Identifying the early islet cellular processes of autoimmune type 1 diabetes (T1D) in humans is challenging given the absence of symptoms during this period and the inaccessibility of the pancreas for sampling. In this article, we study temporal events in pancreatic islets in LEW.1WR1 rats, in which autoimmune diabetes can be induced with virus infection, by performing transcriptional analysis of islets harvested during the prediabetic period. Single-cell RNA-sequencing and differential expression analyses of islets from prediabetic rats reveal subsets of β- and α-cells under stress as evidenced by heightened expression, over time, of a transcriptional signature characterized by interferon-stimulated genes, chemokines including Cxcl10, major histocompatibility class I, and genes for the ubiquitin-proteasome system. Mononuclear phagocytes show increased expression of inflammatory markers. RNA-in situ hybridization of rat pancreatic tissue defines the spatial distribution of Cxcl10+ β- and α-cells and their association with CD8+ T cell infiltration, a hallmark of insulitis and islet destruction. Our studies define early islet transcriptional events during immune cell recruitment to islets and reveal spatial associations between stressed β- and α-cells and immune cells. Insights into such early processes can assist in the development of therapeutic and prevention strategies for T1D.

Glucokinase (GK, gene symbol GCK) maturity-onset diabetes of the young (MODY) is caused by heterozygous inactivating mutations in GK and impaired glucose sensing. We investigated effects of dorzagliatin, a novel allosteric GK activator, on insulin secretion rates (ISRs) and β-cell glucose sensitivity (βCGS) in GCK-MODY and recent-onset type 2 diabetes. In a double-blind, randomized, crossover study, 8 participants with GCK-MODY and 10 participants with type 2 diabetes underwent 2-h 12 mmol/L hyperglycemic clamps following a single oral dose of dorzagliatin 75 mg or matched placebo. Effects of dorzagliatin on wild-type and mutant GK enzyme activity were investigated using an NADP+-coupled assay with glucose-6-phosphate dehydrogenase in vitro. In GCK-MODY, dorzagliatin significantly increased absolute and incremental second-phase ISRs versus placebo but not the acute insulin response. Dorzagliatin improved βCGS in GCK-MODY with an upward and leftward shift in ISR-glucose response. Dorzagliatin increased basal ISRs in type 2 diabetes, with smaller changes in second-phase ISRs versus GCK-MODY. In vitro, dorzagliatin directly reduced the glucose half saturation concentration of wild-type GK and selected GK mutants to varying degrees. Dorzagliatin directly restored enzyme activity of select GK mutants and enhanced wild-type GK activity, thereby correcting the primary defect of glucose sensing in GCK-MODY.

Diabetic kidney disease (DKD) is the leading cause of end-stage kidney disease (ESKD). Prognostic biomarkers reflective of underlying molecular mechanisms are critically needed for effective management of DKD. A three-marker panel was derived from a proteomics analysis of plasma samples by an unbiased machine learning approach from participants (N = 58) in the Clinical Phenotyping and Resource Biobank study. In combination with standard clinical parameters, this panel improved prediction of the composite outcome of ESKD or a 40% decline in glomerular filtration rate. The panel was validated in an independent group (N = 68), who also had kidney transcriptomic profiles. One marker, plasma angiopoietin 2 (ANGPT2), was significantly associated with outcomes in cohorts from the Cardiovascular Health Study (N = 3,183) and the Chinese Cohort Study of Chronic Kidney Disease (N = 210). Glomerular transcriptional angiopoietin/Tie (ANG-TIE) pathway scores, derived from the expression of 154 ANG-TIE signaling mediators, correlated positively with plasma ANGPT2 levels and kidney outcomes. Higher receptor expression in glomeruli and higher ANG-TIE pathway scores in endothelial cells corroborated potential functional effects in the kidney from elevated plasma ANGPT2 levels. Our work suggests that ANGPT2 is a promising prognostic endothelial biomarker with likely functional impact on glomerular pathogenesis in DKD.

