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.

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.

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."

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.

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.

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.

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.

There is significant interest in antigen-specific approaches to delaying type 1 diabetes in preclinical stages and supporting tolerance after diagnosis. We conducted a phase I trial of a nonintegrating DNA plasmid constructed to secrete the type 1 diabetes antigen preproinsulin (PPI) and the immune modulatory cytokines transforming growth factor-β1 (TGF-β1), interleukin-10 (IL-10), and IL-2. In this placebo-controlled, double-masked study of 47 adults with stage 3 type 1 diabetes, we showed that the drug is safe and well tolerated, with most reported adverse events (AEs) categorized as grade 1 and with no clinically significant difference in AEs among treatment groups. There were no untoward metabolic or immune effects. We found pharmacodynamic evidence of treatment, as demonstrated by a dose-dependent type 1 interferon (IFN) signature. Plasmid DNA, representing a pharmocokinetic measure, was detected in the two highest dosing groups. We did not find global or antigen-specific immune cell changes following treatment with a DNA plasmid expressing PPI, IL-2, IL-10, and TGF-β1, and we did not detect immune changes driven by IL-2, IL-10, or TGF-β1. Our results support further trials of this novel tolerizing antigen construct.

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.

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.

Recent advances in RNA sequencing (RNA-seq) techniques allow the identification of tissue-specific alternative splicing and can thereby provide new insights into molecular mechanisms of energy metabolism. Full-length transcriptomics based on single-molecule real-time sequencing (SMRT-seq) enable precise detection of isoforms with 99% accuracy in an unbiased manner. In this proof-of-concept study, we integrated SMRT-seq, bulk RNA-seq, and comprehensive metabolic phenotyping to investigate reduced mitochondrial function in the skeletal muscle of individuals with type 2 diabetes. Muscle biopsies were taken from nine individuals with type 2 diabetes and nine age- and BMI-matched glucose-tolerant men. Whole-body insulin sensitivity (WBIS) was assessed by hyperinsulinemic-euglycemic clamps, and muscle mitochondrial respiration was assessed by high-resolution respirometry. In muscle samples, SMRT-seq was used to create full-length reads and isoforms, which were mapped to the genome. Short-read sequencing was used to compare isoform expression between the groups. Participants with diabetes exhibited lower WBIS and fatty acid-driven and complex I-linked respiration compared with control participants. SMRT-seq revealed ∼67,000 isoforms originating from ∼14,000 unique genes. Although isoform numbers per gene did not differ, SMRT-seq-based mapping enabled refined data set clustering compared with conventional short-read sequencing and identified four splicing variants of the ATP5F1A gene encoding a subunit for ATP synthase. Among these, two novel transcripts were expressed exclusively in control participants. This study identified splicing variants of ATP synthase that were differentially expressed between participants with type 2 diabetes and those with normal glucose tolerance, which may contribute to the reduced fatty acid oxidation in diabetes.

Pancreatic islet α-cells are increasingly recognized as amino acid sensors for the organism. Building on our prior work in β-cells, we sought to determine whether the mitochondrial phosphoenolpyruvate (PEP) cycle is involved in α-cell amino acid sensing. Three different methods were used to probe the PEP cycle, including pyruvate kinase activators (TEPP-46), and mice with α-cell-specific deletion (KO) of pyruvate kinase M (PKM1/2-αKO) or mitochondrial PEP carboxykinase (PCK2-αKO). The mitochondrial fuel leucine, in the presence of glutamine, antagonized alanine/arginine-stimulated Ca2+ influx and glucagon secretion under hypoglycemic conditions. Both PKM1/2 and PCK2 deletion prevented leucine from closing α-cell KATP channels. The Ca2+ response to amino acids was suppressed by pyruvate kinase activation with TEPP-46 and enhanced by α-cell deletion of PKM1/2 or PCK2-all without changing glucagon secretion. Using diazoxide/KCl to probe the pathways downstream of membrane depolarization, we identified a further role of the PEP cycle in homeostatically regulating Ca2+ levels. In sum, α-cell pyruvate kinase and the mitochondrial PEP cycle senses leucine and inhibits KATP channels similarly to β-cells, while restricting amino acid-stimulated membrane depolarization and Ca2+ influx. However, none of the amino acids tested, including alanine/arginine, regulate glucagon secretion by modulating membrane depolarization or Ca2+ influx.

Insulin autoantibodies (IAAs) are commonly measured by radiobinding assays (RBAs), which detect both high- and low-affinity binding. Improved assays that preferentially detect high-affinity IAA are likely to provide greater specificity and thereby increase the diagnostic accuracy of IAA for early-stage type 1 diabetes (T1D). This study aimed to develop and validate a novel bridging ELISA for IAA detection. The bridging ELISA detects IAAs by their bivalent cross-linking of two proinsulin moieties in fluid phase. Validation was performed by using samples from 227 patients with newly diagnosed stage 3 T1D and 1,021 control participants. Additionally, 202 children positive for IAA by RBA from general population screening were tested by the bridging ELISA and electrochemiluminescence (ECL) assay. At 99.5% specificity, ELISA detected IAA in 65.2% of patients with stage 3 T1D vs. 60.8% by RBA. Among children identified as having RBA-positive IAA in general population screening, 80.3% of those with multiple islet autoantibodies and 48.1% of those with a single IAA had ELISA-IAA positive findings (P < 0.0001). For children with a single IAA by RBA, ELISA detected 78.9% of those with ECL-IAA-positive findings vs. 27.9% of those with ECL-IAA negative findings (P < 0.0001). Samples that were IAA negative by ELISA showed lower antibody affinity. The ELISA-IAA assay demonstrates high sensitivity and specificity and could become a practical tool for T1D population screening and clinical diagnosis across laboratories.

