Diabetes

Recent epidemiologic studies indicate that adult-onset type 1 diabetes (AOT1D) is more common than childhood-onset type 1 diabetes, yet it remains clinically underrecognized. Because little is known about the emergence of islet autoantibodies in AOT1D, we conducted a retrospective study using electronic medical records from the United States Military Health System and longitudinal serum samples from 169 individuals with AOT1D and 40 healthy controls obtained from the Department of Defense Serum Repository. Among 643 prediagnostic samples from individuals with AOT1D, IA-2 autoantibodies were the most prevalent (50%), followed by GADA (46%), IA-2{beta} (34%), ZnT8-R (27%), and ZnT8-W (15%). Overall, 85% (144/169) of subjects were seropositive for at least one autoantibody prior to diagnosis. Analysis of the earliest available sample from all of the AOT1D cases, grouped into 5-year intervals preceding diagnosis, demonstrated a progressive increase in seropositivity over time: 38% of subjects were seropositive more than 20 years before diagnosis, increasing to 44% at 20-15 years, 59% at 15-10 years, 73% at 10-5 years, and 91% within 5 years of diagnosis. Among the 144 seropositive individuals, positivity for two or more autoantibodies was the most common pattern, occurring in 50% (72/144) of cases. Isolated GADA positivity (22%) and isolated IA-2/IA-2{beta} positivity (24%) occurred at similar frequencies, whereas isolated ZnT8 positivity was uncommon (4%). Temporal analysis showed that isolated GADA positivity appeared earliest, with a median onset of 7.9 years before diagnosis, whereas multiple-autoantibody positivity, IA-2 positivity, and ZnT8 positivity emerged later, with median onsets of 4.6, 4.5, and 1.9 years before diagnosis, respectively. These findings extend observations from pediatric type 1 diabetes to adults and demonstrate that AOT1D-associated autoimmunity often begins decades before clinical diagnosis, highlighting a potentially important window for risk stratification and preventive intervention.

Type 2 diabetes is characterized by failure of pancreatic {beta} cells to adapt insulin secretion to metabolic demand, due to impaired {beta}-cell function and/or reduced {beta}-cell mass. The unfolded protein response (UPR) is central to this adaptation by maintaining endoplasmic reticulum homeostasis and supporting insulin biosynthesis, secretion, proliferation, and survival. NCK1, is an adaptor protein that regulates diverse cellular processes, including insulin biosynthesis and UPR activation, positioning it at the crossroads of several processes essential for {beta}-cell function. Moreover its silencing is reported to enhance adaptive PERK signaling and {beta}-cell survival in vitro, suggesting that it could represent an important regulator of {beta}-cell adaptation. Here, we explored this potential role for NCK1 using {beta}-cell-specific knockout mice (NCK1{beta}KO) and human islets of both sexes. NCK1 expression was positively regulated by glucose yet reduced in islets from individuals living with type 2 diabetes. Loss of {beta}-cell NCK1 impaired insulin gene expression, insulin content, and glucose-stimulated insulin secretion in vitro, and disrupted UPR activation. In vivo, {beta}-cell NCK1 deletion led to sex-dependent adaptation to maintain glucose homeostasis. Under high-fat/high-sucrose diet, both NCK1{beta}KO male and female mice increased pancreatic insulin content, but only males showed improved insulin secretion associated with islet expansion and {beta}-cell proliferation. Females, in contrast, exhibited impaired insulin secretion despite preserved insulin stores, associated with increased numbers of small islets and altered PERK pathway activation. These findings identify NCK1 as a regulator of {beta}-cell insulin synthesis, secretion, and UPR signaling, and reveal sex-specific adaptive mechanisms to {beta}-cell stress. Reduced NCK1 in islets from people living type 2 diabetes may disrupt {beta}-cell adaptation to metabolic stress and contribute to diabetes.

The NACHT, LRR and PYD domains-containing protein 3 (NLRP3) inflammasome is a protein complex that senses metabolic disturbances and in response processes IL-1beta. Prolonged activation of the NLRP3 inflammasome by metabolic stress induces chronic low-grade inflammation and contributes to the development type 2 diabetes and its comorbidities. Here, we developed a mouse model of type 2 diabetes that features impaired proinsulin processing and the ability to form islet amyloid plaques, two determinants of human type 2 diabetes pathology. We show that at advanced ages, these mice develop amyloidosis and inflammation in their insulin-producing pancreatic islets. Beta-cell mass and function were impaired in these mice and severe hyperglycemia developed within 6 months. Oral application of OLT1177, a selective inhibitor of the NLRP3 inflammasome, prevented the development of hyperglycemia. Our data show that inhibition of the NLRP3 inflammasome in a humanized mouse model of severe type 2 diabetes prevents the development of amyloid-associated hyperglycemia.

The decline in first-phase insulin response (FPIR) during the presymptomatic period of type 1 diabetes (T1D) is well established. In-situ functional studies with pancreas tissue slices showed that {beta}-cell loss of glucose-responsiveness was independent of T-cell infiltration into islets in recent-onset T1D cases. However, the mechanisms driving {beta}-cell dysfunction before the onset of T1D remain unclear. In pancreas tissue from donors across the natural history of T1D, we utilized an in-situ, whole-islet phenotypical and transcriptomic approach to unravel novel targets in the glucose-stimulus coupled secretion pathway that are similarly impaired in T-cell infiltrated and non-infiltrated islets. Specifically, we observed that islets from autoantibody positive (single(s) or multiple(m) AAb+) donors exhibited activation of post-transcriptional gene regulation along with reduced protein translation, processing in the endoplasmic reticulum (ER), and ER stress. Disrupted mitochondrial metabolism and bioenergetics were prominent in islets from multiple AAb+ and T1D donors with disease durations [≤]7 years. In addition, T1D islets presented reduced mitochondrial protein import, quality control, and dynamics, together with downregulated genes in insulin secretory pathways. During infiltration, these pathways remain dysregulated while immune/inflammatory transcripts were increased. These studies identified novel mechanisms of {beta}-cell dysregulation before symptomatic onset and independent of T-cell infiltration in T1D pathogenesis.

