Protein tyrosine phosphatase N2 (PTPN2) is a type 1 diabetes (T1D) candidate gene identified from human genome-wide association studies. PTPN2 is highly expressed in human and murine islets and becomes elevated upon inflammation and models of T1D, suggesting that PTPN2 may be important for β-cell survival in the context of T1D. To test whether PTPN2 contributed to β-cell dysfunction in an inflammatory environment, we generated a β-cell-specific deletion of Ptpn2 in mice (PTPN2-β knockout [βKO]). Whereas unstressed animals exhibited normal metabolic profiles, low- and high-dose streptozotocin-treated PTPN2-βKO mice displayed hyperglycemia and accelerated death, respectively. Furthermore, cytokine-treated Ptpn2-KO islets resulted in impaired glucose-stimulated insulin secretion, mitochondrial defects, and reduced glucose-induced metabolic flux, suggesting β-cells lacking Ptpn2 are more susceptible to inflammatory stress associated with T1D due to maladaptive metabolic fitness. Consistent with the phenotype, proteomic analysis identified an important metabolic enzyme, ATP-citrate lyase, as a novel PTPN2 substrate.

Glucagon is generally defined as a counterregulatory hormone with a primary role to raise blood glucose concentrations by increasing endogenous glucose production (EGP) in response to hypoglycemia. However, glucagon has long been known to stimulate insulin release, and recent preclinical findings have supported a paracrine action of glucagon directly on islet β-cells that augments their secretion. In mice, the insulinotropic effect of glucagon is glucose dependent and not present during basal euglycemia. To test the hypothesis that the relative effects of glucagon on hepatic and islet function also vary with blood glucose, a group of healthy subjects received glucagon (100 ng/kg) during fasting glycemia or experimental hyperglycemia (∼150 mg/dL) on 2 separate days. During fasting euglycemia, administration of glucagon caused blood glucose to rise due to increased EGP, with a delayed increase of insulin secretion. When given during experimental hyperglycemia, glucagon caused a rapid, threefold increase in insulin secretion, as well as a more gradual increase in EGP. Under both conditions, insulin clearance was decreased in response to glucagon infusion. The insulinotropic action of glucagon, which is proportional to the degree of blood glucose elevation, suggests distinct physiologic roles in the fasting and prandial states.

Diabetes affects the kidneys, and the presence of albuminuria reflects widespread vascular damage and is a risk factor for cardiovascular disease (CVD). Still, the pathophysiological association between albuminuria and CVD remains incompletely understood. Recent advances in noninvasive imaging enable functional assessment of coronary artery pathology and present an opportunity to explore the association between albuminuria and CVD. In this cross-sectional study, we evaluated the presence of subclinical coronary artery pathology in people with type 2 diabetes, free of overt CVD. Using multimodal imaging, we assessed the coronary microcalcification activity (18F-sodium fluoride positron emission tomography/computed tomography [PET/CT], plaque inflammation [64Cu-DOTATATE PET/CT], and myocardial flow reserve [82Rb PET/CT]). The study population consisted of 90 participants, stratified by albuminuria; 60 had historic or current albuminuria (urine albumin-to-creatinine ratio [UACR] ≥30 mg/g]), and 30 had normoalbuminuria (UACR <30 mg/g). We demonstrated that any albuminuria (historic or current) was associated with a more severe phenotype, in particular, higher levels of microcalcifications and impaired myocardial microvascular function; however, coronary inflammation activity was similar in people with and without albuminuria. Our findings establish a potential underlying mechanism connecting cardiovascular and kidney diseases and could indicate the initial stages of the cardiorenal syndrome.

