Postprandial hypotension (PPH) occurs frequently in type 2 diabetes. Metformin has cardiovascular effects independent of its glucose-lowering capacity, which may modulate the risk of PPH. We investigated the effects of metformin on postprandial blood pressure, including PPH events, heart rate, glucose, insulin, glucagon-like peptide 1 (GLP-1), and gastric emptying, in individuals with type 2 diabetes. Metformin attenuated postprandial decrease in blood pressure and reduced PPH events, in association with augmentation of plasma GLP-1, slowed gastric emptying, and increased heart rate, in type 2 diabetes. These findings establish novel cardiovascular effects of metformin that may mitigate the risk of PPH in type 2 diabetes.

Identification of circulating proteins that may play a role in the pathogenesis of type 1 diabetes can provide promising targets for biomarker and drug target identification. Supported by multiple lines of evidence, circulating abundances of CTSH, IL27RA, SIRPG, and PGM1 were associated with the risk of type 1 diabetes. Tissues and cell types with enrichment of target protein-coding gene expression were identified. CTSH, IL27RA, SIRPG, and PGM1 may be explored as biomarkers or drug targets for type 1 diabetes.

Renin-angiotensin system (RAS) activation plays an important role in the progression of diabetic kidney disease (DKD). However, systemic RAS blockade alone is insufficient to reverse DKD progression. We hypothesized that intrarenal renin-angiotensin system (iRAS) activation plays a crucial role in the progression of DKD. We sought to elucidate the role of the iRAS in DKD progression. Selective deletion of angiotensinogen in renal tubules ameliorated the pathological features of DKD. Our study indicates that iRAS inactivation may be a potential approach for preventing DKD disease severity and its progression.

The COVID-19 pandemic has profoundly affected human health; however, the mechanisms underlying its impact on metabolic and vascular systems remain incompletely understood. Clinical evidence suggests that SARS-CoV-2 directly disrupts vascular homeostasis, with perfusion abnormalities observed in various tissues. The pancreatic islet, a key endocrine miniorgan reliant on its microvasculature for optimal function, may be particularly vulnerable. Studies have proposed a link between SARS-CoV-2 infection and islet dysfunction, but the mechanisms remain unclear. Here, we investigated how SARS-CoV-2 spike S1 protein affects human islet microvascular function. Using confocal microscopy and living pancreas slices from organ donors without diabetes, we show that a SARS-CoV-2 spike S1 recombinant protein activates pericytes, key regulators of islet capillary diameter and β-cell function, and induces capillary constriction. These effects are driven by a loss of ACE2 from pericytes' plasma membrane, impairing ACE2 activity and increasing local angiotensin II levels. Our findings highlight islet pericyte dysfunction as a potential contributor to the diabetogenic effects of SARS-CoV-2 and offer new insights into the mechanisms linking COVID-19, vascular dysfunction, and diabetes.

There is an increasing need for new biomarkers to improve prediction of chronic kidney disease (CKD) in individuals with type 2 diabetes (T2D). We aimed to identify blood-based epigenetic biomarkers associated with incident CKD and develop a methylation risk score (MRS) predicting CKD in individuals with newly diagnosed T2D. DNA methylation was analyzed epigenome wide in blood from 487 individuals with newly diagnosed T2D, of whom 88 developed CKD during an 11.5-year follow-up. Weighted Cox regression was used to associate methylation with incident CKD. Weighted logistic models and cross-validation (k = 5) were performed to test whether the MRS could predict CKD. Methylation at 37 sites was associated with CKD development based on a false discovery rate of <5% and absolute methylation differences of ≥5% between individuals with incident CKD and those free of CKD during follow-up. Notably, 15 genes annotated to these sites, e.g., TGFBI, SHISA3, and SLC43A2 (encoding LAT4), have been linked to CKD or related risk factors, including blood pressure, BMI, and estimated glomerular filtration rate. Using an MRS including 37 sites and cross-validation for prediction of CKD, we generated receiver operating characteristic (ROC) curves with an area under the curve (AUC) of 0.82 for the MRS and AUC of 0.87 for the combination of MRS and clinical factors. Importantly, ROC curves including the MRS had significantly better AUCs versus the one only including clinical factors (AUC = 0.72). The combined epigenetic biomarker had high accuracy in identifying individuals free of future CKD (negative predictive value of 94.6%). We discovered a high-performance epigenetic biomarker for predicting CKD, encouraging its potential role in precision medicine, risk stratification, and targeted prevention in T2D.