Aims/hypothesisDisruption of the circadian glucocorticoid rhythm occurs in human settings including chronic stress, sleep restriction, circadian misalignment, ageing and autonomous cortisol secretion; in mild autonomous cortisol secretion (MACS) and Cushings syndrome, loss of the normal cortisol trough is clinically informative, and flatter diurnal cortisol profiles are associated with cardiometabolic disease. We previously showed that flattening of glucocorticoid rhythms in mice induces rapid and sustained hyperinsulinemia without hyper or hypo-glycaemia, implying that glucocorticoid rhythms may directly regulate the relationship between circulating glucose and systemic insulin output. Here we tested the hypothesis that beta cell glucocorticoid receptor (GR) signalling is required for the compensatory hyperinsulinaemia that maintains glucose homeostasis during glucocorticoid rhythm flattening, and that this reflects glucocorticoid-dependent reprogramming of beta cell stimulus-secretion coupling. MethodsGlucocorticoid rhythms were flattened in male C57BL/6J mice by subcutaneous corticosterone pellet implantation, which elevates trough levels and reduces peak amplitude while preserving the daily mean hormone concentration. Fasting plasma insulin and blood glucose were measured longitudinally and compared with placebo-implanted controls and high-fat diet-fed mice. Beta cell secretory function was assessed by static and dynamic glucose-stimulated insulin secretion in isolated islets, and beta cell excitability by GCaMP6f Ca{superscript 2} imaging in islets from Ins1-Cre;GCaMP6f mice. To test the requirement for beta cell GR in mature beta cells while avoiding developmental effects of constitutive GR deletion, we generated adult-inducible beta cell-specific GR knockout mice (MIP-CreERT;Nr3c1fl/fl; {beta}GRKO). Combined beta cell and hepatic GR knockout mice (double-GRKO) were used to examine an additional extra-pancreatic contribution to systemic insulin availability. Glucose tolerance and insulin sensitivity were assessed by intraperitoneal glucose and insulin tolerance tests. As a secondary question, a possible contribution of altered insulin clearance was examined from plasma C-peptide:insulin ratios and hepatic insulin-degrading enzyme (IDE) abundance. ResultsGlucocorticoid rhythm flattening produced sustained hyperinsulinaemia with maintained euglycaemia, distinct from the delayed hyperinsulinaemia and hyperglycaemia observed in high-fat diet-fed mice. Islets from glucocorticoid-flattened mice exhibited increased insulin secretion at subthreshold (3 mmol/l) glucose, enhanced secretory responses to stimulatory glucose and increased Ca{superscript 2} responses, indicating a lowered glucose threshold for beta cell activation that persisted ex vivo. Beta cell-specific deletion of GR markedly attenuated the hyperinsulinaemic response to glucocorticoid flattening (insulin AUC reduced [~]40% vs controls; p < 0.001) and produced progressive hyperglycaemia and impaired glucose tolerance, despite unchanged or improved insulin sensitivity. A reduced plasma C-peptide:insulin molar ratio (p = 0.007) and decreased hepatic IDE abundance (p = 0.032) indicated that reduced insulin clearance contributes additionally to the elevated circulating insulin, and combined beta cell and hepatic GR deletion lowered circulating insulin further than beta cell GR deletion alone. The absence of hypoglycaemia despite persistent hyperinsulinaemia is consistent with concurrent insulin resistance. Conclusions/interpretationBeta cell GR signalling is required for the compensatory hyperinsulinaemia that maintains glucose homeostasis when glucocorticoid rhythmicity is disrupted, acting through glucocorticoid-dependent lowering of the glucose threshold for insulin secretion; reduced insulin clearance contributes additionally to the rise in circulating insulin. These findings identify beta cell GR signalling as a key determinant of glucose homeostasis during disrupted glucocorticoid rhythmicity. Clinically, the work is most relevant not simply to nonspecific chronic stress, but to human states in which the cortisol rhythm is measurably flattened or the nocturnal trough is lost, including MACS, Cushings syndrome, sleep restriction, shift work/circadian misalignment and ageing. RESEARCH IN CONTEXTO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIFlattened or disrupted glucocorticoid rhythmicity in humans is observed most directly in MACS and Cushings syndrome, where loss of the late-night cortisol nadir is clinically informative, and more broadly as flatter salivary cortisol slopes or elevated evening cortisol in ageing, sleep restriction and circadian misalignment; these patterns are associated with type 2 diabetes, cardiovascular disease and mortality. C_LIO_LIFlattening of glucocorticoid rhythms in mice induces rapid and sustained hyperinsulinaemia without hypoglycaemia, indicating that circulating insulin can be elevated independently of glucose. C_LIO_LIHepatic insulin clearance, mediated in part by insulin-degrading enzyme and CEACAM1, is a major determinant of circulating insulin levels. C_LI What is the key question?O_LIHow does disruption of glucocorticoid rhythmicity increase circulating insulin while maintaining glycaemic control, and is beta cell glucocorticoid receptor signalling required for this adaptive response? C_LI What are the new findings?O_LIGlucocorticoid rhythm flattening lowers the glucose threshold for beta cell activation through enhanced Ca{superscript 2} excitability, an effect that persists in isolated islets and indicates in vivo reprogramming of beta cell function. C_LIO_LIBeta cell-specific deletion of the glucocorticoid receptor blunts the hyperinsulinaemic response to glucocorticoid flattening and produces hyperglycaemia and impaired glucose tolerance despite unchanged or improved insulin sensitivity. C_LIO_LIReduced insulin clearance, associated with decreased hepatic insulin-degrading enzyme abundance, contributes additionally to the elevated circulating insulin, but is not required for maintenance of glucose homeostasis. C_LI How might this impact on clinical practice in the foreseeable future?O_LIIdentifying beta cell glucocorticoid receptor signalling as a requirement for glucose homeostasis during disrupted glucocorticoid rhythmicity may inform strategies for understanding hyperinsulinaemia and steroid-associated metabolic dysfunction in human conditions marked by loss of the cortisol trough or flatter diurnal cortisol profiles, particularly MACS, Cushings syndrome, shift work/circadian misalignment and ageing. C_LI