Clinical studies investigating the benefit of glucose control on the progression of diabetic neuropathy (DN) have come to controversial results in patients with type 2 diabetes (T2D). This study aimed to assess associations of HbA1c levels with parameters of nerve perfusion in patients with T2D with and without DN using dynamic contrast-enhanced magnetic resonance neurography (DCE-MRN) at 3 Tesla. A total of 58 patients with T2D (20 with DN and 38 without DN) took part in this cross-sectional single-center study. Groups were matched for age, BMI, HbA1c, duration of T2D, and renal function. All patients underwent DCE-MRN with subsequent electrophysiologic and serologic testing. The extended Tofts model was used to quantify the sciatic nerve's microvascular permeability (Ktrans), volume fraction of the extracapillary extracellular space, and volume fraction of the plasma space. As a main result, we found that Ktrans correlated positively with HbA1c in patients with DN, while a negative correlation between the two parameters was found in patients without DN. Our results indicate that the effect of glucose control on the capillary permeability of peripheral nerves differs between patients with T2D with and without DN.

NADPH oxidase 4 (Nox4) is a major source of reactive oxygen species (ROS) in retinal endothelial cells (ECs) and is upregulated under hyperglycemic and hypoxic conditions. However, the role of endothelial Nox4 upregulation in long-term retinal blood vessel damage in diabetic retinopathy (DR) remains undefined. Here, we attempted to address this question using humanized EC-specific Nox4 transgenic (hNox4EC-Tg) and EC-specific Nox4 knockout (Nox4EC-KO) mouse models. Our results show that hNox4EC-Tg mice at age of 10-12 months exhibited increased tortuosity of retinal blood vessels, focal vascular leakage, and acellular capillary formation. In vitro study revealed enhanced apoptosis in brain microvascular ECs derived from hNox4EC-Tg mice, concomitant with increased mitochondrial ROS, elevated lipid peroxidation, decreased mitochondrial membrane potential, and reduced mitochondrial respiratory function. In contrast, EC-specific deletion of Nox4 decreased mitochondrial ROS generation, alleviated mitochondrial damage, reduced EC apoptosis, and protected the retina from acellular capillary formation and vascular hyperpermeability in a streptozotocin-induced diabetes mouse model. These findings suggest that sustained upregulation of Nox4 in the endothelium contributes to retinal vascular pathology in diabetes, at least in part, through impairing mitochondrial function. Normalization of Nox4 expression in ECs may provide a new approach for prevention of vascular injury in DR.

In rodents, susceptibility to diet-induced obesity requires microglial activation, but the molecular components of this pathway remain incompletely defined. Prostaglandin PGE2 levels increase in the mediobasal hypothalamus during high-fat-diet (HFD) feeding, and the PGE2 receptor EP4 regulates microglial activation state and phagocytic activity, suggesting a potential role for microglial EP4 signaling in obesity pathogenesis. To test the role of microglial EP4 in energy balance regulation, we analyzed the metabolic phenotype in a microglia-specific EP4 knockout (MG-EP4 KO) mouse model. Microglial EP4 deletion markedly reduced weight gain and food intake in response to HFD feeding. Corresponding with this lean phenotype, insulin sensitivity was also improved in HFD-fed MG-EP4 KO mice, though glucose tolerance remained surprisingly unaffected. Mechanistically, EP4-deficient microglia showed an attenuated phagocytic state marked by reduced CD68 expression and fewer contacts with pro-opiomelanocortin (POMC) neuron processes. These cellular changes observed in the MG-EP4 KO mice corresponded with an increased density of POMC neurites extending into the paraventricular nucleus (PVN). These findings reveal that microglial EP4 signaling promotes body weight gain and insulin resistance during HFD feeding. Furthermore, the data suggest that curbing microglial phagocytic function may preserve POMC cytoarchitecture and PVN input to limit overconsumption during diet-induced obesity.