Advancements in fundus imaging are revealing disruptions in the neurovascular unit in diabetic retinopathy (DR). In the era of anti-vascular endothelial growth factor treatment, a thorough characterization of neurodegeneration is imperative until patients with DR are sufficiently treated. Here, we demonstrate that extracellular mitochondria exacerbate retinal pigment epithelium (RPE) degeneration and inflammation in DR. Extracellular mitochondria increased in the vitreous of patients with DR and were associated with visual impairment but not with proliferative diabetic retinopathy or diabetic macular edema. Animal experiments demonstrated detrimental effects of extracellular mitochondria on RPE and photoreceptors. Lysosomal cell death induced by extracellular mitochondria in RPE cells required mitochondrial DNA but not its pattern recognition receptors. Furthermore, biochemical screening identified candidates for DNA receptors. Among them, DNA-dependent protein kinase was necessary for extracellular mitochondria-induced cell death in both in vitro and in vivo experiments. Extracellular mitochondria further induced interleukin-1β and tumor necrosis factor-α expression in RPE cells in a Toll-like receptor 9-dependent manner. RNA sequencing suggested that extracellular mitochondria exacerbate inflammation by promoting the proliferation and migration of macrophages, at least in part. In summary, extracellular mitochondria are designated as a novel exacerbating factor of RPE degeneration in DR.

Little is known about the population-based mismatch between phenotypic and genetic BMI (BMI-PGM) and its association with type 2 diabetes. We therefore used data from the China Kadoorie Biobank and UK Biobank and calculated BMI-PGM for each participant as the difference between the percentile for adjusted BMI at baseline and the percentile for adjusted polygenic risk score for BMI. Participants were categorized into discordantly low (BMI-PGM < the first quartile), concordant (the first quartile ≤ BMI-PGM < the third quartile), and discordantly high (BMI-PGM ≥ the third quartile) groups. We calculated adjusted hazard ratios (HRs) for the association of BMI-PGM and type 2 diabetes using Cox proportional hazard models in each cohort, and combined HRs using random-effects meta-analyses. During a median follow-up of 12 years for both cohorts, BMI-PGM was associated with the risk of type 2 diabetes, with the discordantly low group showing reduced risk and the discordantly high group showing elevated risk compared with the concordant group, independent of BMI and other conventional risk factors. In addition, normal-weight individuals with discordantly high BMI-PGM faced a higher risk of type 2 diabetes than overweight individuals. These findings suggest that BMI-PGM may play a potential role in reassessing the risk of type 2 diabetes, particularly among normal-weight populations.

Loss-of-function mutations in ATP-sensitive potassium (KATP) channels cause hyperexcitability and insulin hypersecretion, resulting in congenital hyperinsulinism (CHI). Paradoxically, despite the initial insulin hypersecretion, many CHI cases, as well as KATP knockout (KO) animals, eventually "crossover" to undersecretion and even diabetes. Here, we confirm that Sur1 KO islets exhibit higher intracellular concentration of calcium ion ([Ca2+]i) at all concentrations of glucose but show decreased glucose-stimulated insulin secretion. However, when [Ca2+]i is artificially elevated by increasing extracellular [Ca2+], insulin secretion from Sur1 KO islets increases to the same levels as in wild-type (WT) islets. This indicates that a right-shift in [Ca2+]i dependence of insulin secretion, rather than loss of insulin content or intrinsic secretability, is the primary cause for the crossover. Chronic pharmacological inhibition of KATP channel activity by slow release of glibenclamide in pellet-implanted mice causes a very similar crossover to glucose intolerance and impaired insulin secretion seen in Sur1 KO animals. Whole-islet and single-cell transcriptomic analysis reveal markedly reduced Trpm5 in both conditions. Glibenclamide pellet-implanted Trpm5 KO mice also exhibited significant glucose intolerance. However, this was not as severe as in WT animals, which suggests decreased expression of Trpm5 may play a small role in the disruption of insulin secretion with KATP loss.

