoantibody formation and exhibited accelerated autoimmunity, including nephritis, in the pristane-induced model of autoimmune disease. These findings demonstrate that dysregulation of leukocyte integrin signaling, through alterations in SKAP2, may increase the genetic risk for autoimmunity and T1D.

AimsGestational diabetes mellitus (GDM) is the most common pregnancy-related medical complication. GDM is linked to aberrant immune responses in both mothers and offsprings, specifically, the subsequent development of inflammatory diseases. Whereas prior research has focused on specific immune cell subsets, a comprehensive overview of the impacts of GDM on maternal and fetal immune landscape is lacking. Here, we aim to comprehensively decipher how GDM modulates various immune cell populations in mothers and offsprings. MethodsA prospective, longitudinal case-control study was carried out. Maternal blood from GDM-affected (GDM, n=18) and non-GDM-affected (Ctrl, n=21) mothers were collected at ante-(36-38 weeks of gestation) and post-partum (6-8 weeks post-partum) timepoints. Cord blood from GDM (n=7) and Ctrl (n=11) pregnancies were collected upon C-section. They were analyzed with the state-of-the-art cytometry by time of flight (CyTOF) with a 40-marker panel. Additionally, a publicly available RNA-seq dataset for cord blood mononuclear cells was re-analyzed to confirm results from CyTOF experiments. ResultsCompared to Ctrl, GDM was associated with more activated maternal T cell subsets ante-partum, including increased CD45RO+ and Ki67+ CD4+ T cell populations, which reverted post-partum. GDM-affected maternal innate lymphoid cell (ILC) also exhibited increased granzyme B production ante-partum. On the other hand, in GDM-impacted cord blood, fetal T and B cells were more activated, displaying less naive and more effector phenotypes, further supported by RNA-seq analyses. ConclusionsOur comprehensive analyses revealed that GDM is linked to profound changes in the immune landscapes of the mothers (ante-/post-partum) and foetuses (at birth), casting novel insights towards GDM pathophysiology. Longitudinal immune profiling might be warranted for early detection and stratification of risk, and development of targeted interventions to prevent inflammatory disorders in GDM mothers and their offspring. Research in contextO_LIWhat is already known about this subject? O_LIThe maternal and intrauterine environments are important contributors to long-term health outcomes of mothers and offsprings. C_LIO_LISome maternal and fetal immunity changes have been observed in gestational diabetes mellitus (GDM)-affected pregnancies. C_LIO_LIGDM is associated with increased risk of later-life metabolic and inflammatory diseases in mothers as well as offsprings. C_LI C_LIO_LIWhat is the key question? O_LIWhat are the longitudinal alterations in maternal and fetal immune landscapes in GDM-affected pregnancies? C_LI C_LIO_LIWhat are the new findings? O_LIHigh-dimensional immune profiling provided the most comprehensive overview of alterations in maternal and fetal immune landscapes associated with GDM. C_LIO_LIGDM is associated with skewing of maternal CD4+ T cell and ILC towards activated phenotypes ante-partum. C_LIO_LIGDM is linked to more activated fetal T and B cell profiles. C_LI C_LIO_LIHow might this impact on clinical practice in the foreseeable future? O_LIUnderstanding the complex alterations in the maternal and fetal immune landscape in GDM-affected pregnancy provides insights into the long-term impacts of GDM on the mother and offspring. C_LI C_LI

Polymorphism in endoplasmic reticulum antigen processing peptidases (ERAAP) that trim MHC class I ligands is associated with autoimmune diseases, but the mechanism is unknown. We analyzed the effect of Eraap deficiency in the non-obese diabetic (NOD) mouse model of type 1 diabetes. Eraap-/- NOD mice displayed reduced and delayed diabetes, harbored splenic effector T cells unable to transfer diabetes, and exhibited a strong shift from effector to central memory T cells in attenuated islet infiltrates. Eraap deficiency increased presentation of the immunodominant epitope insulin B15-23 by beta cells but at the same time provided complete protection from diabetes to chimeras reconstituted with bone marrow encoding a CD8+ T cell recognizing this epitope. These results underline the strong impact of self-antigen presentation to CD8+ T cells in diabetes. At the same time, they highlight the complex consequences of interfering with MHC-I antigen presentation in autoimmunity and advise caution in therapeutic modulation of ERAP activity in this context.