Endogenous insulin secretion is a key regulator of postprandial hepatic glucose metabolism, but this process is dysregulated in diabetes. Subcutaneous insulin delivery alters normal insulin distribution, causing relative hepatic insulin deficiency and peripheral hyperinsulinemia, a major risk factor for metabolic disease. Our aim was to determine whether insulin's direct effect on the liver is preeminent even when insulin is given into a peripheral vein. Postprandial-like conditions were created (hyperinsulinemia, hyperglycemia, and a positive portal vein to arterial glucose gradient) in healthy dogs. Peripheral (leg vein) insulin infusion elevated arterial and hepatic levels 8.0-fold and 2.8-fold, respectively. In one group, insulin's full effects were allowed. In another, insulin's indirect hepatic effects were blocked with the infusion of triglyceride, glucagon, and inhibitors of brain insulin action (intracerebroventricular) to prevent decreases in plasma free fatty acids and glucagon, while blocking increased hypothalamic insulin signaling. Despite peripheral insulin delivery the liver retained its full ability to store glucose, even when insulin's peripheral effects were blocked, whereas muscle glucose uptake markedly increased, creating an aberrant distribution of glucose disposal between liver and muscle. Thus, the healthy liver's striking sensitivity to direct insulin action can overcome the effect of relative hepatic insulin deficiency, whereas excess insulin in the periphery produces metabolic abnormalities in nonhepatic tissues.

Exercise profoundly influences glycemic control by enhancing muscle insulin sensitivity, thus promoting glucometabolic health. While prior glycogen breakdown so far has been deemed integral for muscle insulin sensitivity to be potentiated by exercise, the mechanisms underlying this phenomenon remain enigmatic. We have combined original data from 13 of our studies that investigated insulin action in skeletal muscle either under rested conditions or following a bout of one-legged knee extensor exercise in healthy young male individuals (n = 106). Insulin-stimulated glucose uptake was potentiated and occurred substantially faster in the prior contracted muscles. In this otherwise homogenous group of individuals, a remarkable biological diversity in the glucometabolic responses to insulin is apparent both in skeletal muscle and at the whole-body level. In contrast to the prevailing concept, our analyses reveal that insulin-stimulated muscle glucose uptake and the potentiation thereof by exercise are not associated with muscle glycogen synthase activity, muscle glycogen content, or degree of glycogen utilization during the preceding exercise bout. Our data further suggest that the phenomenon of improved insulin sensitivity in prior contracted muscle is not regulated in a homeostatic feedback manner from glycogen. Instead, we put forward the idea that this phenomenon is regulated by cellular allostatic mechanisms that elevate the muscle glycogen storage set point and enhance insulin sensitivity to promote the uptake of glucose toward faster glycogen resynthesis without development of glucose overload/toxicity or feedback inhibition.

Despite significant progress in understanding the pathogenesis of type 2 diabetes (T2D), the condition remains difficult to manage. Hence, new therapeutic options targeting unique mechanisms of action are required. We have previously observed that elevated skeletal muscle succinyl CoA:3-ketoacid CoA transferase (SCOT) activity, the rate-limiting enzyme of ketone oxidation, contributes to the hyperglycemia characterizing obesity and T2D. Moreover, we identified that the typical antipsychotic agent pimozide is a SCOT inhibitor that can alleviate obesity-induced hyperglycemia. We now extend those observations here, using computer-assisted in silico modeling and in vivo pharmacology studies that highlight SCOT as a noncanonical target shared among the diphenylbutylpiperidine (DPBP) drug class, which includes penfluridol and fluspirilene. All three DPBPs tested (pimozide, penfluridol, and fluspirilene) improved glycemia in obese mice. While the canonical target of the DPBPs is the dopamine 2 receptor, studies in obese mice demonstrated that acute or chronic treatment with a structurally unrelated antipsychotic dopamine 2 receptor antagonist, lurasidone, was devoid of glucose-lowering actions. We further observed that the DPBPs improved glycemia in a SCOT-dependent manner in skeletal muscle, suggesting that this older class of antipsychotic agents may have utility in being repurposed for the treatment of T2D.

