Diabetes holds significant social importance due to its high incidence rate and multitude of associated complications. The identification of diabetes biomarkers and the understanding of the intricate biological mechanisms underlying diabetes are crucial for the early diagnosis and treatment of diabetes. In this study, we conducted comprehensive omics profiling of CpGs, plasma proteins, and serum metabolites in an National Survey of Physical Traits (NSPT) cohort of 3,451 individuals, among whom 293 were patients with diabetes. Global association analysis identified 175 CpGs, 29 proteins, and 93 metabolites significantly linked to diabetes, among which 43 CpGs and 25 metabolites were validated in an independent cohort comprising 532 individuals. Mendelian randomization and mediation analysis identified 20 causal biomarkers and 190 signaling pathways linking biomarkers from different layers. By integrating the cross-omics evidence, we provide a list of putative causal biomarkers of diabetes to serve as a valuable resource for the diabetes community. Cross-omics integration prioritized biomarkers for therapeutic targeting, highlighting COLEC11 as an example of a potential target and whose function was further validated in vitro. The early-prediction model using the prioritized biomarkers improved the area under the receiver operating characteristic curve by 27.5% compared with the baseline model, using clinical features alone. Our findings provide a comprehensive list of prioritized multiomics biomarkers and elucidate specific signaling pathways in diabetes, contributing significantly to the selection of therapeutic target and the understanding of diabetes pathophysiology.

Pancreatic β-cells can self-renew in the adult pancreas through replication, but the contribution of ductal progenitors to endocrine regeneration has been the subject of debate for two decades. While these mechanisms are not mutually exclusive, some lineage-tracing strategies suggest that intraductal endocrine cells cannot dynamically derive from ducts. Combining one such approach with a novel in vivo model in which live pancreatic slices are transplanted into the anterior chamber of the eye (ACE) of recipient mice, we show long-term growth of preexisting islets and real-time generation of neogenic insulin-expressing cells from ductal areas. Our results represent a departure from historical approaches to address these questions, which have been based on either static analyses of pancreatic tissue or "before and after" lineage-tracing designs. The slice-in-ACE model reveals the dynamic processes at play during regeneration and demonstrates the active formation of insulin-producing cells within the ductal network.

We investigated serum metabolites in monozygotic (MZ) and dizygotic (DZ) twins discordant for type 1 diabetes (T1D) to explore potential environmental factors, with a focus on differences in gut microbiota-associated metabolites that may influence T1D. Serum samples from 39 twins discordant for T1D were analyzed using a semi-targeted metabolomics approach via liquid chromatography-high-resolution tandem mass spectrometry. Statistical analyses identified significant metabolites (P < 0.1) within three groups: all twins (combined group [All]), MZ twins, and DZ twins. Thirteen metabolites exhibited significant differences between individuals with T1D and those without T1D. Across all groups, 3-indoxyl sulfate and 5-hydroxyindole were significantly reduced in individuals with T1D. Carnitine was reduced, and threonine, muramic acid, and 2-oxobutyric acid were significantly elevated in both All and MZ groups. Allantoin was significantly reduced and 3-methylhistidine was significantly elevated in All and DZ groups. These findings suggest metabolite dysregulation associated with gut dysbiosis was observed. However, further validation of our findings in a larger cohort is needed.

Despite stimulating glucagon secretion, the mechanisms by which protein ingestion lowers glucose excursions remain unclear. We investigated this using the triple stable isotope glucose tracer technique to measure postprandial glucose fluxes. Eleven healthy adults completed three trials, ingesting 25 g glucose (25G; 100 kcal), 50 g glucose (50G; 200 kcal), or 25 g glucose plus 25 g whey protein (25WG; 200 kcal). Glucose excursions were lowest for 25WG. Glucagon increased approximately threefold with 25WG but was suppressed with 25G and 50G. Insulin and glucose-dependent insulinotropic polypeptide (GIP) were higher for 25WG versus 25G, whereas glucagon-like peptide 1 (GLP-1) was similar. Compared with 50G, 25WG produced a greater GIP but similar GLP-1 response, with a trend toward higher early-phase insulin. Endogenous glucose production (EGP) was less suppressed with 25WG (∼50%) versus 25G (∼70%) or 50G (∼80%). Compared with 25G, 25WG did not enhance glucose disposal (Rd) but reduced early-phase (30-60 min) glucose absorption. These findings confirm that protein-glucose coingestion robustly stimulates glucagon while enhancing GIP and insulin, leading to lower postprandial glucose excursions. Despite greater insulin secretion, the net glycemic benefit seems to stem from reduced early glucose absorption rather than increased Rd. This provides novel insights into the mechanisms by which protein improves postprandial glucose handling despite interfering with EGP suppression.