Background: The glucagon-like peptide-1 receptor (GLP1R) is a key regulator of glucose metabolism and appetite and a major therapeutic target for type 2 diabetes (T2D) and obesity. Genetic studies have implicated the GLP1R locus in both body mass index (BMI) and T2D, but it remains unclear whether their underlying genetic associations are the same. Methods: We analyzed 431,107 participants of genetically inferred European ancestry from the Million Veteran Program. Within 500 kb of GLP1R, we performed locus-wide linear regression models for BMI and logistic regression models for T2D, adjusted for age, sex, and 10 principal components. We identified primary and secondary BMI sentinel variants using conditional analyses and evaluated their associations with T2D. Bayesian fine-mapping was used to construct credible sets of GLP1R locus for BMI and T2D. Results: Conditioning on the primary sentinel variant rs12213929 (upstream of GLP1R, {beta} = 0.11; 95% CI 0.09-0.14; p = 1.94E-17), we identified a secondary variant (rs13216992, intron of GLP1R) independently associated with BMI ({beta} = 0.10; 95% CI 0.07-0.13; p = 7.88E-14). The two sentinel variants showed low linkage disequilibrium (r2 = 0.03). A two-variant allelic burden score (0-4; sum of the rs12213929 G-allele count and rs13216992 C-allele count) showed that participants with 4 risk alleles had 0.47 kg/m2 higher BMI than those with 0 risk alleles (95% CI 0.39-0.55; p < 2E-16). Both variants were associated with higher T2D risk, but with distinct patterns after BMI adjustment: the rs12213929-T2D association persisted after adjustment for BMI (OR = 1.02; 95% CI 1.01-1.03; p = 0.0004), whereas the rs13216992-T2D association was fully attenuated (OR = 1.00; 95% CI 0.99-1.01; p = 0.68). Fine-mapping identified a compact 95% BMI credible set of 17 variants and a broader 95% T2D credible set of 42 variants, with all BMI credible variants contained within the T2D set. Conclusions: The GLP1R locus harbors at least two independent BMI-associated variants that exhibit heterogeneous relationships with T2D: rs12213929 influences T2D risk partly through BMI-independent pathways, whereas rs13216992 appears to act predominantly via adiposity. These findings refine the genetic architecture at this key therapeutic target gene and provide a foundation for functional and pharmacogenomic studies to determine whether GLP1R variation can inform precision prevention and treatment of obesity and T2D.

ObjectiveHbA1c thresholds used to define dysglycemia in autoantibody-positive individuals at risk for type 1 diabetes do not account for age-related increases in HbA1c and may overestimate progression risk in adults. We evaluated whether age-adjusted HbA1c or a higher HbA1c threshold improves risk stratification across age groups. Research Design and MethodsWe analyzed 5,024 autoantibody-positive relatives (3,720 children and 1,304 adults) participating in the TrialNet Pathway to Prevention study. Age-related HbA1c effects were modelled using 6,273 adults from the population-based Exeter 10,000 cohort. Progression risk was compared using the standard dysglycemia threshold (HbA1c [&ge;] 5.7% [39 mmol/mol]), age-adjusted HbA1c, and an alternative threshold of HbA1c [&ge;]6.0% (42 mmol/mol). ResultsUsing HbA1c [&ge;] 5.7%, children had higher 1-year progression risk than adults among single autoantibody-positive participants (38% [95% CI 28, 47] vs. 13% [7.2, 19]) and multiple autoantibody-positive participants (55% [49, 60] vs. 38% [27, 47]; both p<0.001). Age adjustment reduced these differences; progression risk was similar among single autoantibody-positive participants (38% [28, 47] vs. 27% [13, 39]; p=0.32), with attenuated differences among multiple autoantibody-positive participants. An HbA1c threshold [&ge;]6.0% yielded comparable progression risk between adults and children across autoantibody subgroups. In post hoc analyses, adults aged <30 years had progression risk similar to children (p=0.1). ConclusionsAge-related variation in HbA1c influences dysglycemia classification in adults at risk for type 1 diabetes. Age-adjusted HbA1c or a higher HbA1c threshold ([&ge;]6.0% [42 mmol/mol]) in adults [&ge;]30 years identifies individuals with progression risk comparable to children and may improve age-specific risk stratification in prevention seungs.

