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Optimizing Single-Cell Long-Read Sequencing for Enhanced Isoform Detection in Pancreatic Islets

Alternative splicing is an essential mechanism for generating protein diversity by producing distinct isoforms from a single gene. Dysregulation of splicing that affects pancreatic function and immune tolerance has been linked to both types 1 and 2 diabetes. Next-generation sequencing technologies, with their short read lengths, are limited in their ability to accurately detect splice variants. Long-read sequencing technologies offer the potential to overcome these limitations by providing full-length transcript information; however, their application in single-cell RNA sequencing has been hindered by technical challenges, including insufficient read lengths and higher error rates. Furthermore, cell types that produce high levels of a single transcript, such as islet endocrine cells, can obscure identification of lower-abundance transcripts. In this study, we optimized a protocol for single-cell long-read sequencing in pancreatic islets to improve read length and transcript detection. Our findings demonstrate that 5′ library preparation protocols outperform 3′ protocols, resulting in better transcript identification. Furthermore, we show that targeted depletion of insulin transcripts enhances the detection of informative reads, highlighting the utility of transcript-depletion strategies. This optimized protocol enables isoform-specific gene expression analysis and reveals differential transcript usage across the various cell types in pancreatic islets. By leveraging this approach, we gain deeper insights into the transcriptomic complexity and cellular heterogeneity within pancreatic islets.Article HighlightsThis study addresses the limitations of current single-cell long-read RNA sequencing technologies in detecting full-length transcripts and isoform diversity, particularly in pancreatic islets.We demonstrate that optimizing single-cell library preparation protocols reproducibly enhances read length and transcript identification in pancreatic islets.Combined with targeted insulin depletion and extended reverse transcription, 5′ capture methods significantly improved read length and isoform detection compared with standard protocols, while maximizing the number of informative reads.These improvements yield longer reads in single-cell experiments, substantially enhancing transcript identification and enabling more accurate analysis of isoform diversity.

Diabetic Corneal Neuropathy Precedes and Is Associated With Diabetic Retinopathy

Diabetic corneal neuropathy (DCN) and diabetic retinopathy (DR) are microvascular complications and share common pathophysiological mechanisms. However, the relationship between them remains poorly defined. In this cross-sectional study, we aimed to investigate the association among DCN, DR, and tear mediators in 1,654 eyes from 822 participants, comprising 634 patients with type 2 diabetes and 188 healthy participants. Our data demonstrated that compared with control participants, all patients with diabetes had significantly impaired corneal nerve metrics, increased dendritic cell length and density, and larger corneal microneuromas, even in the absence of DR. Patients with nonproliferative DR (NPDR) and proliferative DR (PDR) showed significantly reduced corneal nerve parameters compared with those with no DR. Furthermore, patients with PDR presented significantly worse ocular surface clinical manifestations than patients with no DR, patients with NPDR, and control participants. Cumulative link mixed models demonstrated that corneal sensitivity and corneal nerve parameters were significantly associated with the severity of DR. Tear substance P concentrations were significantly lower across all stages of DR compared with control participants. Tear MMP-9, substance P, and IGFBP-3 levels were significantly associated with corneal nerve and ocular surface parameters. This study demonstrates that DCN precedes the onset of DR and worsens with the severity of DR. Corneal nerve status could be an early indicator and predictor of DR.Article HighlightsBoth diabetic corneal neuropathy (DCN) and diabetic retinopathy (DR) are microvascular complications and share common pathophysiological mechanisms. The links between them are poorly understood.We investigated the relationship between DCN and DR by studying imaging features, clinical manifestations, and tear biomarkers.Our study demonstrated that DCN precedes the onset of DR, and DCN worsens with the severity of DR stage. Corneal sensitivity and corneal nerve parameters are significantly associated with DR severity. Tear MMP-9, substance P, and IGFBP-3 were significantly altered in DR.Corneal nerve status could be an early indicator and predictor of DR.