Aims/hypothesisHeterogeneity in the pathophysiology of type 2 diabetes is increasingly being realised. The currently available rodent models of type 2 diabetes all have limitations and do not accurately reflect all human type 2 diabetes subtypes. NOD.BR-H2k /Wicker mice (NODk), derived from the non-obese diabetic (NOD) mouse, are type 1 diabetes resistant. However, transgene induced beta-cell stress in male NODk mice induces hyperinsulinaemia followed by diabetes. Here we have investigated the propensity of NODk mice to develop a Western-diet (WD) induced hyperinsulinaemic subtype of type 2 diabetes. Comparator mouse strains used were BALB/c and B10.BR-H2k /SgSnJ mice (B10k). MethodsIn the longer-term studies (14-24 weeks), NODk, B10k and BALB/c mice were randomised to receive Chow or WD from 4 weeks of age, followed by serial measurement of body weight and fed-state blood glucose. IPGTT and IPITT tests were conducted at 13 weeks of age. Blood and pancreas were harvested for further analyses at 14 and 24 weeks of age, or sooner if diabetes developed (blood glucose concentrations [&ge;]20 mmol/l on two consecutive days). In the acute studies, metabolic characteristics of the three strains at 8 weeks of age, continued on Chow or after a 5-day WD challenge (WDC) were assessed, along with harvesting pancreas on day 5 for ex vivo islet insulin secretion, electron microscopy, and bulk islet transcriptomics analyses. ResultsMale WD-fed NODk mice became markedly hyperinsulinaemic, gained excess weight and developed a severe type 2 diabetes phenotype. Emergence of diabetes was associated with islet endocrine cell apoptosis and loss of beta-cell mass, without evidence of insulitis. Insulin resistance on IPITT testing, however, was not evident in Chow-fed NODk mice. In contrast, male B10k mice already had poor glucose tolerance on Chow diet and, despite having a hypoinsulinaemic phenotype, were resistant to WD-induced diabetes. BALB/c mice developed very mild glucose intolerance and hyperinsulinaemia in response to the WD. Female NODk mice were diabetes resistant. At 8 weeks of age, male Chow-fed NODk mice were mildly hyperinsulinaemic despite relative hypoglycaemia compared to the other strains. The acute 5-day WDC markedly increased hyperinsulinaemia in NODk mice. Transcriptomics analyses identified robust strain-specific differences, including altered islet cell differentiation, energy metabolism, endoplasmic reticulum to golgi vesicle transport and insulin processing. Conclusions/interpretationNODk mice, which exhibit mild hyperinsulinaemic hypoglycaemia on Chow diet and rapidly develop marked hyperinsulinaemia on WD, are type 2 diabetes prone. In contrast, B10k mice have poor glucose tolerance on Chow diet and no or limited capacity to increase insulinaemia in response to WD, are diabetes resistant. These findings support the hypothesis that hyperinsulinaemia is upstream to insulin resistance in the pathogenesis of severe insulin resistant subset of type 2 diabetes for which the WD-fed NODk mouse is a suitable new mouse model. Research in ContextO_ST_ABSWhat is already known about this subject?C_ST_ABSO_LIWhich of insulin hypersecretion and insulin resistance are upstream in the pathogenesis of the severe insulin resistant subtype of type 2 diabetes continues to be debated C_LIO_LIRodent models of type 2 diabetes do not accurately reflect all human subtypes of type 2 diabetes C_LIO_LINODk mice, derived from the non-obese diabetic (NOD) mouse, are type 1 diabetes resistant, but with transgene induction of islet beta-cell stress develop hyperinsulinaemia, followed by type 2 diabetes C_LI What is the key question?O_LICould Western-diet fed NODk mice be developed as a model of severe insulin resistant type 2 diabetes and shed light on its upstream pathogenesis? C_LI What are the new findings?O_LIMale NODk mice tend to hyperinsulinaemic hypoglycaemia on Chow diet, rapidly develop marked hyperinsulinaemia on Western-diet feeding, and then develop type 2 diabetes C_LIO_LIMale B10k mice (one of two comparator strains (B10k and BALB/c)) have poor glucose tolerance on Chow diet, limited capacity to increase insulinaemia in response to Western-diet feeding, but are resistant to develop Western-diet induced type 2 diabetes C_LIO_LIIsolated islet findings show strain differences that favour intrinsic hyper-responsiveness and hypo-responsiveness of islet beta-cells of NODk and B10k mice, underpinning their respective metabolic phenotypes C_LI How might this impact on clinical practice in the foreseeable future? O_LIThe findings are in support of the insulin hypersecretion hypothesis for severe insulin resistant type 2 diabetes, such that therapies to limit islet beta-cell hyperresponsiveness to prevent and treat this subtype of diabetes warrant investigation C_LI