Obesity affects one in three adults and is complicated by adipose inflammation, lipotoxicity and cell death. We previously identified RIPK1 as a genetic determinant of human obesity risk and adipose inflammation. Because RIPK1 is the apical kinase in the necroptosis pathway upstream of RIPK3 and the executioner protein MLKL, and emerging evidence links MLKL to lipid metabolism, MLKL has surfaced as a potential metabolic regulator. However, conflicting findings in Mlkl knockout mice fed a high fat diet have left its therapeutic relevance unresolved. MLKL has not been previously targeted through therapeutic knockdown in vivo in the context of diet-induced obesity. Here, we evaluated two independent MLKL antisense oligonucleotides (ASOs) in high fat diet (HFD)-fed C57BL/6J mice. In a 24-week progression model, MLKL ASO markedly reduced body weight, fat mass and hepatic steatosis compared with controls, while preserving lean mass. MLKL knockdown also lowered the respiratory exchange ratio, indicating a shift toward increased fat oxidation. In the intervention model, once obesity was established after 12 weeks of HFD feeding, both MLKL ASOs, and similarly, two independent RIPK1 ASOs, reversed weight gain and improved systemic glucose control. In vitro, MLKL-CRISPR/Cas9 knockout blocked 3T3-L1 adipogenesis, indicating a requirement for MLKL during adipocyte differentiation. However, in mature adipocytes, MLKL siRNA reduced palmitic acid-induced lipid accumulation, increased isoprenaline-stimulated lipolysis, and prevented TNF-mediated suppression of insulin-mediated AKT signalling and glucose uptake. Collectively, these findings demonstrate that partial MLKL suppression reprograms whole-body energy metabolism, enhances insulin sensitivity and limits diet-induced adiposity. MLKL, therefore, represents a promising and mechanistically novel therapeutic target for obesity and insulin resistance.

Age-defined type 1 diabetes (T1D) endotypes, T1DE1 and T1DE2, are characterized by reproducible differences in pancreatic immunopathology and clinical course. In particular, these endotypes differ in the extent and composition of lymphocytic insulitis and in the extent of loss of insulin-producing {beta} cell mass, at diagnosis. However, blood-based biomarkers that may distinguish these endotypes and inform the underlying immune-islet biology axis at diagnosis remain limited. Here, we characterized the clinical features and profiled circulating microRNAs (miRNAs) in plasma from two independent INNODIA cohorts of individuals with newly diagnosed stage 3 T1D (discovery, n=115; replication, n=147), stratified into age-defined endotypes (T1DE1, <7 years; T1DE2, [&ge;]13 years; and intermediate T1DInt, 7-12 years). Differential-expression and age-adjusted models were coupled to orthogonal ddPCR validation. Putative miRNAs cellular sources were inferred using reference miRNA expression atlases. Biological context was explored via correlations of miRNAs with whole-blood transcriptomics. Clinically, T1DE1 was associated with lower {beta}-cell function and higher first-year C-peptide decline, alongside distinct islet autoantibody patterns, consistent with an immunologically aggressive endotype. Small RNA-seq analysis and ddPCR validation identified a reproducible signature in which miR-150-5p, a B-and T-lymphocyte related miRNA, and miR-375-3p, a {beta} cell enriched molecule, were consistently increased in T1DE1 compared with T1DE2 across both cohorts. MiR-150-5p retained robust association with T1DE1 even after age adjustment, and neither miRNA was associated with age in non-T1D pediatric datasets, supporting T1D endotype specificity. The increased circulating miR-150-5p signal was not explained by differences in peripheral blood B-or T-cell frequencies in high-parameter flow-cytometry subsets, and its levels correlated inversely with whole-blood expression of the immune-associated miR-150-5p target genes MPPE1 and RABGAP1L. Finally, applying a rule-based combined classifier (miR-150-5p and miR-375-3p "high") achieved re-stratification of T1D individuals, including those in the intermediate age group, into two miRNA-defined groups with distinct {beta} cell functional trajectories. Collectively, these data suggest circulating miR-150-5p and miR-375-3p as non-invasive biomarkers linked to endotype-associated biology at T1D diagnosis, with potential utility for endotype-centered stratification and trial enrichment.