Despite elucidation of the molecular genetic basis of several lipodystrophy syndromes, molecular defects in some ultra-rare subtypes of familial lipodystrophies remain unidentified. We analyzed whole-exome sequencing (WES) data of four affected and two unaffected females from an undiagnosed autosomal dominant familial partial lipodystrophy (FPL) pedigree and identified only one novel heterozygous variant, p.Ala1603Tyr, in NOTCH3 meeting the filtering criteria. Further analysis of WES data of 222 patients with unexplained FPL identified two unrelated patients with FPL with novel heterozygous (p.Cys1600Tyr and p.Gln1552Pro) NOTCH3 variants. All variants were clustered in the heterodimerization domain of the negative regulatory region of NOTCH3. RNA sequencing and proteomics analysis of skin fibroblasts revealed significantly higher RNA and protein expression of NOTCH3 and activation of widespread senescence pathways in the patients with FPL versus control study participants. NOTCH3 is highly expressed in adipose tissue and plays many crucial roles in developmental patterning, cell fate decisions, regulation of cell survival, and proliferation. We conclude that gain-of-function missense variants in the negative regulatory region of NOTCH3 cause a novel subtype of FPL by activation of senescence pathways. This novel variety of FPL should be considered for patients without obesity but with early- or childhood-onset diabetes.

Lipedema is a lipodystrophic disease that is typically characterized by a marked increase in lower-body subcutaneous adipose tissue that is purported to have increased inflammation and fibrosis, have impaired microvascular/lymphatic circulation, and be resistant to reduction by weight loss therapy. However, these outcomes have not been adequately studied. We evaluated body composition, insulin sensitivity, metabolic health, and adipose tissue biology in women with obesity and lipedema (Obese-LIP) before and after moderate (∼9%) diet-induced weight loss. At baseline, people with Obese-LIP had ∼23% greater leg fat mass, ∼11% lower android-to-gynoid ratio, and ∼48% greater insulin sensitivity (all P < 0.05) than women matched on age, BMI, and whole-body adiposity. In Obese-LIP, macrophage content and expression of genes involved in inflammation and fibrosis were greater, whereas lymph/angiogenesis-related genes were lower in thigh than abdominal subcutaneous adipose tissue. Weight loss improved insulin sensitivity and decreased total fat mass, with similar relative reductions in abdominal and leg fat masses, but without changes in markers of inflammation and fibrosis. These results demonstrate that affected adipose tissue in women with lipedema is characterized by increased inflammation and fibrogenesis, and alterations in lymphatic and vascular biology. Moderate diet-induced weight loss improves metabolic function and decreases lower-body adipose tissue mass.

Transplanted islet functional β-cell mass is measured by β-cell secretory capacity derived from the acute insulin response to glucose-potentiated arginine (AIRpot); however, data are limited beyond 1 year posttransplantation for individuals with type 1 diabetes. We evaluated changes in β-cell secretory capacity in a single-center longitudinal analysis and examined relationships with measures of islet cell hormone metabolism and clinical measures of graft function (mixed-meal tolerance test [MMTT] C-peptide, BETA-2 score, and continuous glucose monitoring [CGM]). Eleven individuals received purified human pancreatic islets over one or two intraportal infusions to achieve insulin independence and were observed over a median of 6 (interquartile range 5-7) years. β-Cell secretory capacity remained stable over 3 years before declining. Fasting glucagon and proinsulin secretory ratios under glucose potentiation were inversely correlated with AIRpot. A functional β-cell mass of 40% normal predicted insulin independence and was strongly predicted by ratio of MMTT C-peptide to glucose and BETA-2 score. A functional β-cell mass of >20% normal predicted excellent glycemic outcomes, including ≤1% time in range <60 mg/dL, ≤2% time in range >180 mg/dL, and ≥90% time in range 70-180 mg/dL. β-Cell replacement approaches should target a functional β-cell mass >40% normal to provide sufficient islet reserve for sustained insulin independence. Ratio of MMTT C-peptide to glucose and BETA-2 score can inform changes in functional β-cell mass in the clinical setting.

The biomarkers connecting obesity and cardiometabolic diseases are not fully understood. We aimed to 1) evaluate the associations between BMI, waist circumference (WC), and ∼5,000 plasma proteins (SomaScan V4), 2) identify protein signatures of BMI and WC, and 3) evaluate the associations between the protein signatures and cardiometabolic health, including metabolically unhealthy obesity and type 2 diabetes incidence in the Singapore Multi-Ethnic Cohort Phase 1 (MEC1). Among 410 BMI-associated and 385 WC-associated proteins, we identified protein signatures of BMI and WC and validated them in an independent data set across two time points and externally in the Atherosclerosis Risk in Communities (ARIC) study. The BMI and WC protein signatures were highly correlated with total and visceral body fat, respectively. Furthermore, the protein signatures were significantly associated with cardiometabolic risk factors and metabolically unhealthy obesity. In prospective analyses, the protein signatures were strongly associated with type 2 diabetes risk in MEC1 (odds ratio per SD increment in WC protein signature 2.84; 95% CI 2.47-3.25) and ARIC (hazard ratio 1.98; 95% CI 1.88-2.08). Our protein signatures have potential uses in the monitoring of metabolically unhealthy obesity.

