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.

Chronic overnutrition promotes excessive hepatic triglyceride accumulation, subsequently leading to insulin resistance and systemic metabolic dysfunction. Inorganic pyrophosphatase 1 (PPA1), an enzyme that hydrolyzes inorganic pyrophosphate, plays a key role in driving synthetic biochemical reactions. Here, we identified PPA1 as a novel regulator of systemic energy expenditure that functions by controlling hepatic production of fibroblast growth factor 21 (FGF21). FGF21 is a hormone predominantly secreted by the liver that protects against obesity by enhancing whole-body energy expenditure. Although nutritional states and various transcription factors are known to regulate hepatic FGF21 expression, the underlying mechanisms remain elusive. In this study, we demonstrate that hepatic-specific deletion of PPA1 effectively attenuates high-fat diet-induced obesity, reduces hepatic lipid deposition, and improves systemic insulin sensitivity in vivo. PPA1 ablation in the liver significantly elevates circulating FGF21 levels and increases whole-body energy expenditure by promoting adipose tissue browning and thermogenesis. Knockdown of hepatic FGF21 expression partially counteracts the protective effect conferred by PPA1 deficiency. Mechanistically, hepatic PPA1 deficiency elevates FGF21 through the GCN2/eIF2α/ATF4 pathway, a process that is dependent on the loss of its enzymatic activity. Our findings not only establish PPA1 as a critical regulator of systemic energy metabolism but also identify it as a novel modulator of FGF21, highlighting its potential as a therapeutic target for obesity and related metabolic disorders.

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.

GLP-1 receptor agonists have emerged as key pharmacological tools in the treatment of type 2 diabetes and obesity. While their anorexigenic effects are well characterized, the mechanisms by which GLP-1 modulates pancreatic β-cell function remain only partially understood. In this article, we argue that GLP-1 receptor agonists should be viewed as integrative regulators of β-cell function and explore the multifaceted actions of GLP-1 analogs on β-cell signaling. GLP-1 influences key secretagogues and intracellular mediators (calcium, glutamate, γ-aminobutyric acid, serotonin, and urocortin-3), with complex roles in insulin exocytosis. Additionally, we discuss the interplay between calcium and cAMP, and how GLP-1 modulates both pathways to coordinate insulin secretion. Emerging evidence suggests that GLP-1 analogs affect mitochondrial morphology and redox homeostasis. Considering the relatively low expression of classical antioxidant enzymes in β-cells, and their reliance on both glycolytic and mitochondrial metabolism to sustain insulin secretion, the influence of GLP-1 on mitochondrial dynamics and reactive oxygen species may play a central role in sustaining cell function and viability. Despite recent advances, critical gaps persist in the literature, particularly regarding organelle cross talk, intracellular calcium stores, and the modulation of vesicular content. Drawing on current evidence, we propose that three mechanistic dimensions (intracellular neurotransmitters, mitochondrial remodeling, and redox control) represent key areas where clarifying GLP-1 actions could most effectively advance the field. Further investigation into these mechanisms is essential for a comprehensive understanding of GLP-1 actions in β-cells. This knowledge may help refine current incretin-based therapies and identify novel molecular targets for the treatment of metabolic disorders.

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.

Type 2 diabetes increases cardiovascular risk, with endothelial dysfunction playing a key role. Prolonged disease duration exacerbates cardiovascular risk, but the underlying mechanisms remain unclear. We previously demonstrated that red blood cells (RBCs) from individuals with type 2 diabetes impair endothelial function via reduced miRNA (miR)-210-3p. We investigated whether disease duration influences RBC-induced endothelial dysfunction and its link to miR-210-3p. RBCs were isolated from diabetic db/db mice of various ages and from humans with newly diagnosed (<1 year) or long-lasting type 2 diabetes (>7 years). Endothelial-dependent relaxation (EDR), miR-210-3p levels, its target protein glycerol-3-phosphate dehydrogenase 2 (GPD2), and oxidative stress marker 4-hydroxynonenal (4-HNE) were assessed. RBCs from 14- and 22-week-old, but not 7-week-old, db/db mice impaired EDR. These RBCs showed similarly reduced miR-210-3p levels and increased vascular GPD2 and 4-HNE expression. RBCs from individuals with long-lasting type 2 diabetes, but not from the newly diagnosed group, impaired EDR. After ≥7 years, RBCs from initially newly diagnosed individuals impaired EDR, which was rescued by miR-210-3p mimic transfection. In contrast, RBCs from healthy subjects did not impair EDR after follow-up. These findings underscore the pivotal role of disease duration for RBC-mediated vascular dysfunction, linked to miR-210-3p downregulation. RBC miR-210-3p may serve as a biomarker for diabetes-related vascular disease.

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.