Exercise is a cornerstone therapy for diabetes because working skeletal muscles take up glucose at dramatically greater rates than postprandial insulin-stimulated glucose uptake and, notably, do so without a requirement for insulin. This remarkable ability of working muscles is preserved in diabetes, when muscles become resistant to insulin. However, the mechanism of insulin-independent glucose uptake by working muscles is not fully understood. Here we describe a previously unrecognized glucose uptake pathway in muscle, which we refer to as "mSGLT" based on shared properties with the Sodium Glucose Linked Transporter family. In contrast to the abundant GLUT4 transporter, mSGLT is not regulated by insulin, requires Na,K-ATPase-2 activity, and transports the hexose -methyl-D-glucoside (MDG), a glucose derivative that is handled by SGLTs but not GLUT4. The mSGLT pathway and GLUT transport pathways are independent and additive. In addition to exercise, mSGLT imports glucose under other conditions of adrenergic stimulation, which inhibits pancreatic insulin release and reduces the insulin sensitivity of muscle. SGLT2-specific antibodies recognize a protein in muscle of similar size to the kidney SGLT2; this protein localizes to the muscle t-tubules, together with Na,K-ATPase-2 and MAP17, the regulatory subunit of SGLT2. However, skeletal muscles do not express a full-length transcript of Slc5a2 (SGLT2), and SGLT2-specific inhibitors do not inhibit mSGLT with high affinity. The novel transporter may be a muscle variant of Slc5a2 that results from post-transcriptional or post-translational mechanisms. mSGLT and its regulation offer potential muscle-specific therapeutic targets for treating hyperglycemia and other conditions when insulin-stimulated glucose disposal into muscle is impaired.

Effector CD8+ T cells are key cellular drivers of type 1 diabetes (T1D) pathogenesis, yet questions remain regarding the molecular defects leading to altered cytotoxicity, their signature in peripheral tissues, and their receptor specificity. Thus, we analyzed human pancreatic lymph nodes (pLN) using mass cytometry and single cell RNA sequencing (scRNAseq) with combined proteomic and T cell receptor (TCR) profiling. Cytometric analysis revealed an enriched population of T stem-cell memory (TSCM)-like cells (CD8+CD45RA+CD27+CD28+CCR7+CXCR3+ T cells) in T1D pLNs. scRNAseq profiling indicated an elevated inflammatory cytokine gene signature (IFITM3, LTB) along with regulators of terminal differentiation (BCL6, BCL3), coupled with reduced expression of exhaustion-associated genes (DUSP2, NR4A2, TSC22D3) in CD8+ T cells in T1D pLN. Additionally, effector CD8+ T cells expressed features of progenitor exhausted cells (BCL2) in T1D pLN. Immune Response Enrichment Analysis (IREA) indicated IL-15 signaling as a significant driver of these phenotypes. Integrated TCR and transcriptomic analysis revealed a cluster of diverse naive-like CD8+ T cell clones in T1D pLN. When comparing pLN and pancreatic slice cellular isolates, we observed sharing of effector CD8+ T cells, with upregulation of terminal effector signatures detected within the pancreas relative to paired pLN samples. Multiplex imaging revealed differential localization of TCF1 and TOX expressing T cells in the pancreas, with TCF1+TOX+ cells located in closer proximity to the islets and displaying a mixture of activation and exhaustion-associated phenotypes. Thus, we provide multimodal cellular profiles enriched in T1D tissues for consideration in therapeutic targeting.