OBJECTIVE To characterize the prevalence and penetrance of maturity-onset diabetes of the young (MODY) in a multi-ancestry population using a genotype-first design. RESEARCH DESIGN AND METHODS We analyzed whole-genome sequencing and clinical data from 374,973 unrelated All of Us participants (42.0% non-European ancestry). We identified pathogenic or likely pathogenic (P/LP) variants in 10 established MODY genes and assessed carrier prevalence, diabetes penetrance, and glycemic profiles. We evaluated age-dependent diabetes risk by comparing carriers with non-carriers stratified by type 2 diabetes polygenic risk score (T2D PRS). RESULTS We identified 370 carriers of P/LP MODY gene variants (0.099%; 1 in 1,013), with similar carrier prevalence among European- and African-ancestry participants (0.105% in both groups). Diabetes penetrance was incomplete (13.4% by age 40; 43.5% by age 60) and varied by etiology: highest for GCK (56.0% by age 60), intermediate for HNF genes (HNF1A/HNF1B/HNF4A; 45.4%), and lowest for non-GCK/HNF genes (ABCC8/INS/KCNJ11/NEUROD1/PDX1/RFX6; 29.0%). In multivariable Cox models using non-carriers in the middle 80% of the T2D PRS as the reference, non-GCK/HNF gene variant carriers had modestly increased diabetes risk (HR, 1.57), similar to non-carriers in the top 10% of T2D PRS (HR, 1.64). These associations were observed in both European- and non-European-ancestry individuals. HbA1c profiles differed by etiology, with stable mild hyperglycemia in GCK variant carriers and greater variability among HNF and non-GCK/HNF gene variant carriers. CONCLUSIONS MODY gene variants showed incomplete, etiology-dependent penetrance across ancestries. Carriers of P/LP variants in lower-penetrance genes had diabetes risk comparable to that of non-carriers with high polygenic susceptibility.

Background. Type 2 diabetes mellitus (T2D) is defined by progressive pancreatic {beta}-cell dysfunction whose molecular underpinnings remain incompletely understood. Single-cohort transcriptomic analyses of donor islets have yielded heterogeneous gene lists of limited cross-study reproducibility, constraining both mechanistic interpretation and biomarker development. Methods. We combined two complementary analytical strategies applied to four public human islet transcriptomic cohorts (GSE25724, GSE20966, GSE38642, and GSE164416; n = 7-57 donors per contrast). For the integrative arm, three microarray datasets and one bulk RNA-seq dataset were processed independently and unified through gene-level random-effects meta-analysis, hallmark pathway scoring (GSVA/MSigDB), and iterative module refinement, yielding a two-axis disease framework. For the diagnostic arm, a consensus multi-method machine learning pipeline, combining LASSO penalized logistic regression, Support Vector Machine Recursive Feature Elimination (SVM-RFE), and Random Forest importance scoring, was applied to 184 differentially expressed genes from the RNA-seq cohort, with all normalization steps performed within leave-one-out cross-validation (LOOCV) folds to prevent data leakage. Machine learning classification of the RNA-seq cohort was additionally subjected to external transportability testing in the independent bulk human islet RNA-seq cohort GSE50244 using an overlap-restricted reduced score and a threshold fixed in the discovery cohort. Results. Meta-analysis across all four cohorts identified 337 high-confidence T2D-associated genes (96.1% directional concordance in beta-cell-enriched tissue). These were distilled into two refined 14-gene modules: ImmuneStress (MICB, HLA-DRA, HLA-DPA1, IL1R2, and others) and BetaCellIdentitySecretion (RASGRP1, PPP1R1A, SLC2A2, and others), whose composite IsletDysfunctionScore provided the most stable cross-platform separation of non-diabetic from T2D islets (Hedges' g = 1.80, p = 9.83 x $10^-17$, $\text{I}^2$= 0%). Consistent with progressive disease, IsletDysfunctionScore increased monotonically from non-diabetic to impaired glucose tolerance to T2D. Separately, the machine learning pipeline derived a 10-gene diagnostic panel: GABRA2, SLC2A2, ARG2, DKK3, PRIMA1, TAFA4, HHATL, PARVG, RNU1-70P, and the novel lncRNA ENSG00000284653, that achieved perfect discrimination in LOOCV (AUC = 1.000, sensitivity = 1.000, specificity = 1.000, zero misclassifications across all 57 donors). A leakage-verification experiment confirmed that this performance reflected genuine biological signal: global quantile normalization prior to cross-validation collapsed AUC to 0.380. External testing showed that 8 of the 10 panel genes were measurable in GSE50244. The frozen 8-gene reduced score retained strong discrimination (external AUC = 0.907), with 6 of 8 genes preserving directional concordance, but the discovery-derived threshold did not transfer because the external score distribution was shifted upward and compressed, yielding complete sensitivity but zero specificity at the frozen cutoff Conclusions. Integrating pathway-level meta-analysis with machine learning classification, we present a coherent two-axis model: immune/stress activation and loss of beta-cell identity/secretory competence, together with a compact, biologically interpretable 10-gene diagnostic signature. Panel genes converge on GABA signaling, glucose transport, arginine metabolism, WNT pathway inhibition, and a novel lncRNA, providing both mechanistic hypotheses and high-priority targets for external validation. These findings offer a reproducible transcriptomic scaffold for future mechanistic, biomarker, and clinical translation studies of human islet dysfunction. They also support external transportability of the core biological signal, while indicating that absolute operating thresholds are cohort-dependent and would require recalibration before deployment in independent datasets.