The microtubule network in β-cells attenuates insulin secretion by pulling insulin secretory granules away from the plasma membrane. Thus, high-glucose-induced microtubule remodeling is required for robust glucose-stimulated insulin secretion. We now demonstrate that hormones secreted by α-cells regulate microtubule dynamics in β-cells through receptors for glucagon (GcgR) and glucagon-like peptide 1 (GLP-1R). Activation of GcgR or GLP-1R destabilizes microtubules in β-cells, accompanied by increased insulin secretion. In contrast, inhibiting these receptors attenuates high-glucose-induced microtubule destabilization and decreases secretion. Supporting the physiological significance of this regulation, β-cells in islets with a higher α-cell-to-β-cell ratio exhibit more dynamic microtubules than those with a lower ratio, and a high-fat diet challenge in mice, which can compromise β-cell secretion, attenuates this effect in their islets. Within individual islets, β-cells located near α-cells show faster microtubule remodeling upon glucose stimulation than those more distant from α-cells. Consequently, islets with a higher α-cell-to-β-cell ratio secrete more insulin in response to glucose stimulation and plasma membrane depolarization, results recapitulated by exogenous glucagon stimulation or chemically induced microtubule destabilization in islets with lower α-cell-to-β-cell ratios. These combined results suggest that α-cells use glucagon-mediated and/or GLP-1-mediated paracrine signaling to fine-tune β-cell secretion via microtubule remodeling.

Angiotensin II (AngII) activation, a key driver of diabetes pathogenesis and associated complications, induces kidney injury by promoting oxidative stress and inflammation. Ferroptosis is an iron-dependent regulated cell death, playing a crucial role in kidney injury. This study aimed to explore the contribution of ferroptosis to AngII-induced kidney injury and its regulatory mechanisms. Our findings reveal that chronic AngII stimulation leads to renal dysfunction, characterized by elevated serum creatinine levels, increased urinary protein-to-creatinine ratio, and tubular injury. These changes are associated with ferroptosis in renal tubular epithelial cells (TECs) and a marked upregulation of dipeptidase 1 (DPEP1) expression. Notably, the ferroptosis inhibitor ferrostatin-1 (Fer-1) effectively reversed ferroptosis in TECs, restored tubular integrity, and improved renal function. DPEP1 gene silencing and the DPEP1 inhibitor cilastatin significantly inhibited AngII-induced ferroptosis in TECs. Mechanistically, AngII upregulated DPEP1 expression via the transcription factor SP1. Elevated DPEP1 enhanced ubiquitination of SLC3A2, a key cystine/glutathione transporter. Furthermore, inhibiting DPEP1 with cilastatin in a mouse model effectively reversed ferroptosis and alleviated kidney injury. These findings highlight ferroptosis' key role in AngII-induced kidney injury and suggest DPEP1 targeting as a therapeutic strategy against AngII-driven renal damage.

Although control of metabolism by leptin is primarily viewed as centrally mediated, leptin has also been shown to directly regulate adipocyte function. However, the impact of the peripheral effects of leptin on systemic metabolism, especially in the context of obesity, remains unclear. To address this question, we selectively restored adipocyte leptin receptor (LEPR) expression in obese male and female LEPR-conditional knockout mice. Adipocyte LEPR restoration did not affect body weight but selectively increased brown adipose tissue (BAT) mass in male mice. This was associated with increased energy expenditure, smaller BAT adipocytes, lower triglycerides content, and increased markers of browning and lipolysis exclusively in males. Additionally, adipocyte LEPR restoration enhanced the expression of markers of endothelial cells and angiogenesis in male mouse BAT, supporting increased local vascularization. Improved BAT function in males was also associated with lower HbA1c, better insulin sensitivity, reduced systolic blood pressure, decreased arterial stiffness, and improved endothelial function. Lastly, adipocyte LEPR restoration lowered circulating proinflammatory cytokines and reduced tissue inflammation in the aorta and heart, again in males only. These findings reveal a critical role for adipocyte leptin signaling in regulating BAT function and emphasize its importance in maintaining glycemic and cardiovascular health in males with obesity.

The parasympathetic nervous system modulates hormone and digestive enzyme secretion from the pancreas. However, the mechanisms of neuroeffector transmission within the final parasympathetic pathway in the pancreas have not been elucidated. Here, we demonstrate that intrapancreatic cholinergic neurons are bona fide postganglionic neurons that functionally couple vagal input to target cells in the pancreas. In living pancreatic slices from various mice expressing genetically encoded sensors and actuators, we found that intrapancreatic neurons responded to cholinergic input via nicotinic and muscarinic M1 acetylcholine receptors. However, only muscarinic receptor signaling was necessary and sufficient to elicit responses in exocrine and endocrine target cells. We established that muscarinic receptor signaling in intrapancreatic neurons is linked to the potassium M-current, thus producing the sustained reverberating activity required to efficiently modulate insulin and glucagon secretion and elicit oscillatory responses in acinar cells. Whereas intrapancreatic neurons triggered responses in acinar cells without additional stimulation, they only primed and amplified hormone secretion already stimulated by changes in glucose levels. This mechanistic insight into how intrapancreatic neurons regulate pancreas function challenges canonical models of parasympathetic neurotransmission and is critical to understanding autonomic control of the pancreas.