Short-chain fatty acids (SCFAs) have been extensively studied for potential beneficial roles in glucose homeostasis and risk of diabetes; however, most of this research has focused on butyrate, acetate, and propionate. The effect on metabolism of branched SCFAs (BSCFAs; isobutyrate, isovalerate, and methylbutyrate) is largely unknown. In a cohort of 219 non-Hispanic White participants and 126 African American participants, we examined the association of BSCFA with dysglycemia (prediabetes and diabetes) and oral glucose tolerance test-based measures of glucose and insulin homeostasis, as well as with demographic, anthropometric, lifestyle, and lipid traits, and other SCFAs. We observed a bimodal distribution of BSCFAs, with 25 individuals having high levels (H-BSCFA group) and 320 individuals having lower levels (L-BSCFA group). The prevalence of dysglycemia was lower in the H-BSCFA group compared with the L-BSCFA group (16% vs. 49%; P = 0.0014). This association remained significant after adjustment for age, sex, race, BMI, and levels of other SCFAs. Consistent with the lower rate of dysglycemia, fasting and postprandial glucose levels were lower and the disposition index was higher in the H-BSCFA group. Additional findings in H-BSCFA versus L-BSCFA included lower fasting and postprandial C-peptide levels and lower insulin clearance without differences in insulin levels, insulin sensitivity, insulin secretion, or other variables examined, including diet and physical activity. As one of the first human studies associating higher BSCFA levels with lower odds of dysglycemia and improved glucose homeostasis, this study sets the stage for further investigation of BSCFA as a novel target for prevention or treatment of diabetes.

Vascular inflammation is known to cause degeneration of retinal capillaries in early diabetic retinopathy (DR), a major microvascular complication of diabetes. Past studies investigating these diabetes-induced retinal vascular abnormalities have focused primarily on the role of molecular or biochemical cues. Here we show that retinal vascular inflammation and degeneration in diabetes are also mechanically regulated by the increase in retinal vascular stiffness caused by overexpression of the collagen-cross-linking enzyme lysyl oxidase (LOX). Treatment of diabetic mice with LOX inhibitor β-aminopropionitrile (BAPN) prevented the increase in retinal capillary stiffness, vascular intracellular adhesion molecule-1 overexpression, and leukostasis. Consistent with these anti-inflammatory effects, BAPN treatment of diabetic mice blocked the upregulation of proapoptotic caspase-3 in retinal vessels, which concomitantly reduced retinal capillary degeneration, pericyte ghost formation, and the diabetes-induced loss of contrast sensitivity in these mice. Finally, our in vitro studies indicate that retinal capillary stiffening is sufficient to increase the adhesiveness and neutrophil elastase-induced death of retinal endothelial cells. By uncovering a link between LOX-dependent capillary stiffening and the development of retinal vascular and functional defects in diabetes, these findings offer a new insight into DR pathogenesis that has important translational potential.

In type 1 diabetes, the reduced glucagon response to insulin-induced hypoglycemia has been used to argue that β-cell secretion of insulin is required for the full glucagon counterregulatory response. For years, the concept has been that insulin from the β-cell core flows downstream to suppress glucagon secretion from the α-cells in the islet mantle. This core-mantle relationship has been supported by perfused pancreas studies that show marked increases in glucagon secretion when insulin was neutralized with antisera. Additional support comes from a growing number of studies focused on vascular anatomy and blood flow. However, in recent years this core-mantle view has generated less interest than the argument that optimal insulin secretion is due to paracrine release of glucagon from α-cells stimulating adjacent β-cells. This mechanism has been evaluated by knockout of β-cell receptors and impairment of α-cell function by inhibition of Gi designer receptors exclusively activated by designer drugs. Other studies that support this mechanism have been obtained by pharmacological blocking of glucagon-like peptide 1 receptor in humans. While glucagon has potent effects on β-cells, there are concerns with the suggested paracrine mechanism, since some of the supporting data are from isolated islets. The study of islets in static incubation or perifusion systems can be informative, but the normal paracrine relationships are disrupted by the isolation process. While this complicates interpretation of data, arguments supporting paracrine interactions between α-cells and β-cells have growing appeal. We discuss these conflicting views of the relationship between pancreatic α-cells and β-cells and seek to understand how communication depends on blood flow and/or paracrine mechanisms.