Endogenous Glucagon-Like Peptide 1 Enhanced by Vildagliptin Reduces Triglyceride Appearance During Intraduodenal Fat Infusion in Type 2 Diabetes

Glucagon-like peptide 1 (GLP-1) receptor agonists improve dyslipidemia and reduce cardiovascular risk in type 2 diabetes (T2D), but the role of endogenous GLP-1 in lipid metabolism remains unclear. We evaluated the effect of dipeptidyl peptidase 4 (DPP-4) inhibition on the response of plasma triglycerides (TGs) to intraduodenal lipid and a mixed meal, and the impact of GLP-1 receptor blockade with exendin(9-39) in T2D. Fifteen participants with T2D, managed by diet and/or metformin, were studied on three occasions in a double-blind, randomized, crossover design. Vildagliptin (50 mg) or placebo was administered orally (t = −60 min), followed by intravenous exendin(9-39) from t = −60 to 150 min on one of the two vildagliptin days or 0.9% saline on two other days. A lipid emulsion was infused intraduodenally (2 kcal/min, t = 0–120 min), followed by a mixed meal (t = 120–150 min). Plasma TG levels, quantified by liquid chromatography–tandem mass spectrometry, increased after lipid and meal, with most individual TGs corresponding to those in the lipid emulsion. Vildagliptin reduced TG(54:4) and TG(54:5) concentrations (each P < 0.01), without affecting total TGs. Blocking endogenous GLP-1 during vildagliptin treatment increased plasma total TGs (P < 0.001), associated with elevations of 10 individual TG species (P < 0.05 each). These outcomes suggest that endogenous GLP-1 contributes to the physiological modulation of postprandial TG appearance in T2D.Article HighlightsThe contribution of endogenous glucagon-like peptide 1 (GLP-1) to postprandial lipid metabolism in type 2 diabetes (T2D) is poorly defined.The specific questions we wanted to answer were what is the effect of dipeptidyl peptidase 4 (DPP-4) inhibition on plasma triglyceride (TG) profiles during an intraduodenal lipid infusion and following a mixed meal in T2D, and how does blockage of endogenous GLP-1 signaling affect these responses?We found that plasma TGs increased after intraduodenal lipid infusion and the meal, with most species reflecting the infused lipid emulsion. Vildagliptin selectively reduced TG(54:4) and TG(54:5) species, whereas exendin(9-39) increased total TGs and 10 individual TG species.The implications of our finding are that endogenous GLP-1 plays a physiological role in the regulation of postprandial lipid handling in T2D.

Distinct Enterovirus Antigen Landscape in Children With Islet Autoimmunity

Enteroviruses (EVs) have long been implicated in the development of islet autoimmunity (IA) and type 1 diabetes. However, given the ubiquity of EV infections in children, disease susceptibility is likely driven by host-specific immune responses rather than viral exposure alone. To investigate the host antibody response to EVs, we used virome-wide serological profiling (VirScan) to compare the EV antigen landscapes in IA-positive case children versus IA-negative control children across two independent pediatric cohorts separated by 12 years, using samples collected at the time point of seroconversion. We identified a reproducible and distinct EV-specific antibody signature in IA-positive case samples, with an enriched immunogenic hotspot localized within a highly conserved region in the 3D RNA-dependent RNA polymerase. Additionally, IA-positive male children exhibited significantly heightened antibody responses against a motif in the VP1 capsid protein compared with IA-negative male children (risk ratio 1.24; 95% CI 1.02, 1.52; P = 0.03). Our findings provide paradigm-shifting evidence that differential antiviral humoral responses, rather than the specific types of EV infection, play a central role in IA development, highlighting the need for an updated framework to study host-virus interactions in autoimmune pathogenesis.Article HighlightsChildren positive for islet autoimmunity (IA) in two different Australian cohorts showed a distinct enterovirus (EV) antibody signature against specific regions of the EV genome polyprotein.A specific motif in the 3D region of the EV polyprotein was consistently enriched across cohorts and sexes, making it a potential marker for IA onset.Anti-VP1 motif antibody levels varied by sex, with significantly elevated levels in male children linked to early IA onset, highlighting possible sex-specific antiviral immunity.Findings support that host immune responses against EVs drive IA development, calling for a new framework to study host-virus interactions in IA.

