Diabetic kidney disease (DKD) progression involves NIMA-related kinase 7 (NEK7)-driven podocyte pyroptosis, with hyperglycemia-induced O-GlcNAcylation as a key posttranslational regulator. This study elucidates how O-GlcNAc modification governs NEK7 stability and its pathological role. We used clinical DKD specimens, high-glucose-stimulated podocytes, and streptozotocin-induced diabetic mice to first examine NEK7, O-GlcNAc, O-GlcNAc transferase (OGT), and glutamine fructose-6-phosphate amidotransferase 1 (GFPT1) expression, confirming the pyroptosis role of NEK7 via siRNA knockdown. Bioinformatic analysis predicted O-GlcNAcylation motifs, validated by T302A mutagenesis and coimmunoprecipitation. Protein stability was assessed using cycloheximide chase and ubiquitination assays. Therapeutic efficacy of the GFPT1 inhibitor (6-diazo-5-oxo-l-norleucine) DON was evaluated in vitro and in vivo through biochemical parameters, histopathology, and pyroptosis markers. Chronic hyperglycemia activated the hexosamine biosynthetic pathway (HBP), elevating pathology-associated O-GlcNAc modifications that promoted NEK7 accumulation via posttranslational stabilization. This was accompanied by upregulated O-GlcNAc, OGT, and GFPT1 in DKD glomeruli and high-glucose podocytes. Crucially, threonine 302 was identified as the primary O-GlcNAcylation site of NEK7. This modification reduced proteasomal degradation, extended NEK7 half-life, and enhanced NLRP3 inflammasome activation and interleukin release. Pharmacological HBP inhibition using DON normalized O-GlcNAcylation, suppressed pyroptosis, and mitigated renal injury. We report the discovery of the glucose/O-GlcNAc/NEK7/NLRP3 axis driving podocyte pyroptosis in DKD, proposing threonine 302 as a potential therapeutic target. These findings establish a novel posttranslational modification mechanism and suggest a dual-target therapeutic strategy for DKD management.

Somatostatin is a powerful inhibitor of insulin secretion and β-cell electrical activity, but the effects are weak in intact islets, possibly because of high intraislet somatostatin levels. We used optogenetics in conjunction with hormone secretion measurements, electrophysiology, and cytoplasmic free Ca2+ concentration ([Ca2+]i) imaging to interrogate the relative roles of paracrine and electrical control of β-cells by δ-cells. We confirm that optoactivation and optoinhibition of δ-cells stimulated and inhibited their electrical activity and somatostatin secretion, respectively. Unexpectedly, neither optoactivation nor optoinhibition of δ-cells had any effect on insulin secretion at 1 or 20 mmol/L glucose. Paradoxically, optoactivation of δ-cells at 6 mmol/L glucose increased insulin secretion by 113%, an effect that correlated with β-cell action potential firing. In [Ca2+]i imaging experiments, optoactivation of δ-cells induced islet-wide β-cell [Ca2+]i transients and synchronized the oscillatory pattern induced by 7 mmol/L glucose. Conversely, optoinhibition of δ-cells and somatostatin secretion reduced rather than increased β-cell electrical activity and [Ca2+]i in the <10% of β-cells situated <20 µm from δ-cells. We propose that δ-cells, in addition to subserving an inhibitory paracrine effect, play a role in the rapid propagation of electrical signals across the islet, possibly contributing to the coordination of β-cell activity.

Prediction of kidney function decline is challenging in patients with type 2 diabetes mellitus (T2DM). Urinary Dickkopf-3 (uDKK3), a profibrotic tubular protein, is a promising biomarker for detecting tubular injury and predicting the progression of chronic kidney disease. This study assessed whether uDKK3 measurements improve risk prediction in patients with T2DM treated at the primary care level. Elevated uDKK3 levels were associated with kidney function decline, on top of established biomarkers (estimated glomerular filtration rate and albuminuria). uDKK3 also identified patients at increased risk for cardiovascular events. uDKK3 may help identify high-risk patients in primary care who could benefit from intensified treatment and/or referrals to specialists.