Endothelial cells (EC) play essential roles in retinal vascular homeostasis. This study aimed to characterize retinal EC heterogeneity and functional diversity using single-cell RNA sequencing. Systematic analysis of cellular compositions and cell-cell interaction networks identified a unique EC cluster with high inflammatory gene expression in diabetic retina; sphingolipid metabolism is a prominent aspect correlated with changes in retinal function. Among sphingolipid-related genes, alkaline ceramidase 2 (ACER2) showed the most significant increase. Plasma samples of patients with nonproliferative diabetic retinopathy (NPDR) with diabetic macular edema (DME) or without DME (NDME) and active proliferative DR (PDR) were collected for mass spectrometry analysis. Metabolomic profiling revealed that the ceramide levels were significantly elevated in NPDR-NDME/DME and further increased in active PDR compared with control patients. In vitro analyses showed that ACER2 overexpression retarded endothelial barrier breakdown induced by ceramide, while silencing of ACER2 further disrupted the injury. Moreover, intravitreal injection of the recombinant ACER2 adeno-associated virus rescued diabetes-induced vessel leakiness, inflammatory response, and neurovascular disease in diabetic mouse models. Together, this study revealed a new diabetes-specific retinal EC population and a negative feedback regulation pathway that reduces ceramide content and endothelial dysfunction by upregulating ACER2 expression. These findings provide insights into cell-type targeted interventions for diabetic retinopathy.

More than 30% of patients with type 1 diabetes develop diabetic kidney disease (DKD), which significantly increases mortality risk. The Diabetes Control and Complications Trial (DCCT) and follow-up study, Epidemiology of Diabetes Interventions and Complications (EDIC), established that glycemic control measured by HbA1c predicts DKD risk. However, the continued high incidence of DKD reinforces the urgent need for additional biomarkers to supplement HbA1c. Here, we assessed biomarkers induced by methylglyoxal (MG), a metabolic by-product that forms covalent adducts on DNA, RNA, and proteins, called MG adducts. Urinary MG adducts were measured in samples from patients with type 1 diabetes enrolled in DCCT/EDIC who did (case patients; n = 90) or did not (control patients; n = 117) develop DKD. Univariate and multivariable analyses revealed that measurements of MG adducts independently predict DKD before established DKD biomarkers such as glomerular filtration rate and albumin excretion rate. Elevated levels of MG adducts bestowed the greatest risk of developing DKD in a multivariable model that included HbA1c and other clinical covariates. Our work establishes a novel class of biomarkers to predict DKD risk and suggests that inclusion of MG adducts may be a valuable tool to improve existing predictors of complications like DKD prior to overt disease, and to aid in identifying at-risk individuals and personalized risk management.

The important role of m6A RNA modification in β-cell function has been established; however, how it regulates pancreatic development and endocrine differentiation remains unknown. Here, we generated transgenic mice lacking RNA methyltransferase-like 3 (Mettl3) specifically in Pdx1+ pancreatic progenitor cells and found the mice with the mutation developed hyperglycemia and hypoinsulinemia at age 2 weeks, along with an atrophic pancreas, reduced islet mass, and abnormal increase in ductal formation. At embryonic day 15.5, Mettl3 deletion had caused a significant loss of Ngn3+ endocrine progenitor cells, which was accompanied by increased Sox9+ ductal precursor cells. We identified histone deacetylase 1 (Hdac1) as the critical direct m6A target in bipotent progenitors, the degeneration of which caused abnormal activation of the Wnt/Notch signaling pathway and blocked endocrine differentiation. This transformation could be manipulated in embryonic pancreatic culture in vitro through regulation of the Mettl3-Hdac1-Wnt/Notch signaling axis. Our finding that Mettl3 determines endocrine lineage by modulating Hdac1 activity during the transition of bipotent progenitors might help in the development of targeted endocrine cell protocols for diabetes treatment.