Deciphering the heterogeneity of type 2 diabetes in prognosis and treatment effect is essential. We used a novel dimensionality reduction approach to describe the type 2 diabetes phenotype continuum and visualize the difference in lifestyle intervention efficacy in Chinese patients. Based on 17,816 participants with newly diagnosed type 2 diabetes (aged ≥40 years) from a nationwide cohort, 12 key phenotypes were residualized for age and sex to construct a two-dimensional tree structure. The tree demonstrated the continuous type 2 diabetes spectrum and region-specific characteristics, with a mixed phenotypic trunk and three extreme phenotypic branches. When mapping data from 325 participants with type 2 diabetes from a randomized controlled trial onto the original tree, lifestyle intervention induced a migration toward the left part of tree, indicating an overall metabolic improvement. Specifically, diet intervention was more effective for glycemic control in the upper part of the tree and featured moderate diabesity and elevated insulin, whereas exercise intervention was more effective for glycemic control in the left side of the tree and featured less adiposity and better overall metabolic status. In summary, this analysis depicted the tree structure representing the underlying pathophysiological features of patients with newly diagnosed type 2 diabetes and identified tree regions with different sensitivity to diet or exercise intervention. The results have the potential to aid lifestyle intervention selection.

Type 1 diabetes (T1D) is caused by the selective autoimmune ablation of pancreatic β-cells. Emerging evidence reveals β-cell secretory dysfunction arises early in T1D development and may contribute to diseases etiology; however, the underlying mechanisms are not well understood. Our data reveal that proinflammatory cytokines elicit a complex change in the β-cell's Golgi structure and function. The structural modifications include Golgi compaction and loss of the interconnecting ribbon resulting in Golgi fragmentation. We further show that Golgi structural alterations coincide with persistent altered cell surface glycoprotein composition. Our data demonstrate that inducible nitric oxide synthase (iNOS)-generated nitric oxide (NO) is necessary and sufficient for β-cell Golgi restructuring. Moreover, the unique sensitivity of the β-cell to NO-dependent mitochondrial inhibition results in β-cell-specific Golgi alterations that are absent in other cell types, including α-cells. Examination of human pancreas samples from autoantibody-positive and T1D donors with residual β-cells further revealed alterations in β-cell, but not α-cell, Golgi structure that correlate with T1D progression. Collectively, our studies provide critical clues as to how β-cell secretory functions are specifically impacted by cytokines and NO that may contribute to the development of β-cell autoantigens relevant to T1D.

Diabetic wounds represent a significant clinical and economic burden, affecting both patients and health care systems. While current therapeutic approaches, such as negative pressure wound therapy, offer benefits, their limitations necessitate alternative strategies. Newly discovered dental pulp stem cell-derived intracellular vesicles have emerged as a promising candidate in regenerative medicine due to their therapeutic potential. In vitro assessments using HUVECs, HaCaTs, and RAW264.7 cells revealed that intracellular vesicles enhance cell migration, angiogenesis, and proliferation while suppressing the cGAS-STING pathway. Additionally, intracellular vesicles promoted M2 macrophage polarization and maintained mitochondrial function. In a diabetic mouse wound model, both intracellular vesicles and negative pressure wound therapy individually improved wound healing, but their combination exhibited a synergistic effect, resulting in faster wound closure, enhanced angiogenesis, and reduced inflammation. The combined treatment also exhibited excellent biocompatibility. These findings highlight the therapeutic potential of intracellular vesicles as an adjunct to negative pressure wound therapy for diabetic wound treatment.