The accumulation of mitochondria in thermogenic adipose tissue (i.e., brown and beige fat) increases energy expenditure, which can aid in alleviating obesity and metabolic disorders. However, recent studies have shown that knocking out key proteins required to maintain mitochondrial function inhibits the energy expenditure in thermogenic fat, and yet the knockout (KO) mice are unexpectedly protected from developing obesity or metabolic disorders when fed a high-fat diet (HFD). In the current study, nonbiased sequencing-based screening revealed the importance of Yin Yang 1 (YY1) in the transcription of electron transport chain genes and the enhancement of mitochondrial function in thermogenic adipose tissue. Specifically, YY1 adipocyte-null (YAKO) mice showed lower energy expenditure and were intolerant to cold stress. Interestingly, YAKO mice showed alleviation of HFD-induced metabolic disorders, which can be attributed to a suppression of adipose tissue inflammation. Metabolomic analysis revealed that blocking YY1 directed glucose metabolism toward lactate, enhanced the uptake of glutamine, and promoted the production of anti-inflammatory spermidine. Conversely, blocking spermidine production in YAKO mice reversed their resistance to HFD-induced disorders. Thus, although blocking adipocyte YY1 impairs the thermogenesis, it promotes spermidine production, alleviates adipose tissue inflammation, and therefore leads to an uncoupling of adipose tissue energy expenditure from HFD-induced metabolic disorders.

Murine pancreatic endocrinogenesis has been extensively studied, but human data remain scarce due to limited sample availability. Here, we first built a large collection of human embryonic and fetal pancreases covering the first trimester of pregnancy to explore human endocrinogenesis. Using an experimental pipeline combining in toto staining, tissue clearing, and light-sheet fluorescence microscopy, we show that insulin-, glucagon-, and somatostatin-positive cells appear simultaneously at Carnegie stage (CS) 16. This contrasts with rodents, in which glucagon-positive cells appear first, followed by insulin-positive and, finally, somatostatin-positive cells and highlights interspecies differences. We also detected bihormonal endocrine cells in 7 of 9 human pancreases between CS16 and CS18, which were no longer detected at later stages. We observed that cell distribution within human fetal islets resembles adult mouse islets, with a core of β-cells surrounded by α- and δ-cells, differing from a more complex arrangement in adult human islets. This, in connection with the small size of human fetal islets when compared with adult islets, suggests that adult human islets may form by fusion of preexisting islets, in contrast to the mouse fission model. Together, our study provides a detailed and comprehensive description of the spatiotemporal dynamics of human pancreatic endocrinogenesis.

Glucose tolerance improves significantly upon consuming a second, identical meal later in the day (second-meal phenomenon). We previously established that morning hyperinsulinemia primes the liver for increased afternoon hepatic glucose uptake (HGU). Although the route of insulin delivery is an important determinant of the mechanisms by which insulin regulates liver glucose metabolism (direct hepatic vs. indirect insulin action), it is not known whether insulin's delivery route affects the second-meal response. To determine whether morning peripheral insulin delivery (as occurs clinically, i.e., subcutaneously) can enhance afternoon HGU, conscious dogs were treated in the morning with insulin delivered either via the portal vein or peripherally (leg vein), while glucose was infused to maintain euglycemia. Consequently, arterial insulin levels increased similarly in both groups, but relative hepatic insulin deficiency occurred with peripheral insulin delivery. In the afternoon, all animals were challenged with the same hyperinsulinemic-hyperglycemic clamp to simulate identical postprandial-like conditions. The substantial enhancement of HGU in the afternoon caused by morning portal vein insulin delivery was lost when insulin was delivered peripherally. This indicates that morning insulin does not cause the second-meal phenomenon via its indirect actions on the liver but, rather, through direct activation of hepatic insulin signaling.

Thermogenesis of brown adipose tissue (BAT) provides metabolic benefits against pathologic conditions, such as type 2 diabetes, obesity, cardiovascular disease, and cancer. The thermogenic function of BAT relies on mitochondria, but whether mitochondrial remodeling is required for the beneficial effects of BAT remains unclear. We recently identified FAM210A as a BAT-enriched mitochondrial protein essential for cold-induced thermogenesis through the modulation of OPA1-dependent cristae remodeling. Here, we report a key role of FAM210A in the systemic response to a high-fat diet (HFD). We discovered that an HFD suppressed FAM210A expression, associated with excessive OPA1 cleavage in BAT. Ucp1-Cre-driven BAT-specific Fam210a knockout (Fam210aUKO) similarly elevated OPA1 cleavage, accompanied by whitening of BAT. When subjected to an HFD, Fam210aUKO mice gained similar fat mass as sibling control mice but developed glucose intolerance, insulin resistance, and liver steatosis. The metabolic dysfunction was associated with overall increased lipid content in both the liver and BAT. Additionally, Fam210aUKO leads to inflammation in white adipose tissue. These data demonstrate that FAM210A in BAT is necessary for counteracting HFD-induced metabolic dysfunction but not obesity.