Exofacial Epitope–Specific Antibodies Detect GLUT4 Translocation in Adult Human, Rat, and Mouse Skeletal Muscle

Skeletal muscle glucose transporter 4 (GLUT4) translocation to the plasma membrane determines glucose uptake in response to insulin and exercise and is disrupted in insulin resistance, making its experimental measurement critical. Confocal light microscopy is widely used for this purpose because of its ability to provide quantitative, high-resolution spatial information from small tissue amounts. However, conventional immunofluorescence colocalization microscopy lacks sensitivity and specificity in the detection of GLUT4 translocation. We validated the use of exofacial epitope–specific GLUT4 antibodies to quantify sarcolemmal GLUT4 translocation in fixed, nonpermeabilized adult human and rodent muscle fibers. Across human, mouse, and rat muscles, these antibodies sensitively detected stimulus-induced GLUT4 translocation, and labeling was abolished in muscle-specific GLUT4-knockout muscle, confirming specificity. Importantly, this study includes the first unambiguous visualization of endogenous GLUT4 translocation in intact human skeletal muscle fibers after insulin stimulation and exercise. In TBC1D4-knockout rats, insulin-stimulated GLUT4 translocation was absent despite wild-type–level GLUT4 expression, confirming an essential role for TBC1D4 in this process. Thus, exofacial GLUT4 antibodies provide a straightforward, sensitive, and specific approach to quantify endogenous GLUT4 translocation in fixed adult skeletal muscle.Article HighlightsReliable quantification of glucose transporter 4 (GLUT4) translocation in intact skeletal muscle is essential for understanding insulin and exercise responses but remains technically challenging.We aimed to test whether exofacial GLUT4 antibodies can specifically detect sarcolemmal GLUT4 translocation in fixed, nonpermeabilized muscle fibers from humans and rodents.GLUT4 translocation in response to insulin, AMPK activation, and exercise was detectable in human and rodent muscles. Insulin-stimulated translocation correlated with 2-deoxyglucose uptake and was abolished in TBC1D4-knockout muscle.Exofacial GLUT4 antibodies enable straightforward, specific quantification of endogenous GLUT4 translocation in rodent and human muscles in healthy and insulin-resistant states.

Staying Functional Through Connection and Adaptation: When Islets Inspire Islet Biologists

In response to the lockdowns and travel bans during the coronavirus disease 2019 pandemic, Peter C. Butler at the University of California, Los Angeles (UCLA), started a virtual islet biology seminar series. After the authors of this article joined him as co-organizers, this initiative became the Islet Research Seminar Series (IRSS). Like islets of Langerhans adapt to their changing environment, the islet biology community quickly embraced this new format. The IRSS evolved into a lasting scientific forum that convenes weekly and is attended by islet biologists from the U.S., Canada, Europe, and Israel. The series covers a range of topics in islet biology, with presentations from scientists representing all career stages. It has proven particularly valuable for trainees and early-stage investigators in exposing them to a variety of topics in islet biology without travel required and facilitating more spontaneous interactions with senior scientists than at in-person meetings. While the online format is not meant to replace live scientific conferences, we believe that the IRSS plays a unique role in keeping the islet biology community connected and abreast of the most recent scientific discoveries in our field. The success of this platform stands as a testament to the scientific community to adapt and thrive through challenges. This article is dedicated to Peter C. Butler, UCLA, who initiated the IRSS.

ISL1 Restricts Progenitor Programs and Promotes β-Cell Maturation, Revealing Sex Differences in Diabetes Progression

Pancreatic islet cells differentiate from a common progenitor pool through tightly regulated transcriptional and epigenetic programs. ISL1, a LIM homeodomain transcription factor, is essential for islet development, but its molecular functions remain poorly defined. Here, we demonstrate that ISL1 is critical for maintaining endocrine cell identity and enabling terminal differentiation, particularly of α- and β-cells. Using conditional Isl1 deletion in endocrine precursors, combined with single-cell RNA sequencing and chromatin profiling (H3K27ac and H3K27me3), we reveal disruption of the transcriptional and epigenetic landscape in Isl1-deficient islets. Loss of Isl1 results in the failure to establish α-cell identity, loss of δ- and γ-cell lineages, and the persistence of immature β-cells with impaired functional profiles in Isl1CKO mice. Longitudinal single-cell analysis shows that Isl1CKO endocrine cells exhibit sustained progenitor-like states and defective β-cell maturation. These defects are accompanied by activation of stress and diabetes-associated transcriptional programs, along with sex-specific responses that may influence disease onset and progression. Mechanistically, ISL1 represses intermediate progenitor programs and facilitates chromatin remodeling necessary for endocrine lineage commitment and terminal maturation. Our findings highlight a previously underappreciated role for ISL1 in preserving endocrine cell fate and function and offer insight into how its dysregulation may contribute to diabetes.Article HighlightsISL1 is a known maturity-onset diabetes of the young candidate and type 2 diabetes susceptibility gene, yet its molecular role in pancreatic endocrine maturation has remained unresolved.Deletion of Isl1 in endocrine progenitors results in islets composed of dysfunctional α-cells lacking glucagon production and immature β-cells with impaired basal insulin secretion, ultimately accelerating diabetes progression.ISL1 functions as a transcriptional repressor guiding chromatin remodeling and transcriptional transitions toward hormone-producing endocrine cells.The metabolic phenotype resulting from Isl1 deletion is associated with sustained progenitor-like states and activation of diabetes- and stress-associated pathways, with distinct sex-specific responses observed between male and female mice.