Long noncoding RNAs (lncRNAs) play essential roles in cellular processes, often exhibiting cell type-specific expression and influencing kidney function. While single-cell RNA sequencing (scRNA-seq) has advanced our understanding of cellular specificity, past studies focus solely on protein-coding genes. We hypothesize that lncRNAs, due to their cell-specific nature, have crucial functions within particular renal cells and thereby play essential roles in renal cell function and disease. Using single-nucleus RNA-seq (snRNA-seq) data from kidney samples of five healthy individuals and six patients with diabetic kidney disease (DKD), we explored the noncoding transcriptome. Cell type-specific lncRNAs were identified, and their differential expression in DKD was assessed. Integrative analyses included expression quantitative trait loci (eQTL), genome-wide association studies (GWAS) for estimated glomerular filtration rate (eGFR), and gene regulatory networks. Functional studies focused on TCF21 antisense RNA inducing promoter demethylation (TARID), a lncRNA with podocyte-specific expression, to elucidate its role in podocyte health. We identified 174 lncRNAs with cell type-specific expression across kidney cell types. Of these, 54 lncRNAs were differentially expressed in DKD. Integrative analyses, including eQTL data, GWAS results for eGFR, and gene regulatory networks, pinpointed TARID, a podocyte-specific lncRNA, as a key candidate upregulated in DKD. Functional studies confirmed TARID's podocyte-specific expression and revealed its central role in actin cytoskeleton reorganization. Our study provides a comprehensive resource of single-cell lncRNA expression in the human kidney and highlights the importance of cell type-specific lncRNAs in kidney function and disease. Specifically, we demonstrate the functional relevance of TARID in podocyte health.

Ribosome profiling (Ribo-seq) measures translational regulation and reveals novel or unannotated open reading frames (nuORFs) otherwise difficult to identify. Recent reports demonstrate that nuORFs regulate gene expression and immune recognition, highlighting their emerging biological roles. Pancreatic β-cells are critical for maintaining euglycemic conditions, and β-cell impairment contributes to diabetes development. Identification of nuORF and protein/peptide products in human β-cells could reveal novel mechanisms that regulate β-cell function during homeostatic and disease conditions. Here, we applied a proteogenomic approach to human β-cells to define previously unknown protein/peptide products. First, we applied cell type-specific Ribo-seq to map the translatome of human stem cell-derived β-cells (sBCs). Pathways crucial for β-cell function and antigen presentation were subject to translational regulation. We detected a recently described immunogenic neoantigen, INS-DRiP, presumably originating from a downstream start site in INS mRNA. Moreover, our analysis revealed 965 novel nuORFs in sBCs, with a majority showing protein-level support. Comparison with primary human islets further validated nuORF translation and highlighted β-cell specificity. We identified a novel, primate-specific regulatory upstream ORF within TYK2, which is crucial for β-cell function and interferon response and has many variants strongly associated with type 1 diabetes. Finally, we used immunopeptidomics, HLA-binding prediction models, and T-cell coculture assays to validate the presentation and immunogenicity of preproinsulin peptides and nuORFs. Our findings underscore the importance of translational regulation in β-cell function and provide an important resource to the diabetes research community.

Dorzagliatin is a dual-acting allosteric activator of glucokinase (GCK). Dorzagliatin improved second-phase insulin secretion in individuals with type 2 diabetes and heterozygous carriers of GCK mutations. We investigated the effects of dorzagliatin on pancreatic insulin, glucagon, and glucagon-like-peptide 1 (GLP-1) secretion in individuals with impaired glucose tolerance (IGT) and normal glucose tolerance (NGT). In a double-blind, randomized, crossover, single-dose study, 9 participants with IGT and 10 with NGT underwent 2-h 12 mmol/L hyperglycemic clamp following a single dose of dorzagliatin 50 mg or matched placebo. Plasma insulin, C-peptide, glucagon, and total GLP-1 levels were measured at regular intervals. There were no differences in first-phase insulin after the dorzagliatin dose in either group. Dorzagliatin significantly increased second-phase insulin secretion rate and β-cell glucose sensitivity by 1.3-fold compared with placebo in IGT but remained similar in NGT. Dorzagliatin increased basal plasma insulin in the NGT group only. Glucagon (area under the curve0-120 min = 161 ± 58 vs. 234 ± 70 pmol*min/L [mean ± SD]; P = 0.01) was suppressed after dorzagliatin in the NGT group but not the IGT group. Plasma glucagon was positively correlated with total GLP-1 levels. Dorzagliatin did not affect insulin sensitivity in either group. Dorzagliatin has different actions on β- and α-cells depending on glucose tolerance, increasing second-phase insulin secretion in IGT while enhancing glucose-suppression of glucagon secretion in NGT.