Pancreatic beta cells produce and secrete insulin to maintain glucose homeostasis. Due to their high secretory activity, beta cells rely heavily on endoplasmic reticulum (ER) function and are particularly susceptible to ER stress, which contributes to beta cell dysfunction in diabetes. However, the transcriptional mechanisms linking ER stress to beta cell failure remain poorly understood. In this study, we investigated the role of the transcription factor Mef2a in ER stress-mediated beta cell dysfunction using primary mouse islet cells. ER stress induced by thapsigargin increased Mef2a expression and activated canonical unfolded protein response (UPR) pathways. Overexpression of Mef2a reduced beta cell proliferation, suppressed expression of key beta cell transcription factors including Pdx1, MafA, NeuroD1, and Nkx6.1, and impaired glucose-stimulated insulin secretion. Mef2a overexpression also altered mitochondrial respiration, characterized by reduced glucose-coupled respiration and increased maximal respiratory capacity. In contrast, Mef2a knockdown attenuated ER stress induced activation of ATF6 and IRE1/XBP1 dependent UPR genes. Importantly, reducing Mef2a expression preserved beta cell identity gene expression and improved insulin secretion during ER stress induced by thapsigargin or tunicamycin. Together, these findings identify Mef2a as a stress-responsive regulator that contributes to ER stress-mediated beta cell dysfunction and suggest that modulating Mef2a activity may help preserve beta cell function during metabolic stress.

Type 1 diabetes (T1D) is a consequence of {beta}-cell death. ER stress precedes T1D onset and prolonged ER stress in {beta}-cells can lead to {beta}-cell apoptosis. We reported that lipid signaling generated by the Ca2+-independent phospholipase A2{beta} (iPLA2{beta}), encoded by Pla2g6, participates in ER stress-mediated {beta}-cell apoptosis. {beta}-Cell membranes are enriched in arachidonic acid containing glycerophospholipids and the iPLA2{beta} catalyzes the hydrolysis of arachidonic acid in ER stressed {beta}-cells. Metabolism of arachidonic acid leads to the generation of various proinflammatory lipids, raising the possibility that they contribute to ER stress and {beta}-cell death leading to T1D. However, molecular mechanisms by which such {beta}-cell-iPLA2{beta}-derived lipid (iDL) signaling contributes to {beta}-cell apoptosis are not understood. It is well known that ER stress-mediated {beta}-cell apoptosis is associated with induction of transcription factors, NF{kappa}B and STAT1. We report here that both induce Pla2g6 and, unexpectedly, we find that iPLA2{beta}, which lacks DNA-binding motifs, associates with NFkB, Stat1, and Pla2g6 promoter regions. Consistently, p65-NF{kappa}B and pSTAT1 induction is reduced with select inhibition or knockdown of iPLA2{beta}. Surprisingly, iPLA2{beta} expression is also reduced by select inhibition of iPLA2{beta}, raising the possibility of feedback regulation by iDLs. In support, we find that select iDLs, recognized to be proinflammatory, enhance association of iPLA2{beta} with Pla2g6, Nfkb, and Stat1 promoter regions leading to induction of all three gene products and {beta}-cell apoptosis. Our findings reveal previously unrecognized transcriptional regulation by iDL signaling and, iPLA2{beta} itself, that leads to gene products that promote {beta}-cell apoptosis. Analogous findings in human islets validate this mechanism raising the possibility that targeting select lipid signaling can reduce ER stress in {beta}-cells and ameliorate T1D development.

Background Glycated haemoglobin (HbA1c) underpins type 2 diabetes (T2D) and prediabetes management worldwide and reflects both glycaemia and erythrocyte biology. A missense variant in PIEZO1 (rs563555492T), carried by 1 in 12 South Asians, has been associated with a nonglycaemic reduction in HbA1c. We aimed to further characterise this association and evaluate its clinical consequences. Methods We undertook genetic and linked health data analyses across two cohorts: 19,898 (37.4% female) South Indians from the Madras Diabetes Research Foundation (MDRF) and 43,011 (54.4% female) British Bangladeshis and British Pakistanis in Genes & Health. In MDRF, we tested associations with glycaemic and erythrocytic traits using additive genetic models. In Genes & Health we modelled diagnosis of prediabetes, T2D, and diabetic eye disease using flexible parametric survival models. Ten-year absolute risks were estimated for a population aged 40-50 years. Findings PIEZO1 rs563555492T was associated with erythrocytic traits and lower HbA1c, but not with fasting glucose, postprandial glucose, or C-peptide. This variant reduced risk of prediabetes (HR 0.63, 95% CI 0.58-0.69) and T2D (0.85, 0.78-0.93) diagnosis, and increased risk of diabetic eye disease among individuals with T2D (1.20, 1.01-1.43). Modelling suggested approximately 1,019 missed prediabetes and 303 missed T2D diagnoses per 100,000 adults over 10 years. Interpretation An ancestry-enriched PIEZO1 variant is associated with lower HbA1c independent of glycaemia, reduced prediabetes and T2D diagnosis suggesting delayed detection, and increased complication risk. Reliance on HbA1c may systematically underestimate glycaemic risk in a substantial minority of South Asians. Funding The Wellcome Trust; NIHR