Studies of diabetic glomerular injury have raised the possibility of developing useful early biomarkers and therapeutic approaches for the treatment of type 2 diabetic nephropathy (T2DN). In this study, we found that FGF13 expression is induced in glomerular endothelial cells (GECs) during T2DN progression. Endothelial-specific deletion of Fgf13 potentially alleviates T2DN damage, while Fgf13 overexpression has the opposite effect. Mechanistically, Fgf13 deficiency results in improved mitochondrial homeostasis and endothelial barrier integrity in T2DN. Moreover, FGF13-sensitive alteration of Parkin safeguards mitochondrial homeostasis in endothelium of T2DN through promotion of mitophagy and inhibition of apoptosis. Additionally, it is confirmed that the beneficial effects of Fgf13 deficiency on T2DN are abolished by endothelial-specific double deletion of Fgf13 and Prkn. The effects of Fgf13 deficiency on mitophagy and apoptosis through Parkin-dependent regulation may be distinct and separable events under diabetic conditions. These data show that the bifunctional role of Fgf13 deficiency in promoting mitophagy and inhibiting apoptosis through Parkin can shape mitochondrial homeostasis regulation in GECs and T2DN progression. As a potential therapeutic target for prevention and control of T2DN, a mechanistic understanding of the biofunction of FGF13 may also be relevant to the pathogenesis of other FGF13- and Parkin-associated diseases.

Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), originally identified to be a lipid droplet-associated protein in adipocytes, positively associates with insulin sensitivity. Recently, we discovered that it is expressed abundantly in human endothelial cells and regulates vascular function. The current study was designed to characterize the physiological effects and molecular actions of endothelial CIDEC in the control of vascular phenotype and whole-body glucose homeostasis. To achieve this, we generated a humanized mouse model expressing endothelial-specific human CIDEC (E-CIDECtg). E-CIDECtg mice exhibited protection against high-fat diet-induced glucose intolerance, insulin resistance, and dyslipidemia. Moreover, these mice displayed improved insulin signaling and endothelial nitric oxide synthase activation, enhanced endothelium-dependent vascular relaxation, and improved vascularization of adipose tissue, skeletal muscle, and heart. Mechanistically, we identified a novel interplay of CIDEC-vascular endothelial growth factor A (VEGFA)-vascular endothelial growth factor receptor 2 (VEGFR2) that reduced VEGFA and VEGFR2 degradation, thereby increasing VEGFR2 activation. Overall, our results demonstrate a protective role of endothelial CIDEC against obesity-induced metabolic and vascular dysfunction, in part, by modulation of VEGF signaling. These data suggest that CIDEC may be investigated as a potential future therapeutic target for mitigating obesity-related cardiometabolic disease.

Environmental phenols are ubiquitous endocrine disruptors and putatively diabetogenic. However, data during pregnancy are scant. We investigated the prospective associations between pregnancy phenol concentrations and gestational diabetes mellitus (GDM) risk. In a nested matched case-control study of 111 individuals with GDM and 222 individuals without GDM within the prospective PETALS cohort, urinary bisphenol A (BPA), BPA substitutes (bisphenol F and bisphenol S [BPS]), benzophenone-3, and triclosan were quantified during the first and second trimesters. Cumulative concentrations across the two times were calculated using the area under the curve (AUC). Multivariable conditional logistic regression examined the association of individual phenols with GDM risk. We conducted mixture analysis using Bayesian kernel machine regression. We a priori examined effect modification by Asian/Pacific Islander (A/PI) race/ethnicity resulting from the case-control matching and highest GDM prevalence among A/PIs. Overall, first-trimester urinary BPS was positively associated with increased risk of GDM (adjusted odds ratio comparing highest vs. lowest tertile [aORT3 vs. T1] 2.12 [95% CI 1.00-4.50]). We identified associations among non-A/Ps, who had higher phenol concentrations than A/PIs. Among non-A/PIs, first-trimester BPA, BPS, and triclosan were positively associated with GDM risk (aORT3 vs. T1 2.91 [95% CI 1.05-8.02], 4.60 [1.55-13.70], and 2.88 [1.11-7.45], respectively). Triclosan in the second trimester and AUC were positively associated with GDM risk among non-A/PIs (P < 0.05). In mixture analysis, triclosan was significantly associated with GDM risk. Urinary BPS among all and BPA, BPS, and triclosan among non-A/PIs were associated with GDM risk. Pregnant individuals should be aware of these phenols' potential adverse health effects.