β-Cell extracellular vesicles (EVs) play a role as paracrine effectors in islet health, yet mechanisms connecting β-cell stress to changes in EV cargo and potential impacts on diabetes remain poorly defined. We hypothesized that β-cell inflammatory stress engages neutral sphingomyelinase 2 (nSMase2)-dependent EV formation pathways, generating ceramide-enriched small EVs that could impact surrounding β-cells. Consistent with this, proinflammatory cytokine treatment of INS-1 β-cells and human islets concurrently increased β-cell nSMase2 and ceramide abundance, as well as small EV ceramide species. Direct chemical activation or genetic knockdown of nSMase2, chemical treatment to inhibit cell death pathways, or treatment with a glucagon-like peptide-1 (GLP-1) receptor agonist also modulated β-cell EV ceramide. RNA sequencing of ceramide-enriched EVs identified a distinct set of miRNAs linked to β-cell function and identity. EV treatment from cytokine-exposed parent cells inhibited peak glucose-stimulated insulin secretion in wild-type recipient cells; this effect was abrogated when using EVs from nSMase2 knockdown parent cells. Finally, plasma EVs in children with recent-onset type 1 diabetes showed increases in multiple ceramide species. These findings highlight nSMase2 as a regulator of β-cell EV cargo and identify ceramide-enriched EV populations as a contributor to EV-related paracrine signaling under conditions of β-cell inflammatory stress and death.

We evaluated whether a binary metabolic end point for change (Δ) from baseline to 1-year postrandomization could be useful in type 1 diabetes (T1D) prevention trials. Using 2-h oral glucose tolerance testing data from the stage 1 participants in the recent abatacept prevention trial and similar participants in the observational TrialNet Pathway to Prevention (PTP) study, we assessed Δmetabolic measures, plotted glucose and C-peptide response curves, and categorized vectors for Δ from baseline to 1 year as metabolic treatment failure versus success. Analyses were validated using the teplizumab prevention study. PTP participants with Δglucose >0 and ΔC-peptide <0 from baseline to 1 year were at substantially higher risk for stage 3 T1D than those with Δglucose <0 and ΔC-peptide >0 (P < 0.0001). Based on this, we compared placebo versus treatment groups in both trials for failure (Δglucose >0 with ΔC-peptide <0) versus success (Δglucose <0 with ΔC-peptide >0) after 1 year. Using this end point, a favorable metabolic impact of abatacept was found after 12 months of treatment. An analytic approach using a binary metabolic end point of failure versus success at a fixed time interval appears to detect treatment effects at least as well as standard primary end points with shorter follow-up.

Growth differentiation factor 15 (GDF15) is an anorectic and weight loss-inducing hormone that responds to stimuli such as endoplasmic reticulum stress, exercise, metformin, and, more recently, dietary lipids. Given its potential as an antiobesogenic agent, we examined how endogenous GDF15 responds to an Intralipid infusion in different organs to regulate food intake in vivo. We found that an acute Intralipid infusion into the upper small intestine (USI) inhibited food intake and increased plasma GDF15, as well as kidney and hepatic Gdf15 expression in chow-fed but not high-fat (HF)-induced hyperphagic male rats. Kidney Gdf15 knockdown blunted Intralipid-induced increases in kidney and plasma GDF15 levels as well as its feeding-lowering effects, while hepatic Gdf15 expression remained unaffected. Lastly, we knocked down GDNF family receptor α-like (Gfral) in the area postrema, which negated the feeding-lowering effect of Intralipid despite a rise in plasma GDF15 levels in chow rats. In summary, we report that kidney GDF15 is necessary for USI intralipid sensing to trigger an area postrema axis to inhibit food intake. We propose that HF feeding impairs acute lipid sensing to lower feeding by negating the lipid-regulatory effect on kidney GDF15.