GDF5 Exacerbates Tubulointerstitial Injury by Inducing Partial Epithelial–Mesenchymal Transition of Tubular Epithelial Cells in Diabetic Kidney Disease

Adipokines serve crucial functions in diabetic kidney disease (DKD) pathogenesis. Growth differentiation factor 5 (GDF5) is highly expressed in adipose tissue, but its specific role in DKD is unknown. In this study, we observed elevated GDF5 expression in both patients with DKD and db/db mice, suggesting a potential association between GDF5 and DKD progression. Elevated plasma GDF5 levels are associated with an increased risk of incident chronic kidney disease in patients with type 2 diabetes. In animal studies, adipose-specific overexpression of GDF5 increased circulating GDF5 and exacerbated renal injury in db/db mice, characterized by increased tubulointerstitial injury and inflammation infiltration. Conversely, adipose-specific knockdown reduced circulating GDF5 and alleviated renal injury. In vitro studies demonstrated that GDF5 induces partial epithelial–mesenchymal transition in renal tubular epithelial cells via activation of the SMAD1/5/8 signaling pathway, as evidenced by reduced E-cadherin expression and increased Snail1 levels. Notably, the supernatant from GDF5-treated injured HK-2 cells was found to enhance the secretion of proinflammatory cytokines by macrophages. These findings suggest that adipose-derived GDF5 acts as a novel mediator contributing to tubulointerstitial injury in DKD.Article HighlightsElevated growth differentiation factor 5 (GDF5) expression is correlated with disease progression in both patients with diabetic kidney disease and db/db mice.Adipose-specific GDF5 overexpression exacerbates, whereas its knockdown alleviates, renal tubulointerstitial injury in vivo.GDF5 directly induces partial epithelial–mesenchymal transition in tubular cells by activating the SMAD1/5/8 signaling pathway.Tubular cells exposed to GDF5 release factors that promote proinflammatory cytokine secretion in macrophages.

Activation of the Pancreatic “Metabolic Synapse” Aggravates Type 2 Diabetes Mellitus by Inducing PANoptosis in β-Cells

Pancreatic β-cells play a central role in type 2 diabetes mellitus (T2DM), yet the interactions between β-cells and stromal components within the islet microenvironment remain poorly defined. We investigated the contribution of pancreatic fibroblasts to β-cell dysfunction and T2DM progression. We used single-cell sequencing technology and in vitro experiments to investigate the mechanisms by which bariatric surgery ameliorates T2DM. We introduce the novel concept of a “metabolic synapse” to describe the interaction between pancreatic fibroblasts and β-cells. Our findings reveal that pancreatic fibroblasts secrete excessive glutamate in the early stages of T2DM. Elevated glutamate concentrations within the islet microenvironment subsequently activate N-methyl-d-aspartic acid receptors (NMDARs), triggering PANoptosis in pancreatic β-cells and accelerating T2DM progression. Consistent with this, significant changes in NMDAR expression were observed in human pancreatic samples from patients with T2DM. These findings uncover a previously unrecognized fibroblast–β-cell communication pathway in the islet niche, provide mechanistic insights into T2DM pathogenesis, and highlight the glutamate–NMDAR axis as a potential therapeutic target for nonsurgical intervention.Article HighlightsWe identify a fibroblast–β-cell “metabolic synapse” in type 2 diabetes that couples stromal glutamate overflow to–β-cell N-methyl-d-aspartic acid receptor (NMDAR) activation.Single-cell maps and coculture assays show diabetogenic stress drives fibroblasts to hypersecrete glutamate, whereas β-cells upregulate NMDARs, triggering PANoptosis.In rodents, pharmacologic NMDAR blockade attenuates β-cell PANoptosis, preserves islet function, and improves glycemic control.Human pancreatic samples cohorts reveal fibrosis regression and stage-wise NMDAR upregulation, highlighting the glutamate–NMDAR axis as a therapeutic target.