Despite the successes of human genome-wide association studies, the causal genes underlying most metabolic traits remain unclear. We used outbred heterogeneous stock (HS) rats, coupled with expression data and mediation analysis, to identify quantitative trait loci (QTLs) and candidate gene mediators for adiposity, glucose tolerance, serum lipids, and other metabolic traits. Physiological traits were measured in 1,519 male HS rats, with liver and adipose transcriptomes measured in >410 rats. Genotypes were imputed from low-coverage whole-genome sequencing. Linear mixed models were used to detect physiological and expression QTLs (pQTLs and eQTLs, respectively), using both single nucleotide polymorphism (SNP)- and haplotype-based models for pQTL mapping. Genes with cis-eQTLs that overlapped pQTLs were assessed as causal candidates through mediation analysis. We identified 14 SNP-based pQTLs and 19 haplotype-based pQTLs, of which 10 were in common. Using mediation, we identified the following genes as candidate mediators of pQTLs: Grk5 for fat pad weight and serum triglyceride pQTLs on Chr1, Krtcap3 for fat pad weight and serum triglyceride pQTLs on Chr6, Ilrun for a fat pad weight pQTL on Chr20, and Rfx6 for a whole pancreatic insulin content pQTL on Chr20. Furthermore, we verified Grk5 and Ktrcap3 using gene knockdown/out models, thereby shedding light on novel regulators of obesity.

Excessive adiposity is the main cause of obesity and type two diabetes (T2D). Variants in human IMP2/IGF2BP2 gene are associated with increased risk of T2D. However, little is known about its role in adipogenesis and in insulin resistance. Here, we investigate the function of IMP2 during adipocyte development. Mice with Imp2 deletion in mesenchymal stem cells (MSC) are resistant to diet-induced obesity without glucose and insulin tolerance affected. Imp2 is essential for the early commitment of adipocyte-derived stem cells (ADSC) into preadipocytes, but the deletion of Imp2 in MSC is not required for the proliferation and terminal differentiation of committed preadipocytes. Mechanistically, Imp2 binds Wnt receptor Fzd8 mRNA and promotes its degradation by recruiting CCR4-NOT deadenylase complex in an mTOR-dependent manner. Our data demonstrate that Imp2 is required for maintaining white adipose tissue homeostasis through controlling mRNA stability in ADSC. However, the contribution of IMP2 to insulin resistance, a main risk of T2D, is not evident.

Many coronavirus disease 2019 (COVID-19) risk factors, including obesity and diabetes, are associated with an abnormal basal metabolic rate (BMR). We aimed to evaluate whether BMR could impact the susceptibility to or severity of COVID-19. We performed genetic correlation and Mendelian randomization (MR) analyses to assess genetic correlations and potential causal associations between BMR (n = 448,348) and three COVID-19 outcomes: severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection, COVID-19 hospitalization, and critical COVID-19 (n = 1,086,211-2,597,856). A multivariable MR (MVMR) analysis was used to estimate the direct effect of BMR on COVID-19 independent of BMI and type 2 diabetes. BMR has positive genetic correlations with the COVID-19 outcomes (genetic correlations 0.213-0.266). The MR analyses indicated that genetic liability to BMR confers causal effects on SARS-CoV-2 infection (odds ratio 1.14, 95% CI 1.09-1.20, P = 1.65E-07), hospitalized COVID-19 (1.31, 1.18-1.46, P = 8.69E-07), and critical COVID-19 (1.04, 1.19-1.64, P = 4.89E-05). Sensitivity analysis of MR showed no evidence of directional pleiotropy or heterogeneity, indicating the robustness of its results. The MVMR analysis showed that the causal effects of BMR on hospitalized COVID-19 and critical COVID-19 were dependent on BMI and type 2 diabetes but that BMR may affect the SARS-CoV-2 infection risk independently of BMI and type 2 diabetes (odds ratio 1.09, 95% CI 1.03-1.15, P = 4.82E-03). Our study indicates that a higher BMR contributes to amplifying the susceptibility to and severity of COVID-19. The causal effect of BMR on the severity of COVID-19 may be mediated by BMI and type 2 diabetes.