ObjectiveThe aryl hydrocarbon receptor (AhR) pathway primarily mediates pollutant responses by activating xenobiotic metabolism enzymes like cytochrome P450 1A1 and 1A2 (CYP1A). Although AhR has also been implicated in systemic metabolic dysfunction and is inducible in pancreatic islets, its role in islet physiology remains unclear. MethodsWe analyzed a publicly available bulk human islet transcriptomic dataset to identify pathways associated with CYP1A1 expression. We also assessed islet responses to the pollutant 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and glucolipotoxicity (GLT) in vitro using two mouse models: a global Cyp1a1/1a2 double knockout (CypKO) model, which disrupts canonical AhR-CYP1A signaling in whole islets, and a {beta}-cell-specific Ahr knockout ({beta}AhrKO) model, which abolishes AhR signaling selectively in {beta}-cells. We then examined the role of {beta}-cell Ahr in early adaptation to high-fat diet (HFD) feeding in vivo. ResultsXenobiotic and nutrient metabolism pathways were enriched in donors with high CYP1A1 expression. Global Cyp1a1/1a2 deletion increased susceptibility of female mouse islets to TCDD-induced impairments in insulin secretion but had minimal effects on GLT responses in either sex. In contrast, {beta}-cell Ahr deletion did not affect islet responses to TCDD, but exacerbated GLT-induced islet dysfunction in male islets and increased baseline insulin secretion in both vehicle- and GLT-exposed female islets in vitro. Lastly, {beta}-cell Ahr deletion prevented adaptive HFD-induced hyperinsulinemia in both sexes in vivo. ConclusionIslet AhR signaling shapes responses to chemical and nutrient stressors in a context- and sex-dependent manner. While the canonical AhR-CYP1A axis supports female islet resilience to TCDD, {beta}-cell AhR signaling more broadly regulates nutrient stress responses in both sexes.

SLAM-associated protein (SAP) is required for T follicular helper (Tfh)-B cell interactions that underlie germinal center formation, but it is unclear if SAP governs islet-reactive CD4+ T cell-B cell interactions and downstream pro-inflammatory CD8+ T cell destruction of islets in type 1 diabetes (T1D). To address this question, we utilized the VH125SD.NOD mouse model, whereby 1-3% of all B cells bind insulin. Germline SAP loss in this model led to reduced T1D incidence and impaired germinal center B cell formation, yet did not alter T follicular helper cell formation or phenotype. SAP loss reduced pro-inflammatory and activated insulin-autoreactive B-T interactions and limited anti-insulin B cell proliferation, activation, and upregulation of co-stimulatory molecules otherwise enhanced in the pancreas. Anti-insulin extrafollicular antibody and memory responses following immunization were preserved in VH125SD.SAP-/-.NOD mice, but activated atypical anti-insulin B cell responses were reduced. Ultimately, SAP loss led to reduced pro-inflammatory CD8+ T cell formation and islet-reactive progenitor exhausted CD8+ T cells in pancreata. These data highlight the essential role of SAP in mediating proinflammatory, anti-insulin B-T interactions to support T1D. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=161 SRC="FIGDIR/small/741363v1_ufig1.gif" ALT="Figure 1"> View larger version (45K): org.highwire.dtl.DTLVardef@1f5eb40org.highwire.dtl.DTLVardef@27f133org.highwire.dtl.DTLVardef@4c7047org.highwire.dtl.DTLVardef@5f612a_HPS_FORMAT_FIGEXP M_FIG C_FIG

Importance: Higher coffee intake has been associated with lower risk of type 2 diabetes (T2D), but the underlying biological pathways remain incompletely understood. Objective: To examine associations of coffee intake with insulin sensitivity, adiposity, and T2D risk, and assess whether coffee intake modifies associations between pathway-specific genetic susceptibility and incident T2D. Design, Setting, and Participants: Cross-sectional analyses among 806 participants without T2D in the VITamin D and OmegA-3 TriaL (VITAL) clinical sub-cohort, who underwent repeated dietary assessment, clinical phenotyping, and dual-energy X-ray absorptiometry imaging at baseline and year-2. Prospective analyses among 333,053 UK Biobank participants without T2D at baseline who had dietary and genetic data and were followed for a median of 13.3 years. Exposures: Coffee intake assessed by food frequency questionnaires. In UK Biobank, 12 pathway-specific polygenic scores (pPS) representing distinct T2D pathophysiological mechanisms were evaluated. Main Outcomes and Measures: The primary outcomes, in VITAL, were HbA1c, oral glucose tolerance test-derived measures of glucose response and insulin sensitivity, beta-cell function, and overall, truncal, and visceral adiposity; in UK Biobank, was incident T2D. Results: In VITAL, higher coffee intake was associated with higher insulin sensitivity (standardized beta; per cup/day, 0.046; P = .004) and lower visceral adipose tissue mass (beta -0.047; P = .006), after adjusting for demographic, lifestyle, and clinical factors, including body mass index. In UK Biobank, higher coffee intake was associated with lower T2D incidence (hazard ratio per cup/day, 0.96; 95% CI, 0.95-0.97), lower triglyceride-to-HDL cholesterol ratio (beta: -0.01; P = 2.51 x 10-19), and lower visceral adipose tissue mass (beta: -0.01; P = 4.28 x 10-9). Associations of 3 pPS related to insulin resistance and fat distribution with incident T2D were attenuated among participants consuming higher amount of coffee than among non-consumers (P for interaction < .0043). Conclusions and Relevance: Higher coffee intake was associated with greater insulin sensitivity, lower visceral adiposity, and lower risk of T2D. Together with the attenuation of associations between pathway-specific genetic susceptibility and T2D risk among higher coffee consumers, these findings suggest that insulin resistance and visceral adiposity-related pathways may contribute to the association between coffee intake and T2D risk.