Despite the increasing prevalence of type 2 diabetes in youth, its causal associations with circulating biomarkers remain elusive. We first aimed to identify circulating metabolites causally linked to youth-onset type 2 diabetes using Mendelian randomization (MR). By analyzing 675 metabolites from large metabolomic European genome-wide association studies (GWAS) and data on youth type 2 diabetes from the multiancestry Progress in Diabetes Genetics in Youth (ProDiGY) consortium, we identified 34 candidate metabolites. Among these, phosphatidylcholine (pc) ae C42:3 and propionylcarnitine provided the strongest evidence of association with youth-onset type 2 diabetes, based also on positive genetic colocalization and sensitivity analyses accounting for adiposity. Among the 34 candidate metabolites, 23 were retained following colocalization and a replication MR using independent metabolomic GWAS and testing effects on adult type 2 diabetes. Furthermore, we validated associations of six of these metabolites with glucose metabolism-related traits in an observational study in the Avon Longitudinal Study of Parents and Children (ALSPAC). Notably, pc ae C42:3 levels at age 7 years were linked to dysglycemia and insulin resistance in adolescence. These findings underscore the dynamic role of metabolites in glucose metabolism in childhood, offering insights for future screening and treatment strategies.

In mice, glucagon regulates lipid metabolism by activating receptors in the liver; however, its role in human lipid metabolism is incompletely understood. Here we describe three normal-weight individuals from a consanguineous family with early-onset hepatic steatosis and/or cirrhosis. Using exome sequencing, we found they were homozygous for two missense variants in the glucagon receptor gene (GCGR). In cells, the double GCGR mutation reduced cell membrane expression and signaling, resulting in an almost complete loss of function. Carriers of pathogenic GCGR mutations had substantially elevated circulating glucagon and amino acid levels and increased adiposity. Introducing the double GCGR mutation into human induced pluripotent stem cell-derived hepatocytes using CRISPR/Cas9 caused increased lipid accumulation. Our results provide an explanation for increased liver fat seen in clinical trials of GCGR antagonists and reduced liver fat in people with obesity and steatotic liver disease treated with GCGR agonists.

Pancreatic β-cells undergo senescence and loss during aging; however, the underlying mechanisms remain incompletely understood. This study aimed to investigate what sirtuin 6 (SIRT6) does during β-cell aging. Pancreatic β-cell-specific Sirt6 transgenic (TgSIRT6) mice were generated for this study. DNA damage, cell death, and cell proliferation were analyzed in cell and mouse models. SIRT6 protein levels were decreased in pancreatic β-cells during aging. TgSIRT6 mice exhibited less DNA damage and cell death, including apoptosis, necroptosis, and pyroptosis, in β-cells than control mice. TgSIRT6 mice had increased total islet area and mass in pancreas compared with control mice. As a result, TgSIRT6 mice showed better glucose tolerance and glucose-stimulated insulin secretion than control mice. RRAD and GEM-like GTPase 2 (REM2), an endogenouse inhibitor of high-voltage-activated calcium channels, was negatively regulated by SIRT6. Knockdown of Rem2 in INS-1 cells partially rescued the SIRT6 deficiency- and palmitic acid-induced DNA damage, lipid peroxidation, and cell death. Rem2 β-cell-specific knockout mice had less DNA damage and cell death in β-cells than control mice. Our data suggest that SIRT6 is a critical antiaging factor in pancreatic β-cells and is a potential therapeutic target.