Intraretinal hyperreflective foci (HRF) are significant biomarkers for diabetic macular edema. However, HRF at the vitreoretinal interface (VRI) have not been examined in diabetic retinopathy (DR). A prospective observational clinical study with 162 consecutive eyes using OCT imaging showed significantly increased HRF at the VRI during DR progression (P < 0.01), which was reversed by anti-vascular endothelial growth factor (VEGF) therapy. F4/80+ macrophages increased significantly at the VRI in Kimba (vegfa+/+) or Akimba (Akita × Kimba) mice (both P < 0.01), but not in diabetic Akita (Ins2+/-) mice, indicating macrophage activation was modulated by elevated VEGF rather than the diabetic milieu. Macrophage depletion significantly reduced HRF at the VRI (P < 0.01). Furthermore, BrdU administration in Ccr2rfp/+Cx3cr1gfp/+vegfa+/- mice identified a significant contribution of M2-like tissue-resident macrophages (TRMs) at the VRI. Ki-67+ and CD11b+ cells were observed in preretinal tissues of DR patients, while exposure of vitreal macrophages to vitreous derived from PDR patients induced a significant proliferation response in vitro (P < 0.01). Taken together, the evidence suggests that VEGF drives a local proliferation of vitreous resident macrophages (VRMs) at the VRI during DR. This phenomenon helps to explain the derivation and disease-relevance of the HRF lesions observed through OCT imaging in patients.

A recent discovery effort resulted in identification of novel splice variant and secretory granule antigens within the HLA class I peptidome of human islets and documentation of their recognition by CD8+ T cells from peripheral blood and human islets. In the current study, we applied a systematic discovery process to identify novel CD4+ T cell epitopes derived from these candidate antigens. We predicted 145 potential epitopes spanning unique splice junctions and within conventional secretory granule antigens and measured their in vitro binding to DRB1*04:01. We generated HLA class II tetramers for the 35 peptides with detectable binding and used these to assess immunogenicity and isolate T cell clones. Tetramers corresponding to peptides with verified immunogenicity were then used to label T cells specific for these putative epitopes in peripheral blood. T cells that recognize distinct epitopes derived from a cyclin I splice variant, neuroendocrine convertase 2, and urocortin-3 were detected at frequencies that were similar to those of an immunodominant proinsulin epitope. Cells specific for these novel epitopes predominantly exhibited a Th1-like surface phenotype. Among the three epitopes, responses to the cyclin I peptide exhibited a distinct memory profile. Responses to neuroendocrine convertase 2 were detected among pancreatic infiltrating T cells. These results further establish the contribution of unconventional antigens to the loss of tolerance in autoimmune diabetes.

The functional mass of insulin-secreting pancreatic β-cells expands to maintain glucose homeostasis in the face of nutrient excess, in part via replication of existing β-cells. Type 2 diabetes appears when these compensatory mechanisms fail. Nutrients including glucose and fatty acids are important contributors to the β-cell compensatory response, but their underlying mechanisms of action remain poorly understood. We investigated the transcriptional mechanisms of β-cell proliferation in response to fatty acids. Isolated rat islets were exposed to 16.7 mmol/L glucose with or without 0.5 mmol/L oleate (C18:1) or palmitate (C16:0) for 48 h. The islet transcriptome was assessed by single-cell RNA sequencing. β-Cell proliferation was measured by flow cytometry. Unsupervised clustering of pooled β-cells identified different subclusters, including proliferating β-cells. β-Cell proliferation increased in response to oleate but not palmitate. Both fatty acids enhanced the expression of genes involved in energy metabolism and mitochondrial activity. Comparison of proliferating versus nonproliferating β-cells and pseudotime ordering suggested the involvement of reactive oxygen species (ROS) and peroxiredoxin signaling. Accordingly, N-acetyl cysteine and the peroxiredoxin inhibitor conoidin A both blocked oleate-induced β-cell proliferation. Our study reveals a key role for ROS signaling through peroxiredoxin activation in oleate-induced β-cell proliferation.