Adipose Tissue Resistance to the Antilipolytic Effect of Insulin and Niacin in Humans With Obesity

Adipose tissue (AT) lipolysis insulin resistance results in excess free fatty acid (FFA) release. We tested the hypothesis that the ability of insulin to suppress AT lipolysis is unrelated to the ability of niacin to suppress lipolysis, because niacin acts through a different proximal signaling pathway. Ten volunteers (5 women and 5 men) with upper-body obesity and/or type 2 diabetes mellitus (T2DM) underwent two study visits with overnight intravenous infusions of niacin (1.4 mg/min) or saline, followed by a hyperinsulinemic-euglycemic clamp. FFA-palmitate Ra was measured using [U-13C] and [2H9]palmitate infusions; abdominal AT biopsies were performed before and during the insulin clamp. The suppression of FFA-palmitate Ra by insulin on the saline control day and by niacin after an overnight infusion were highly correlated (r = −0.93, P < 0.001). Fasting AT Akt (pAktS473/474-to-panAkt ratio, P = 0.01) and perilipin 1 (PLN1) (pPLN1S552-to-panPLN1 ratio, P = 0.02) phosphorylation were less during niacin treatment than in the saline control study. Because the suppression of lipolysis by insulin and niacin are highly correlated within individuals and because niacin and insulin act through different proximal signaling pathways, we propose dysregulated AT lipolysis in obesity/T2DM is due to dysfunction(s) in distal lipolysis proteins rather than isolated “insulin resistance.”Article HighlightsWe undertook this study to compare adipose tissue lipolysis responses to insulin and niacin in humans.We tested the hypothesis that adipose tissue insulin resistance would be unrelated to adipose tissue niacin resistance.The suppression of lipolysis by insulin and niacin were highly correlated.Dysregulated adipose tissue lipolysis in obesity/type 2 diabetes is due to dysfunction(s) in distal lipolysis proteins rather than isolated “insulin resistance.”

Epigenetic Regulation of VCAM-1 by Lipoxin A4 Is Renoprotective Against Diabetic Kidney Disease

Chronic low-grade inflammation underlies many microvascular complications of diabetes, including diabetic kidney disease (DKD). Lipoxins (LXs), an endogenously produced family of lipid mediators, resolve inflammation and protect against renal scarring as occurs in DKD. This study examined the mechanism by which LXs protect against DKD, focusing on the regulation of VCAM-1 and the recruitment of macrophages to the diabetic glomerulus. LXA4 and two fourth-generation mimetics were assessed in diabetic ApoE knockout mice, followed by in vitro studies in the main renal cell populations, including podocytes, proximal tubular, mesangial, and glomerular endothelial cells. LXs attenuated albuminuria, mesangial expansion, and collagen and fibronectin deposition as both a preventive and delayed intervention in experimental DKD. LXs also consistently attenuated the TNF-α–induced expression of VCAM-1 in all the human and mouse renal cell populations examined. Further analysis identified that the renoprotection was in part mediated by an epigenetic modification of the VCAM-1 gene through H3K4 monomethylation, which did not appear to be dependent on NF-κB activation in human glomerular endothelial cells. LXs protect against DKD by modulating glomerular endothelial cell inflammation and via a novel LX-mediated epigenetic mechanism regulating the VCAM-1 promoter in these cells.Article HighlightsLipoxins (LXs) protect against diabetic kidney disease (DKD) by resolving chronic low-grade inflammation, but the exact mechanism by which this occurs is not known.We investigated the effect of LXs on inflammatory markers and the recruitment of macrophages to the diabetic glomerulus by using LXs as both a preventive and delayed interventional treatment in streptozotocin-induced diabetic ApoE knockout mice.Protection against DKD was associated with reduced glomerular macrophage accumulation. LXs also attenuated the expression of VCAM1 in glomerular endothelial cells.LXs protect against DKD in part by a mechanism that reduces VCAM1 gene expression via H3K4 monomethylation on the VCAM1 gene.