An intrinsic hallmark of type 1 diabetes is the correlation between appearance of autoantibodies directed against islet cell autoantigens with subsequent development of the disease. We recently studied effects of human monoclonal autoantibodies (mAbs) derived from a patient with prediabetes and demonstrated that a GAD65mAb penetrated and accumulated in β-cells and significantly reduced the insulin secretion rate (ISR). Accordingly, in the current study, we performed more detailed analyses of the effects of this GAD65mAb on rat and human islets. ISR was suppressed by ∼40% after 3 days of exposure. Mechanisms mediating the effects were found to involve inhibition of mitochondrial generation of ATP, which decreased in parallel with that of ISR. As expected, the GAD65mAb inhibited γ-aminobutyric acid secretion. The effects of GAD65mAb were observed in rat and human islets but not in mouse islets, which do not express GAD65. GAD65mAb also reduced insulin secretion in vivo, where decreased insulin levels after intraperitoneal (i.p.) injection of glucose were observed in rats after i.p. injection of GAD65mAb. Thus, it appears that an islet cell autoantibody against GAD65 can directly impact and impair secretory function in islets in vitro and in vivo through a mechanism that involves inhibition of mitochondrial energetics.

Postbariatric hypoglycemia (PBH) is a serious complication of Roux-en-Y gastric bypass (RYGB), characterized by severe hypoglycemia that may lead to loss of consciousness and seizures. The exact mechanism of PBH is poorly understood. One potential mechanism is β-cell expansion. To this end, we investigated β-cell mass in individuals with and without PBH after RYGB using [68Ga]Ga-NODAGA-exendin-4 positron emission tomography/computed tomography imaging (PET/CT). Individuals with PBH (n = 10) and without PBH (n = 9) after RYGB were included. PET/CT imaging was performed after infusion with 102.2 ± 6.9 MBq of the [68Ga]Ga-NODAGA-exendin-4 tracer to quantify pancreatic β-cell mass. The two groups did not differ with respect to sex, age, BMI, and total body weight loss after RYGB. Time between RYGB and inclusion was longer for individuals with PBH compared with those without. β-Cell mass did not differ between the groups. Individuals with PBH had a smaller pancreas than those without. β-Cell mass correlated neither with body weight parameters nor with metabolic parameters. Our data indicating that β-cell mass does not differ between individuals with and without PBH after RYGB argue against expansion of β-cell mass to explain PBH.

Insulin-like growth factor-2 receptor (IGF2R), also known as cation-independent mannose-6-phosphate receptor, is localized in cytosolic vesicles and is unique in its ability to transport enzymes to the lysosome and to clear IGF2 from the cell surface by acting as a scavenger receptor. To evaluate the direct role of IGF2R in β-cell biology, we undertook complementary in vitro knockdown and in vivo knockout approaches. A β-cell line with a stable knockdown of IGF2R (IGF2RKD) exhibited decreased glucose-induced insulin secretion and enhanced cell proliferation. Tamoxifen-inducible β-cell-specific IGF2R knockout mice exhibited impaired glucose tolerance and blunted insulin secretion after high-fat-diet loading that was likely secondary to reduced β-cell mass due to attenuated proliferation. β-cells with IGF2RKD had fewer autophagosomes after starvation and reduced expression of p62, LC3B, and ULK1. Aged mice also had impaired autophagy in βIGF2R-deficient β-cells. Reduced IGF2R function and N6-methyladenosine (m6A) mRNA methylation were observed in islets from both mouse and human type 2 diabetes. Taken together, these data point to IGF2R as an important regulator of insulin secretion, cell proliferation, and autophagy in mammalian β-cells.