Glucose-stimulated β-cells exhibit synchronized calcium dynamics across the islet that recruit β-cells to enhance insulin secretion. Compared with calcium dynamics, the formation and cell-to-cell propagation of electrical signals within the islet are poorly characterized. To determine factors that influence the propagation of electrical activity across the islet underlying calcium oscillations and β-cell synchronization, we used high-resolution complementary metal-oxide-semiconductor multielectrode arrays (CMOS-MEA) to measure voltage changes associated with the membrane potential of individual cells within intact C57BL6 mouse islets. We measured fast (milliseconds, spikes) and slow (seconds, waves) voltage dynamics. Single spike activity and wave signal velocity were both glucose-dependent, but only spike activity was influenced by N-methyl-d-aspartate receptor activation or inhibition. A repeated glucose stimulus revealed a highly responsive subset of cells in spike activity. When islets were pretreated for 72 h with glucolipotoxic medium, the wave velocity was significantly reduced. Network analysis confirmed that in response to glucolipotoxicity the synchrony of islet cells was affected due to slower propagating electrical waves and not due to altered spike activity. In summary, this approach provided novel insight regarding the propagation of electrical activity and the disruption of cell-to-cell communication due to excessive stimulation.

The gold standard for assessing the function of human islets or β-like cells derived from stem cells involves their engraftment under the kidney capsule of hyperglycemic, immunodeficient mice. Current models, such as streptozotocin treatment of severely immunodeficient mice or the NRG-Akita strain, are limited due to unstable and variable hyperglycemia and/or high morbidity. To address these limitations, we developed the IsletTester mouse via CRISPR/Cas9-mediated gene editing of glucokinase (Gck), the glucose sensor of the β-cells, directly in NSG zygotes. IsletTester mice are heterozygous for an Arg345→stop mutation in Gck and present with stable random hyperglycemia (∼250 mg/dL [14 mmol/L]), normal lifespan, and fertility. We demonstrate the utility of this model through functional engraftment of both human islets and human embryonic stem cell-derived β-like cells. The IsletTester mouse will enable the study of human islet biology over time and under different physiological conditions and can provide a useful preclinical platform to determine the functionality of stem cell-derived islet products.

Dysregulation and loss of immune tolerance toward pancreatic β-cell autoantigens are features of type 1 diabetes (T1D). Until recently, life-long insulin injection was the only approved treatment for T1D, but it does not address the underlying disease pathology. The aim for antigen-specific immunotherapy (ASI) is to restore tolerance. ASI holds potential as a new therapeutic strategy for treating autoimmune diseases with well-characterized antigens. Peptide ASI using processing-independent CD4+ T-cell epitopes (PIPs) shows promising results in several autoimmune diseases. Here, we successfully applied the principles of PIP design to the T1D autoantigen glutamate decarboxylase 65 (GAD65). Peptides spanning GAD65 predicted to be pan-HLA-DR binding were selected. Peptide 10 (P10) displayed enriched responses in peripheral blood mononuclear cells from people with T1D. The minimal epitope of the P10 peptide was fine mapped using T-cell hybridomas generated from HLA-DRB1*04:01 transgenic mice. This minimal epitope, P10Sol, was demonstrated, using a novel activation-induced marker assay, to induce tolerance to the parent peptide in the transgenic mice. Finally, we show that GAD65 P10Sol PIP is recognized by CD4+ T cells from people with T1D who possess a range of HLA-DR alleles and, therefore, can be defined as a pan-DR-binding peptide with therapeutic potential.

Differentiation protocols used to generate stem cell-derived islet cells yield heterogenous cell populations. We generated a human embryonic stem cell line that reports insulin- and glucagon-expressing cells in vitro and in vivo without altering their differentiation or function. We showed some insulin- and glucagon-expressing bihormonal cells are cell-autonomously fated to become α-like cells. This reporter cell line can be used to further study and improve stem cell-derived islet differentiation and transplantation.