Obesity-driven type 2 diabetes (T2D) is characterized by pathological alterations in visceral white adipose tissue (vWAT). While microRNAs (miRNAs) are key post-transcriptional regulators, comprehensive human vWAT profiling across metabolic states remains limited. This study characterized vWAT miRNA expression in lean, obese, and obese+T2D individuals to identify regulatory networks associated with metabolic failure. Deep miRNA sequencing was performed on vWAT samples from a discovery cohort, followed by validation via qPCR in an independent replication cohort. Differentially expressed miRNAs across the three groups were bioinformatically integrated with matched mRNA transcriptomic data to construct functional regulatory modules and identify enriched pathways underlying metabolic impairment. Several miRNAs exhibited robust and reproducible differential expression between obesity and obesity with T2D. Integrated miRNA-mRNA analyses revealed coherent regulatory modules involving inflammation, lipid metabolism, insulin signaling, and iron homeostasis. Specifically, miR-141-3p, miR-200b-3p, miR-15b-3p, miR-12136, and miR-585-3p showed consistent differential expression. Notably, miR-141-3p and miR-200b-3p were markedly upregulated and inversely associated with metabolic stress-related genes, including TF and FBXO32. Several miRNAs correlated with clinical markers of metabolic dysfunction, supporting their biomarker potential. By comparing lean, obese, and diabetic populations, this study provides a comprehensive characterization of the vWAT miRNA landscape and identifies specific miRNA-mRNA regulatory circuits that orchestrate the transition from healthy adiposity to pathological adipose tissue dysfunction. These findings pinpoint novel molecular drivers of type 2 diabetes progression and offer potential targets for therapeutic intervention in metabolic endocrine disorders.

The establishment of mixed hematopoietic chimerism is a promising way to induce immune tolerance for islet replacement therapy and to treat the underlying autoimmunity in Type 1 diabetes (T1D). Mixed chimerism not only promotes effective thymic negative selection of autoreactive cells but also restores regulatory T cell (Treg) function and peripheral tolerance. In the current study, we determined that a novel class of donor-derived CD8+CD44+CD122+ Tregs (d-CD8+CD122+ Tregs) plays a crucial role in controlling autoimmunity in non-obese diabetic (NOD) mice with induced mixed chimerism. Using adoptive T cell transfer experiments, we showed that d-CD8+CD122+ Tregs abrogate autoimmunity by selectively depleting the exogenously injected diabetogenic T cells in Recombination-Activating Gene deficient NOD mice. These d-CD8+CD122+ Tregs from NOD chimeras show upregulation of Helios, Programmed cell death protein 1, perforin, granzyme-B, CD39, Folate receptor 4, and downregulation of proinflammatory markers like Scart1 and Scart2. Using in vitro assays, we show that d-CD8+CD122+ Tregs respond specifically to a Complementarity-Determining Region-3 peptide sequence derived from T cell receptors of islet antigen-specific autoreactive T cells. Thus, mixed chimerism might be a method to revitalize CD8+CD122+ Tregs which are decreased in number and functionality in NOD mice. Similarly, we found that individuals with T1D have a deficiency in CD8+CD122+ Tregs, suggesting a potential loss of regulatory function accompanies disease onset. Revitalizing CD8+CD122+ Tregs may offer a new therapeutic strategy of restoring immune tolerance in autoimmune diabetes. One sentence summary Inducing mixed donor chimerism in NOD mice generates donor-derived CD8+CD122+ Tregs that suppress autoimmunity and restore immune tolerance by selectively eliminating autoreactive T cells.