The presence of macrophages surrounding lipotoxic tubular epithelial cells (TECs) is a hallmark of diabetic nephropathy (DN). Nevertheless, the mechanisms of communication between these cell types are not well understood. Previous studies have revealed a unique subset of macrophages that express triggering receptor expressed on myeloid cells 2 (Trem2) in kidneys of human patients and mice with DN. Here, we explored the characteristics and the function of Trem2+ macrophages in the progress of DN. RNA-sequencing of macrophages in kidneys of Trem2 knockout (KO) mice fed a high-fat diet plus streptozotocin (HFD/STZ) revealed functional enrichment of metabolic processes, cytokine production, positive regulation of extracellular signal-regulated kinase (ERK) cascades, and the regulation of phagocytosis. In vivo studies demonstrated that Trem2+ macrophages reduced lipid accumulation and mitigated ferroptosis of TECs in diabetic mice. Mechanistically, Trem2-deficient macrophages amplified the production of interleukin-1β (IL-1β) through activating the ERK signaling pathway. Furthermore, IL-1β triggered CD36 expression via the transcription factor NF-κB. Bioinformatics and functional assays showed NF-κB binds the CD36 promoter, which directly bound to the promoters of CD36 to facilitate its transcription. Inhibition of NF-κB blocked IL-1β-induced CD36 production. This mechanism is exacerbated in Trem2-deficient macrophages, which release excess IL-1β to activate NF-κB in tubular cells, promoting CD36-dependent lipid uptake and ferroptosis. Additionally, we found Trem2 plays a role in enhancing the phagocytosis and clearance of ferroptotic cells by bone marrow-derived macrophages. Altogether, our results suggest Trem2+ macrophages maintain homeostasis of the renal microenvironment and exert a protective function in DN.

Osteoprotegerin (OPG) has emerged as a pivotal factor in metabolic disease pathology; however, its role in hepatic glucose metabolism remains poorly understood. We demonstrated a pronounced reduction in hepatic OPG expression in obese mice, attributable to DNA hypermethylation mediated by DNMT3α. Opg overexpression in the liver reduced the basal metabolic rate and exacerbated glucose metabolism disorders in mice with high-fat diet-induced obesity, whereas Opg knockout yielded opposite effects. Furthermore, mammalian target of rapamycin complex 1 (mTORC1; Raptor)/S6K1/IRS1/AKT signaling was found to be required for the regulation of hepatic glucose metabolism. Mechanistic investigations revealed that OPG interacts with Raptor within the mTORC1 complex, facilitating its phosphorylation at Ser863 and Ser877, influencing the mTORC1 (Raptor)/S6K1/IRS1/AKT signaling pathway, and thereby affecting glucose metabolism and insulin sensitivity. These findings underscore the integral role of OPG in glucose homeostasis and suggest it as a novel therapeutic target in type 2 diabetes.

Heterozygous inactivating mutations in the glucokinase (GCK) gene cause maturity-onset diabetes of the young (GCK-MODY). We identified a novel variant of uncertain significance in the GCK gene (c.77A>T, p.Q26L) in two family members exhibiting contrasting diabetic phenotypes. To explore the diabetogenic potential of the GCK-Q26L mutation and investigate the mono- and polygenetic factors contributing to different phenotypes, whole-exome sequencing and polygenic risk score (PRS) assessments were conducted on three family members. We found that the proband inherited the GCK-Q26L mutation from her father (who had mild, stable hyperglycemia) but exhibited more severe diabetic symptoms, including polydipsia, polyuria, weight loss, ketosis, and significant dyslipidemia. Genetic analysis linked the proband's severe phenotypes to her high PRS for insulin resistance (IR) and type 2 diabetes. A global knock-in mouse model expressing GCK-Q26L presented mild hyperglycemia, impaired glucose tolerance, reduced serum insulin, and impaired glucose-stimulated insulin secretion. Both dorzagliatin and liraglutide improved glucose tolerance and insulin secretion in mutant mice. This study demonstrates that GCK-Q26L is a pathogenic GCK-MODY mutation, and its associated phenotypes are influenced by PRS for IR and type 2 diabetes.