Metabolic Signature of Gestational Diabetes Mellitus and Risk of Adverse Birth Outcomes: A Prospective Birth Cohort Study

Gestational diabetes mellitus (GDM) is a heterogeneous condition diagnosed solely through glucose. It is characterized by profound perturbations in the metabolome, with specific metabolic profiles linked to adverse birth outcomes. Metabolomics can reveal population heterogeneity in health and disease. Here, we used nontargeted metabolomics to systematically profile the circulating metabolome in 2,050 pregnant women during midpregnancy, identifying 30 metabolites that define the GDM metabolic signature (mGDM). Participants were stratified into four groups by distinct glycemic and metabolic profiles, namely normoglycemic non-mGDM, hyperglycemic non-mGDM, normoglycemic mGDM, and hyperglycemic mGDM, and associations with subsequent adverse birth outcomes were assessed. Compared with normoglycemic non-mGDM, normoglycemic mGDM demonstrated nearly a twofold increased risk of preterm birth (odds ratio [OR] 1.93, 95% CI 1.02–3.65) and large for gestational age (OR 2.11, 95% CI 1.53–2.92). Conversely, the hyperglycemic non-mGDM group did not show elevated risks in adverse birth outcomes versus the normoglycemic group. The hyperglycemic mGDM profile was associated with higher risks of preterm birth (OR 2.37, 95% CI 1.04–5.39), large for gestational age (OR 2.28, 95% CI 1.50–3.47), congenital malformations (OR 1.87, 95% CI 1.03–3.39), and neonatal intensive care unit (NICU) admissions (OR 1.69, 95% CI 1.09–2.61). We observed a stepwise increase in adverse outcome risk across the four-level metabolic-glycemic ission). Taken together, our study outlines the metabolic profile of GDM and reveals clinically relevant heterogeneity in adverse pregnancy outcomes by metabolic signature. Integrating blood glucose and metabolomics may improve risk stratification and advance precision maternal care.Article HighlightsWomen diagnosed with gestational diabetes mellitus (GDM) exhibit variability in symptom presentation and pregnancy outcomes. The heterogeneity in metabolic features beyond a GDM diagnosis and their influences on health outcomes is less studied.We aimed to identify the metabolic signatures of GDM, stratify pregnant women by glycemic and metabolic profiles, and further investigate the intergroup heterogeneity and their respective associations with adverse birth outcomes.Maternal metabolic dysregulation, rather than blood glucose alone, exerts more profound associations with adverse outcomes in pregnant women and their offspring.Targeted risk stratification using metabolomics offers a novel opportunity for precision medicine in GDM, potentially improving health management of pregnant women.

Memory Regulatory T Cells as a Biomarker of Early Type 1 Diabetes

Type 1 diabetes (T1D) is the most common chronic autoimmune disease in children, driven by a breakdown in self-tolerance and T cell–mediated immune attack of pancreatic β-cells. There are no biomarkers to effectively diagnose autoimmunity before disease onset and clinical symptom development. Here, we applied deep multiparametric immunophenotyping to compare immune landscapes in 38 patients with new-onset T1D, 24 siblings, and 18 healthy control participants (HCs). Patients with T1D underwent clinical and metabolic evaluations. Immune populations in fresh whole-blood samples were analyzed using a panel of 26 antibodies, detecting 39 different cell populations. Memory regulatory T cells (memory Tregs) were significantly increased in patients with T1D (P < 0.05) and their siblings (P < 0.01) compared with HCs but not between patients with T1D and siblings. Memory Tregs were associated with disease status and age in multivariable analysis. There was a positive correlation between age and memory Tregs in the HC and sibling groups but not in patients with T1D. Baseline memory Treg levels in siblings resembled those of patients with T1D. These findings highlight the existence of an age-independent, disease-specific immune fingerprint that could serve as a minimally invasive biomarker for early diagnosis and personalized immunotherapy. Further studies using functional and single-cells analysis are needed to confirm memory Tregs as a pathogenic trait.Article HighlightsThere are more memory regulatory T cells (Tregs) in individuals with type 1 diabetes (T1D) and siblings than in healthy control (HC) individuals.Individuals with T1D and their siblings share an immunological profile, with siblings displaying an intermediate phenotype that overlaps with both T1D and HC individuals.Memory Tregs increased with age in HC individuals and siblings but not in individuals with T1D.Diabetic ketoacidosis status had no impact on immune cell populations in patients with T1D.