In humans, glucocorticoids (GCs) are commonly prescribed because of their anti-inflammatory and immunosuppressive properties. However, high doses of GCs often lead to side effects, including diabetes and lipodystrophy. We recently reported that adipocyte glucocorticoid receptor (GR)-deficient (AdipoGR-KO) mice under corticosterone (CORT) treatment exhibited a massive adipose tissue (AT) expansion associated with a paradoxical improvement of metabolic health compared with control mice. However, whether GR may control adipose development remains unclear. Here, we show a specific induction of hypoxia-inducible factor 1α (HIF-1α) and proangiogenic vascular endothelial growth factor A (VEGFA) expression in GR-deficient adipocytes of AdipoGR-KO mice compared with control mice, together with an increased adipose vascular network, as assessed by three-dimensional imaging. GR activation reduced HIF-1α recruitment to the Vegfa promoter resulting from Hif-1α downregulation at the transcriptional and posttranslational levels. Importantly, in CORT-treated AdipoGR-KO mice, the blockade of VEGFA by a soluble decoy receptor prevented AT expansion and the healthy metabolic phenotype. Finally, in subcutaneous AT from patients with Cushing syndrome, higher VEGFA expression was associated with a better metabolic profile. Collectively, these results highlight that adipocyte GR negatively controls AT expansion and metabolic health through the downregulation of the major angiogenic effector VEGFA and inhibition of vascular network development.

Enhancing the development of and thermogenesis in brown and beige fat represents a potential treatment for obesity. In this study, we show that Foxj3 expression in fat is stimulated by cold exposure and a β-adrenergic agonist. Adipose-specific Foxj3 knockout impaired the thermogenic function of brown fat, leading to morphological whitening of brown fat and obesity. Adipose Foxj3-deficient mice displayed increased fasting blood glucose levels and hepatic steatosis while on a chow diet. Foxj3 deficiency inhibited the browning of inguinal white adipose tissue (iWAT) following β3-agonist treatment of mice. Furthermore, depletion of Foxj3 in primary brown adipocytes reduced the expression of thermogenic genes and cellular respiration, indicating that the Foxj3 effects on the thermogenic program are cell autonomous. In contrast, Foxj3 overexpression in primary brown adipocytes enhanced the thermogenic program. Moreover, AAV-mediated Foxj3 overexpression in brown fat and iWAT increased energy expenditure and improved systemic metabolism on either a chow or high-fat diet. Finally, Foxj3 deletion in fat inhibited the β3-agonist-mediated induction of WAT browning and brown adipose tissue thermogenesis. Mechanistically, cold-inducible Foxj3 stimulated the expression of PGC-1α and UCP1, subsequently promoting energy expenditure. This study identifies Foxj3 as a critical regulator of fat thermogenesis, and targeting Foxj3 in fat might be a therapeutic strategy for treating obesity and metabolic diseases.

SGLT2 inhibitors have been shown to provide pronounced reductions in cardiorenal outcomes, including cardiovascular death, heart failure, and renal failure. The mechanisms underlying these benefits remain uncertain. We hypothesized that the effects could be attributed to the elevated glycosuria induced by these drugs. Urine concentrations of glucose, creatinine, and ketones were measured at baseline and after 1 year of treatment with either placebo or canagliflozin 100 mg/day, in approximately 2,600 individuals from the Canagliflozin and Renal Events in Diabetes with Established Nephropathy Clinical Evaluation (CREDENCE) trial (enrolling patients with type 2 diabetes, chronic kidney disease (CKD), and albuminuria). Associations between glycosuria and the primary composite end point from CREDENCE, and secondary outcomes were assessed using Cox proportional hazards models. Canagliflozin treatment increased fractional urinary glucose excretion (± SD) from 3 ± 9% at baseline to 30 ± 26% at year 1 (vs. 5 ± 19% with placebo; P < 0.001). Patients in the canagliflozin arm and in the top quartile of urine glucose to creatinine ratio at year 1 were significantly protected for the primary end point (hazard ratio [HR] 0.42; 95% CI 0.30-0.61); similar results were seen for cases of hospitalized heart failure (HR 0.45; 95% CI 0.27-0.73) and all-cause death (HR 0.56; 95% CI 0.39-0.80). These associations persisted when adjustments were made for multiple conventional risk factors. Among patients with type 2 diabetes and CKD treated with canagliflozin, individuals with the highest glycosuria levels had the strongest protection against multiple cardiorenal outcomes.