Diabetic retinopathy (DR) is characterized by microvascular damage and increased vascular permeability in the retina. The investigation of visual outcomes in late-stage DR is limited by challenges of maintaining chronically hyperglycemic mice, and most reports are restricted to early-stage DR. In this study, we used carefully managed diabetic mice to longitudinally investigate associations between vascular leakage and visual acuity during early- and late-stage DR. Diabetes was induced in C57BL/6J mice with streptozotocin, and fluorescence angiography with dual fluorescence (FA-DF) was used to assess retinal vascular leakage dynamics in chronically hyperglycemic mice for 12 months. Retinal vascular leakage was evident 180 days after diabetes induction and before reduced visual acuity, measured using the optokinetic response, and vascular leakage continued to increase during DR progression. Mice were also treated with intravitreal injections of antiangiogenic aflibercept at late-stage DR, and reduced leakage was reliably measured using FA-DF and was associated with improved visual acuity. Inflammatory and vascular phenotypes were assessed using immunostaining, which revealed significantly lower retinal macrophage and vascular densities and reduced capillary diameter in association with anti-VEGF treatment compared with age-matched diabetic controls. In conclusion, this is the first longitudinal quantification of retinal vascular leakage in early, intermediate, and late stages of DR in the same cohort of mice in a minimally invasive fashion to demonstrate the associated effect of antiangiogenic therapy in vivo. Our findings also further confirmed the sensitivity of FA-DF in assessing retinal vascular leakage in conjunction with other functional measures in longitudinal studies in the same animals.

Maternal hyperglycemia is linked to 19 cord blood DNA methylation biomarkers that predict offspring metabolic dysfunction. These methylation changes, associated with maternal glycemic status, improved the prediction of β-cell dysfunction at 7, 11, and 18 years of age compared with clinical factors alone. Validation in human β-cells and pancreatic ductal epithelial cells confirmed that hyperglycemia influences methylation-dependent gene expression. These findings highlight the role of epigenetic modifications at birth as early indicators of diabetes risk, suggesting that in utero hyperglycemic exposure may mediate long-term metabolic outcomes in offspring.

Diabetes has a large medical and public health impact in American Indians. Studies have used genetic data to distinguish type 1 diabetes (T1D) and type 2 diabetes (T2D) and uncover biologic mechanisms underlying T2D clinical heterogeneity. We applied a T1D polygenic score (PS) to 3,084 American Indians (mean age 56 years, 58% women, 39% diabetes). We also calculated partitioned PS for eight clusters of T2D-associated variants and evaluated their association with 20 cardiometabolic traits and five clinical outcomes. The profile of T1D PS for individuals with diabetes was consistent with T2D. A total T2D PS was significantly associated with early age of T2D onset (P = 3.5 × 10-11). Partitioned PS for T2D clusters were significantly associated with cardiometabolic traits for the obesity cluster (increased measures of body fat and total triglycerides but lower HDL cholesterol), while the lipodystrophy cluster was associated with increased fasting insulin, waist-to-hip ratio, triglycerides, and blood pressure, and lower body fat percentage and HDL cholesterol. T2D clusters were not associated with cardiovascular and kidney outcomes. Our findings support a relationship of cluster-specific T2D partitioned PS with cardiometabolic traits described in other populations, but there are opportunities for developing improved clustering methods using genetic variation from American Indians.

Diabetic cardiomyopathy (DbCM) is a chronic metabolic disorder with few effective treatment strategies. Our previous study demonstrated that activated protein C (aPC), a serine protease, exerts cytoprotective effects in DbCM. However, the mechanisms underlying its role in DbCM require further elucidation. We developed a type 1 diabetic mouse model using thrombomodulin gene point mutation mice (TMP/P) with reduced endogenous aPC generation and investigated the protective effects of aPC on DbCM through intraperitoneal injection of PC. Myocardial functions and structure were assessed by echocardiography and histology. Transcriptomic analysis and immunological evaluation were conducted to investigate the downstream targets. The antisenescence role of aPC was reaffirmed by PC treatment in vivo and aPC intervention in cultured neonatal rat ventricular myocytes in vitro. Endogenous aPC levels were reduced and positively correlated with cardiac diastolic function in diabetic mice. Cardiomyocytes manifested a senescent phenotype in DbCM. With impaired aPC activation, TMP/P mice exhibited aggravated diabetes-induced cardiac dysfunction and cardiomyocyte senescence. Mechanistically, aPC alleviated cardiomyocyte senescence in DbCM by acting on PAR1/PAR3 to restore the interaction between P85 and CaMKIIδ, thereby inhibiting CaMKIIδ phosphorylation and its nuclear translocation. In summary, our study highlights that aPC ameliorates cardiomyocyte senescence in DbCM via the PAR1/PAR3-P85-CaMKIIδ axis.