Subcellular lipid accumulation and intermuscular adipose tissue (IMAT) accumulation are associated with insulin resistance, but the impact of combined weight loss and exercise training on localization of lipids and IMAT cellular composition is not known. Twenty-one adults with obesity (18 female and 3 male; 46 ± 2 years; 35.0 ± 0.9 kg/m2) completed a 3-month supervised weight loss and exercise training intervention. Insulin sensitivity was measured using a hyperinsulinemic-euglycemic clamp, and basal and insulin-stimulated vastus lateralis biopsies were collected pre- and postintervention. After the intervention, body weight and body fat decreased (11 ± 1% and 9 ± 1%, respectively), while VO2 peak and insulin sensitivity increased (14 ± 3% and 68 ± 14%, respectively). Lipidomics revealed reduced sarcolemmal and nuclear triglycerides, with unchanged whole-muscle triglycerides. Whole-muscle diacylglycerols increased because of increased nuclear 1,2-diacylglycerols without PKCε, PKCθ, or PKCδ activation. Whole-muscle sphingolipid levels increased because of cytosolic accumulation. Single-nuclei RNA sequencing showed altered IMAT cellular composition, including increased fibro-adipogenic progenitors, vascular cells, and macrophages, and decreased preadipocytes. Bulk muscle RNA sequencing indicated upregulation of genes related to muscle remodeling and cellular respiration, and there were changes in the relationship between nuclear diacylglycerols and gene expression postintervention. These findings dissociate improvements in insulin sensitivity from total muscle diacylglycerol and sphingolipid levels and highlight roles for subcellular lipid redistribution and IMAT remodeling in insulin sensitization.

Hypoglycemia is a preventable adverse treatment effect in diabetes patients, but genetic markers to identify those with increased susceptibility are lacking. We performed a case/control genome-wide association study (GWAS) of hypoglycemia in U.S. Million Veteran Program (MVP) participants with medication-treated diabetes. Case participants had an outpatient random serum/plasma glucose <70 mg/dL or an emergency department visit for hypoglycemia. GWAS was stratified by race/ethnicity, adjusted for age at MVP enrollment, sex, and top 10 population-specific principal components, followed by multipopulation meta-analysis. Secondary analyses examined genetic associations with hypoglycemia stratified by diabetes medication exposure as well as replication in UK Biobank and the Action to Control Cardiovascular Risk in Diabetes clinical trial. The study included 72,244 (22,045 case participants) non-Hispanic White participants, 24,162 (10,441 case participants) non-Hispanic Black participants, and 9,196 (2,800 case participants) Hispanic participants. Four loci had genome-wide significant associations with hypoglycemia in multipopulation meta-analysis: rs12712928 (chromosome 2, SIX2/SIX3 locus), rs1064173 (chromosome 6, HLA-DQB1/DQA2 locus), rs35198068 (chromosome 10, TCF7L2 locus), and rs113748381 (chromosome 17, SCL16A11 locus). All four loci replicated in at least one independent cohort, and the magnitude of associations with hypoglycemia varied by diabetes type. Genome-wide analyses may complement candidate pharmacogenetic studies to identify risk markers of adverse drug effects.

Human-centric models of diabetic cardiomyopathy (DbCM) are needed to provide mechanistic insights and translationally relevant therapeutic targets for patients with diabetes. A systems biology approach using insulin resistant (IR) two-dimensional (2D) human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) and three-dimensional (3D) engineered heart tissue (EHT) provides a comprehensive evaluation of dysregulated pathways and determines suitability as a translationally relevant model of DbCM. Culturing hiPSC-CMs in 2D or 3D EHT in IR media induced insulin resistance and activated multiple pathways implicated in DbCM, including metabolic remodeling, mitochondrial dysfunction, extracellular matrix remodeling, endoplasmic reticulum stress, and blunted response to hypoxia, as assessed using transcriptomics and proteomics. Metabolic flux measurements in both IR 2D and 3D platforms demonstrated increased fatty acid oxidation and lipid storage, whereas glucose metabolism was downregulated. Modeling DbCM in 3D EHTs conferred additional metabolic and functional advantages over the 2D hiPSC-CM, demonstrating impaired contractility and muscle architecture. Metformin treatment improved both contractility and metabolic function, demonstrating the utility of IR EHT for drug assessment. In conclusion, IR 2D and 3D hiPSC-CM models effectively capture key DbCM features. However, 3D EHT provides additional insights into physiological and structural modifications. This highlights the potential of IR EHT for both mechanistic studies and drug screening in DbCM.

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.