Younger-onset type 2 diabetes (T2D) (onset <40 years) represents a growing global health challenge, characterized by heterogenous pathophysiology and accelerated complications. Current one-size-fits-all treatment approaches may be inadequate for this population. To address this heterogeneity, we performed clinical variable-based clustering using BMI, onset age, HbA1c, and HOMA2 indices in 717 participants across discovery and validation cohorts. Three distinct subgroups were identified: mild obesity-related diabetes (MOD), severe insulin-deficient diabetes (SIDD), and severe insulin-resistant diabetes with insulin insufficiency (SIRD-II). Over median follow-up of 2.8 years, SIRD-II demonstrated 11-fold increased risk of progressive chronic kidney disease, while both SIDD and SIRD-II showed threefold increased risk for progressive albuminuria compared with MOD. SIRD-II also demonstrated 3.5-fold and 2.3-fold higher 10-year cardiovascular risk compared with SIDD and MOD respectively. Metabolomic analysis revealed distinct signatures: SIDD exhibited lower levels of lipids, amino acids, and inflammatory markers, while SIRD-II demonstrated elevated glucose, lipids, and branched-chain amino acids, suggesting glucolipotoxicity. Proteomics analysis validated previously reported biomarkers (IGFBP1, RTN4R, PLXNB2) and identified additional molecules (CDHR2, ERBB4, DPP6) that may shed light on disease mechanisms. In conclusion, younger-onset T2D exhibits distinct subgroups with differential pathobiology, molecular signatures, and clinical outcomes, suggesting the need for personalised precision diabetes care.

Type 2 diabetes is a complex disease influenced by both genetic and environmental factors. Although polygenic risk scores (PRS) and family history (FH) are established predictors, their combined utility in East Asian populations remains unclear. In this study, we analyzed 111,899 unrelated participants from the Taiwan Biobank with linkage to nationwide health registry data. We constructed transancestry, East Asian-specific, and European-derived PRS and assessed their associations with type 2 diabetes. The transancestry PRS improved the explained variance in disease risk compared with ancestry-specific PRS and FH. FH, particularly sibling history, also showed a strong association with type 2 diabetes. When combined, PRS and FH contributed independently and additively, improving predictive performance. These findings demonstrate that PRS and FH are independent yet complementary risk indicators and support the application of transancestry PRS models in East Asian populations. Incorporating both genetic and familial risk factors may enhance early detection and facilitate more personalized prevention strategies for type 2 diabetes.

Current imaging technologies cannot detect diabetic retinopathy until there has been significant permanent damage to patients' vision. We hypothesized that hyperglycemia causes retinal hypoxia, and hypoxia may lead to apoptosis of retinal cells. We performed experiments using a mouse model of streptozotocin (STZ)-induced diabetes to investigate the role of hyperglycemia in diabetic eye disease. Our experimental results indicate that diabetic retinas are significantly hypoxic compared with nondiabetic controls. Retinal hypoxia can be detected using HYPOX-4, an early-detection imaging probe, potentially before any detectable changes in the diabetic retina. In the early stages of diabetes, we did not observe any detectable changes in electroretinography response, vascular permeability in fluorescein angiography, or retinal thickness in optical coherence tomographic imaging. In addition, increased HYPOX-4 fluorescence in the diabetic retina was not associated with focal ischemia; rather, increased levels of HYPOX-4 fluorescence were observed throughout the entire diabetic retina. Moreover, hypoxia profiles in STZ-induced diabetic retinas were colocalized with TUNEL-positive apoptotic cells. To confirm the role of hyperglycemia in the diabetic retina, human retinal cells were treated under hyperglycemic conditions, and hypoxia was monitored using the pimonidazole-adduct immunostaining method. Surprisingly, retinal cells became hypoxic under hyperglycemic conditions within the first few hours. We conclude that the diabetic retina becomes hypoxic as a result of hyperglycemia in the early stage of diabetes, which could lead to the degeneration of retinal cells at later stages of the disease. In addition, HYPOX-4 could be used as a powerful early diagnostic imaging method to detect retinal hypoxia in the diabetic retina before any detectable retinopathy.

Indirect calorimetry determines energy expenditure by measuring respiratory gas exchange. The approach is based on the principle that nutrient metabolism consumes O2 and produces CO2 and water in predictable ratios that can be compared with heat production. Due to the impracticality and high cost of direct calorimetry, indirect calorimetry is the major approach for modern metabolic studies in preclinical models. Despite the broad adoption of this method in rodent phenotyping, the field has lacked standardized methods for analyzing, representing, and sharing these data, limiting scientific progress. The recent review in Nature Metabolism by the International Indirect Calorimetry Consensus Committee (IICCC) addresses the lack of standardized methods by providing updated guidelines for data processing and representation and emphasizes the importance of shared data repositories tailored to these studies. These expert consensus standards aim to improve transparency, facilitate data sharing and archiving, and enhance cross-study comparability. We encourage authors submitting to Diabetes to adopt the IICCC guidelines to strengthen rigor and reproducibility in the field.