People with type 2 diabetes frequently use low-calorie sweeteners to manage glycemia and reduce caloric intake. Use of erythritol, a low-calorie sweetener, has increased recently. Higher circulating concentration associates with major cardiac events and metabolic disease in observational data, prompting some concern. As observational data may be prone to confounding and reverse causality, we undertook bidirectional Mendelian randomization (MR) to investigate potential causal associations between erythritol and coronary artery disease (CAD), BMI, waist-hip-ratio (WHR), and glycemic and renal traits in cohorts of European ancestry. Analyses were undertaken using instruments comprising genome-wide significant variants from three cohorts with erythritol measurement. Across instruments, we did not find supportive evidence that increased erythritol increases CAD (b = -0.033 ± 0.02, P = 0.14; b = 0.46 ± 0.37, P = 0.23). MR indicates erythritol may decrease BMI (b = -0.04 ± 0.018, P = 0.03; b = -0.04 ± 0.0085, P = 1.23 × 10-5; b = -0.083 ± 0.092, P = 0.036), with potential evidence from one instrument of increased BMI adjusted for WHR (b = 0.046 ± 0.022, P = 0.035). No evidence of causal association was found with other traits. In conclusion, we did not find supportive evidence from MR that erythritol increases cardiometabolic disease. These findings await confirmation in well-designed prospective studies.

Partial leptin reduction can induce significant weight loss, while weight loss contributes to partial leptin reduction. The cause-and-effect relationship between leptin reduction and weight loss remains to be further elucidated. Here, we show that FGF21 and the glucagon-like peptide 1 receptor (GLP-1R) agonist liraglutide rapidly induced a reduction in leptin. This leptin reduction contributed to the beneficial effects of GLP-1R agonism in metabolic health, as transgenically maintaining leptin levels during treatment partially curtailed the beneficial effects seen with these agonists. Moreover, a higher degree of leptin reduction during treatment, induced by including a leptin neutralizing antibody with either FGF21 or liraglutide, synergistically induced greater weight loss and better glucose tolerance in diet-induced obese mice. Furthermore, upon cessation of either liraglutide or FGF21 treatment, the expected immediate weight regain was observed, associated with a rapid increase in circulating leptin levels. Prevention of this leptin surge with leptin neutralizing antibodies slowed down weight gain and preserved better glucose tolerance. Mechanistically, a significant reduction in leptin induced a higher degree of leptin sensitivity in hypothalamic neurons. Our observations support a model that postulates that a reduction of leptin levels is a necessary prerequisite for substantial weight loss, and partial leptin reduction is a viable strategy to treat obesity and its associated insulin resistance.

Diabetic nephropathy (DN) is the leading cause of end-stage renal disease in the U.S. and has a significant impact on human suffering. Leptin-deficient BTBR (BTBRob/ob) mice develop hallmark features of obesity-induced DN, whereas leptin-deficient C57BL/6J (B6ob/ob) mice do not. To identify genetic loci that underlie this strain difference, we constructed an F2 intercross between BTBRob/ob and B6ob/ob mice. We isolated kidneys from 460 F2 mice and histologically scored them for percent mesangial matrix and glomerular volume (∼50 glomeruli per mouse), yielding ∼45,000 distinct measures in total. The same histological measurements were made in kidneys from B6 and BTBR mice, either lean or obese (Lepob/ob), at 4 and 10 weeks of age, allowing us to assess the contribution of strain, age, and obesity to glomerular pathology. All F2 mice were genotyped for ∼5,000 single nucleotide polymorphisms (SNPs), ∼2,000 of which were polymorphic between B6 and BTBR, enabling us to identify a quantitative trait locus (QTL) on chromosome 7, with a peak at ∼30 Mbp, for percent mesangial matrix, glomerular volume, and mesangial volume. The podocyte-specific gene nephrin (Nphs1) is physically located at the QTL and contains high-impact SNPs in BTBR, including several missense variants within the extracellular immunoglobulin-like domains.