Type 1 diabetes (T1D) is characterized by immune-mediated destruction of pancreatic beta cells, yet the properties that distinguish disease-associated CD8 T cells from other pancreatic resident T cells remain incompletely defined. In this study, we analyzed CD8 T cell receptor (TCR) clonotypes isolated from the pancreas of organ donors with and without T1D and assessed their reactivity to beta cells using stem cell-derived beta-like cells. We found that highly beta cell-reactive CD8 T cells were selectively present in the pancreas of T1D donors but were largely absent from donors without T1D. In contrast, virus-specific CD8 T cells were detected in pancreata of donors with and without T1D and showed no evidence of cross-reactivity to beta-like cells, indicating that pancreatic residency alone does not confer beta cell specificity. Among beta cell-reactive CD8 T cells in T1D, reactivity to native peptides from major islet proteins other than preproinsulin was rare. Thus, despite beta cell specificity as a hallmark of T1D, T cells reactive to native islet proteins other than preproinsulin do not infiltrate the islets. These results identify beta cell reactivity as a key functional feature separating T1D-associated CD8 T cells from other pancreatic T cells. This functional definition of pathogenic T cells offers a framework for understanding selective beta cell loss and for developing approaches to monitor and therapeutically target disease-relevant CD8 T cells.

Human pancreatic islets exhibit greater anatomic and cellular heterogeneity than previously appreciated, raising fundamental questions about how their composition varies with age, sex, region, and islet size and how type 1 diabetes (T1D) alters these relationships. Yet these questions remained largely unresolved due to the bottleneck of manual tissue inspection. Here, we developed an integrated artificial intelligence (AI)-guided imaging, processing, and statistical pipeline enabling unbiased, high-throughput analysis of more than 2 million candidate islets from 106 non-diabetic (ND) and T1D donors. We identified age-, region-, sex-, and islet size-dependent differences in islet distribution and composition between ND and T1D donors. Profound {beta}-cell loss in T1D was accompanied by reciprocal -cell expansion, whereas {delta}-cells and pancreatic polypeptide cells were largely resilient. Cell area and pseudotime analyses uncovered regional and age-dependent trajectories of islet remodeling across T1D progression, along with distinct patterns of cytoarchitectural reorganization of the endocrine pancreas.

Background: Poor glycemic control is associated with cardiovascular disease (CVD) risk. However, it is unknown whether glycemic control is related to abdominal aortic calcification (AAC), a marker of subclinical CVD. We investigated the association between glycated hemoglobin (HbA1c) and moderate-to-high automated AAC among middle-aged to older adults from the general population. Methods:We included UK Biobank Imaging Study participants free of atherosclerotic CVD at baseline. HbA1c was measured at baseline (2006-2010) and categorized as normoglycemia (<39.0 mmol/mol), prediabetes (39.0-47.9 mmol/mol), undiagnosed diabetes (HbA1c [&ge;]48 mmol/mol), and diagnosed diabetes. Machine learning-derived AAC24 (ML-AAC24) scores were estimated using a validated automated algorithm applied to dual-energy X-ray absorptiometry lateral spine images (2014-2022). The associations of HbA1c with moderate-to-high ML-AAC24 (defined as a score [&ge;]2) were assessed using logistic regression adjusting for cardiovascular risk factors. Results: Of the included 48,912 participants (mean {+/-} SD age 55 {+/-} 7.6 years, 52% women), 9.7% had prediabetes (HbA1c 39.0-47.9 mmol/mol [5.7-6.4%]), 0.4% had undiagnosed diabetes, and 2.7% had diagnosed diabetes. Each 1-SD increase in log-transformed HbA1c was associated with higher odds of moderate-to-high ML-AAC24 (adjusted odds ratio [aOR] 1.12, 95% CI: 1.09-1.16). Amongst individuals with normal HbA1c, this association was consistent but somewhat weaker for each 1-SD increase in log-transformed HbA1c (aOR 1.07, 95% CI 1.03-1.10). Compared to participants with normal HbA1c, those with prediabetes (aOR 1.19, 95% CI: 1.08-1.30) or diagnosed diabetes (1.64, 95% CI: 1.39-1.94) had higher odds of moderate-to-high ML-AAC24. These associations were consistent in stratified analyses by sex, age groups, body mass index, smoking status and total cholesterol Conclusions: Linear associations between HbA1c levels and ML-AAC24 were observed in UK adults, even in those with normal HbA1c levels. These findings indicate that AAC may develop early in the dysglycemic continuum, supporting earlier cardiometabolic risk assessment even amongst people with ?normal? levels.