Multiple Mechanisms Contribute to Robust Type 1 Diabetes Protection in Nfkbid - Overexpressing NOD Mice

We previously reported that transgenic overexpression of Nfkbid in NOD mice led to robust type 1 diabetes protection associated with enhanced negative selection of autoreactive T cells and expansion of regulatory T cells (Tregs) with increased suppressive capacity. The goal of this study was to further identify cellular and molecular mechanisms underlying strong protection from type 1 diabetes imbued by overexpressing Nfkbid. Transcript analysis of Tregs from Nfkbid-overexpressing NOD mice show enrichment of cellular replication pathways. Effector T cells from Nfkbid-overexpressing NOD mice have increased indicators of activation-induced exhaustion in pancreatic lymph nodes and islets. This trait is intrinsic to Nfkbid-overexpressing T cells, as ex vivo anti–CD3 stimulation of splenic-derived T cells can reproduce this phenotype. Anti–PD-1 administration breaks the diabetes protective effect. Furthermore, we found that Nfkbid-overexpressing dendritic cells, but not B cells, have an enhanced capacity to stimulate proliferation of diabetogenic AI4 CD8+ T cells (with wild-type levels of Nfkbid). Conversely, B cells, but not dendritic cells, drive enhanced de novo conversion of regulatory T cells from effector T cells. Together, this study documents additional mechanisms that likely synergize to contribute to the diabetes-protective effects of Nfkbid overexpression. Understanding the mechanisms underlying protection from Nfkbid overexpression may lead to novel therapeutic avenues.Article HighlightsT cells from Nfkbid-overexpressing NOD mice have increased evidence of exhaustion, and this feature is intrinsic to T cells following activation.Dendritic cells, but not B cells, overexpressing Nfkbid can drive enhanced CD8+ T-cell activation.Nfkbid-overexpressing B cells, but not dendritic cells, drive enhanced T effector–to–regulator T cell conversion.

Predicting the Timing of the Metabolic Inflection Point in Type 1 Diabetes Progression Using Machine Learning and Survival Analysis Models

An inflection point (IP) marking accelerated β-cell decline occurs ∼1–2 years before type 1 diabetes diagnosis. Precisely determining this timing could optimize clinical intervention. We developed machine learning models to predict proximity to the inferred metabolic IP using oral glucose tolerance test (OGTT) data from islet autoantibody-positive individuals in the TrialNet Pathway to Prevention study. OGTTs were retrospectively labeled by estimating time to the IP using thresholds from 1.2 to 1.6 years. Models were trained on engineered glucose and C-peptide dynamics, slopes, and composite indices, with feature selection by recursive feature elimination (RFE). Classifiers included support vector machines (SVM), random forests, and gradient boosting; a Cox proportional hazards model was also applied to estimate visit-level time to diagnosis and derive time-to-IP predictions. External validation was performed in the independent Diabetes Prevention Trial–Type 1 cohort. The best-performing model—SVM with RFE at the 1.4-year threshold—achieved an area under the curve of 0.77 (95% CI 0.72–0.82). The Cox model provided complementary numeric estimates of time to diagnosis and time to IP, sensitive to the cohort-level lead time offset (Δ). These findings show that machine learning and survival analysis can support early metabolic shift detection, enabling timely risk stratification and personalized monitoring in at-risk individuals.Article HighlightsWe undertook this study to improve early identification of the metabolic inflection point (IP) preceding clinical type 1 diabetes in autoantibody-positive individuals.We aimed to develop and validate machine learning models using oral glucose tolerance test–derived dynamic features to detect proximity to the IP.A support vector machine trained on TrialNet Pathway to Prevention and tested on Diabetes Prevention Trial–Type 1 achieved an area under the curve of 0.77 at 1.4 years prior to diagnosis, with strong calibration and interpretability. Additionally, a Cox proportional hazards model provided numeric estimates of time to IP, offering complementary predictions.These results can support earlier intervention and timely monitoring through personalized oral glucose tolerance test–based risk stratification.