PURPOSE: To assess the predictive performance of panel protein biomarkers as well as an established algorithm that considers repeat biomarker testing for risk prediction of PDAC among a prospective cohort of patients with New-onset diabetes. PATIENTS AND METHODS: A panel of protein biomarkers (CA19-9, CA125, CEA, LRG1, REG3A and TIMP1) were assayed in 6,516 serially collected pre-diagnostic plasma samples from 2,121 NOD patients from the Consortium of Chronic Pancreatitis Diabetes and Pancreatic Cancer (CPDPC)-initiated NOD study who completed the 3-year study follow-up period. The specimen set included 25 pre-diagnostic samples from the 12 PDAC cases diagnosed during study follow-up. We applied a single threshold (ST) method, which considers biomarker levels at a single time point, as well as a previously established parametrical empirical Bayes (PEB) algorithm, which considers prior biomarker measurements, with case calls made based on pre-specified cutoffs corresponding to 1% 1-year risk. Resultant biomarker data as well as case calls were provided to the EDRN Data Management and Coordinating Center as part of a Prospective-sample-collection-Retrospective-Blinded-Evaluation (ProBE)-compliant Phase 3 biomarker validation study. Area under the Receiver Operating Characteristic Curves (AUC), sensitivity, specificity, population-level positive predictive value (PPV), and negative predictive value (NPV) are reported. RESULTS: The 3-year incidence of PDAC in the NOD cohort was 0.57%. When considering PDAC vs non-cancer controls, respective AUCs of individual protein biomarkers ranged from 0.52-0.94, with CA19-9 achieving the highest overall performance of 0.94 (95% CI: 0.86-1.00). At the pre-defined 1% 1-year risk threshold, CA19-9 yielded sensitivity of 83.3% at 97.2% specificity. Additional markers CEA, CA125, and TIMP1 demonstrated sensitivity of 33.3%, 41.7%, and 8.3%, respectively. In a subset of patients, CA19-9 first tested positive at a median (interquartile range [IQR]) of 7 months (4 to 14 months) prior to clinical PDAC diagnosis. Of the two PDAC cases missed by CA19-9 using the ST method, one (diagnosed with stage III PDAC) was detected using the PEBCA19-9 algorithm. CONCLUSION: In the setting of adult new onset diabetes, CA19-9 is a readily available and promising biomarker that can be leveraged for earlier detection of an underlying pancreatic cancer. Additional protein biomarkers may improve sensitivity for earlier detection of PDAC among cases with low CA19-9.

Type 1 diabetes (T1D) is an autoimmune disease characterized by CD8 T cell-mediated destruction of pancreatic {beta} cells; however, the cellular interactions that organize immune activation within human islets remain poorly understood. Here, we integrated thirteen CD45 immune cell single-cell RNA sequencing datasets from human islets spanning non-diabetic donors, stage 3 T1D, and type 2 diabetes (T2D) to comprehensively define immune cell heterogeneity and decipher the intercellular communication networks that drive islet autoimmunity. We identified distinct macrophage states, including CD14 inflammatory macrophages, CD14/TREM2 macrophages, and quiescent-like macrophages, together with CD8 T cells and mast cells. Trajectory and communication analyses revealed CD14 macrophages as central immune hubs that coordinate antigen presentation, costimulatory signaling, and inflammatory chemokine production. Compared with non-diabetic and type 2 diabetic islets, T1D macrophages displayed a disease-specific inflammatory program characterized by enhanced TNF, IL18, CCL3, CCL4, CCL5, and ICOSLG expression, supporting CD8 T cell recruitment and activation. Spatial transcriptomic analysis of human T1D pancreas further demonstrated a {beta}-cell-macrophage-CD8 T cell inflammatory niche, where macrophage-derived CCL3/CCL4/CCL5 and CD8 T cell-expressed CCR5 suggest a chemokine-mediated mechanism of immune targeting. Together, these findings identify CD14 macrophages as key orchestrators of a feed-forward inflammatory circuit driving human islet autoimmunity.

Dysregulated hormone secretion and erosion of endocrine cell identity are features of type 1 and type 2 diabetes, but the transcriptional programs maintaining adult human islet identity and function remain poorly defined. The large MAF transcription factor MAFB is expressed in human - and {beta}-cells, marks their most functionally mature subpopulations, and is downregulated in diabetes, but its role in adult human islets has not been tested directly. Using shRNA-mediated MAFB knockdown (KD) in whole and CD26+ -cell-enriched human pseudoislets, we found that whole pseudoislet MAFB KD impaired glucagon synthesis and secretion while only modestly reducing insulin content and cAMP-potentiated insulin release. Single-cell profiling detected no {beta}-cell transcriptional response beyond MAFB KD itself, consistent with buffering by the related {beta}-cell-enriched MAFA transcription factor. In contrast, -cell-restricted MAFB KD unmasked a cell-autonomous requirement for MAFB in stimulus-secretion coupling. MAFB deficiency also destabilized -cell identity, downregulating canonical -cell and neuroendocrine secretory genes while ectopically inducing mesenchymal and extracellular matrix remodeling programs. In addition, MAFB-dependent downregulation of electron transport chain genes was confined to a large -cell subcluster, manifesting as impaired islet-wide mitochondrial respiration within the broader -cell population. Together, these findings identify MAFB as an essential adult human -cell maintenance factor that links diabetes-associated downregulation to impaired glucagon secretion, -cell identity erosion, and mitochondrial dysfunction. RESEARCH IN CONTEXTO_LIWhat is already known about this subject? O_LIMAFB is expressed in adult human - and {beta}-cells, marks their most functionally mature subpopulations, and is downregulated in type 1 and type 2 diabetes C_LIO_LIIn human stem cell models, MAFB is essential for generating insulin-producing {beta}-like cells, whereas glucagon-producing -like cells are reduced but still formed C_LIO_LINeither model addresses adult human islets: rodent MafB becomes -cell restricted after birth, and stem cell models capture differentiation, not maintenance C_LI C_LIO_LIWhat is the key question? O_LIIs MAFB required to maintain identity and secretory function in adult human islet cells? C_LI C_LIO_LIWhat are the new findings? O_LIMAFB knockdown in primary human pseudoislets impaired glucagon synthesis and secretion but minimally affected {beta}-cells, consistent with buffering by MAFA C_LIO_LIKnockdown in CD26+ -cell-enriched pseudoislets revealed a cell-autonomous requirement for MAFB in stimulus-secretion coupling, and destabilized -cell identity by inducing mesenchymal and extracellular matrix programs C_LIO_LIMAFB loss downregulated electron transport chain genes in the largest -cell subcluster and reduced mitochondrial respiration C_LI C_LIO_LIHow might this impact on clinical practice in the foreseeable future? O_LIPreserving MAFB activity in adult human -cells may represent a strategy to limit -cell dysfunction in diabetes C_LI C_LI

Background: Insulin resistance is a core pathophysiologic feature of metabolic disease, but its reference-standard assessment by steady-state plasma glucose (SSPG) testing is procedurally demanding and labor-intensive, limiting use in routine clinical care and large-scale research. Because OGTT glucose profiles are widely available, we aimed to develop a glucose-only metric to characterize dynamic glucose responses and estimate SSPG-measured insulin resistance. Methods: We developed the Width-Delay Index (WDI), a glucose-only OGTT metric integrating relative exposure width, delayed exposure timing, and glycemic floor. In a dataset of 32 subjects with 16-point venous OGTT profiles and paired SSPG measurements, WDI performance was assessed using leave-one-out cross-validation (LOOCV) for SSPG prediction, together with insulin-resistance discrimination and sparse-sampling robustness analyses. Results: The 15-120 min OGTT window yielded the strongest WDI performance. WDI15-120 predicted SSPG with LOOCV R2 = 0.57 (95% CI, 0.27-0.77), Pearson r = 0.77, and Spearman rho = 0.74. WDI15-120 showed higher predictive performance than standard OGTT glucose measures and insulin-derived indices, including HOMA-IR, Matsuda index, and disposition index. WDI15-120 also discriminated insulin-resistant from insulin-sensitive subjects with AUROC = 0.969. When recalculated from conventional 5-point OGTT sampling, WDI15-120 retained substantial performance, with LOOCV R2 = 0.41 and AUROC = 0.945. Conclusions: WDI provides a simple, glucose-only, physiologically interpretable approach for estimating SSPG-measured insulin resistance from OGTT glucose dynamics.

Type I diabetes (T1D) is an autoimmune disorder in which the insulin producing cells of the pancreas are attacked and destroyed by autoreactive T cells. The innate immune mechanisms that contribute to T1D remain incompletely defined. Genome-wide association studies in humans have identified alleles of the IFIH1 gene, which encodes the intracellular RNA sensor MDA5, that are strongly associated with development of T1D. We previously found that MDA5 signaling drives disease and mortality in a mouse model of Aicardi-Goutieres Syndrome (AGS) caused by mutations in the ADAR1 RNA editing enzyme. Genetic dissection of disease in this ADAR1 mutant mouse model revealed that the double stranded RNA-activated kinase PKR and the RNA sensor ZBP1 are also essential for disease. To test the role of intracellular RNA detection in T1D in the nonobese diabetic (NOD) mouse model, we used CRISPR targeting to generate NOD mice targeted for Ifih1, Eif2ak2 (PKR) and Zbp1. We found that haploinsufficiency for Ifih1 resulted in modest but significant protection from T1D only in male NOD mice, but neither PKR nor ZBP1 contributed to T1D onset or incidence. Moreover, treatment of NOD mice with a pharmacological inhibitor of the integrated stress response (ISR) had no effect on T1D incidence in female NOD mice, but accelerated and exacerbated disease in male NOD mice. Together, our findings demonstrate that MDA5 and the ISR contribute to sex-specific disease incidence in NOD mice.

OBJECTIVE To identify circulating proteins associated with type 2 diabetes (T2D) risk through pathways not fully explained by body mass index (BMI), and to assess therapeutic actionability. RESEARCH DESIGN AND METHODS We applied GWAS-by-subtraction within a genomic structural equation model to European ancestry summary statistics for T2D (74,124 cases, 824,006 controls) and BMI (n = 681,275), partitioning T2D liability into BMI-related and BMI-subtracted components. We then performed proteome-wide Mendelian randomization (MR) using cis-protein quantitative trait loci from four plasma proteomics cohorts: ARIC, deCODE, Fenland, and the UK Biobank Pharma Proteomics Project. Prioritized proteins passed sensitivity analyses with alternative MR methods and were supported by colocalization evidence. Tissue-resolution regulatory support was assessed using cis-eQTL colocalization across GTEx and pancreatic islet, subcutaneous adipose, and whole-blood resources. Actionability was evaluated using the druggable genome and Open Targets. RESULTS GWAS-by-subtraction attenuated the genetic correlation between BMI and BMI-subtracted T2D from 0.54 (SE 0.02) to 0.35 (SE 0.02). Proteome-wide MR prioritized 29 proteins for BMI-subtracted T2D. Thirteen showed eQTL colocalization in at least one tissue, implicating liver and intermediary metabolism (GCDH, NOTCH2), pancreatic islet biology (CTRB2, MANBA), adipose and Wnt signaling (RSPO3, GALNT3), and whole blood regulatory signals (PAM, SNUPN). Sixteen proteins were classified within druggable-genome Tiers 1-3, and five had existing Open Targets compounds. CONCLUSIONS Integrating GWAS-by-subtraction, proteome-wide MR, and colocalization nominated 29 proteins associated with T2D liability not fully explained by BMI. These findings highlight genetically supported targets for follow-up studies of T2D therapies that complement weight-centered approaches.