Habitual physical activity (PA) impacts the plasma proteome and reduces the risk of developing type 2 diabetes (T2D). Using a large-scale proteome-wide approach in Atherosclerosis Risk in Communities study participants, we aimed to identify plasma proteins associated with PA and determine which of these may be causally related to lower T2D risk. PA was associated with 92 plasma proteins in discovery (P < 1.01 × 10-5), and 40 remained significant in replication (P < 5.43 × 10-4). Eighteen of these proteins were independently associated with incident T2D (P < 1.25 × 10-3), including neuronal growth regulator 1 (NeGR1; hazard ratio per SD 0.85; P = 7.5 × 10-11). Two-sample Mendelian randomization (MR) inverse variance weighted analysis indicated that higher NeGR1 reduces T2D risk (odds ratio [OR] per SD 0.92; P = 0.03) and was consistent with MR-Egger, weighted median, and weighted mode sensitivity analyses. A stronger association was observed for the single cis-acting NeGR1 genetic variant (OR per SD 0.80; P = 6.3 × 10-5). Coupled with previous evidence that low circulating NeGR1 levels promote adiposity, its association with PA and potential causal role in T2D shown here suggest that NeGR1 may link PA exposure with metabolic outcomes. Further research is warranted to confirm our findings and examine the interplay of PA, NeGR1, adiposity, and metabolic health.

HLA-DR/DQ haplotypes largely define genetic susceptibility to type 1 diabetes (T1D). The DQB1*06:02-positive haplotype (DR15-DQ602) common in individuals of European ancestry is very rare among children with T1D. Among 4,490 children with T1D in the Finnish Pediatric Diabetes Register, 57 (1.3%) case patients with DQB1*06:02 were identified, in comparison with 26.1% of affected family-based association control participants. There were no differences between DQB1*06:02-positive and -negative children with T1D regarding sex, age, islet autoantibody distribution, or autoantibody levels, but significant differences were seen in the frequency of second class II HLA haplotypes. The most prevalent haplotype present with DQB1*06:02 was DRB1*04:04-DQA1*03-DQB1*03:02, which was found in 27 (47.4%) of 57 children, compared with only 797 (18.0%) of 4,433 among DQB1*06:02-negative case patients (P < 0.001 by χ2 test). The other common risk-associated haplotypes, DRB1*04:01-DQA1*03-DQB1*03:02 and (DR3)-DQA1*05-DQB1*02, were less prevalent in DQB1*06:02-positive versus DQB1*06:02-negative children (P < 0.001). HLA-B allele frequencies did not differ by DQB1*06:02 haplotype between children with T1D and control participants or by DRB1*04:04-DQA1*03-DQB1*03:02 haplotype between DQB1*06:02-positive and -negative children with T1D. The increased frequency of the DRB1*04:04 allele among DQB1*06:02-positive case patients may indicate a preferential ability of the DR404 molecule to present islet antigen epitopes despite competition by DQ602.

Metabolic dysfunction-associated steatotic liver disease (MASLD, formerly known as nonalcoholic fatty liver disease [NAFLD]) and metabolic dysfunction-associated steatohepatitis (MASH, formerly known as nonalcoholic steatohepatitis [NASH]) are leading chronic liver diseases, driving cirrhosis, hepatocellular carcinoma, and mortality. MASLD/MASH is associated with increased senescence proteins, including Activin A, and senolytics have been proposed as a therapeutic approach. To test the role of Activin A, we induced hepatic expression of Activin A in a murine MASLD/MASH model. Surprisingly, overexpression of hepatic Activin A dramatically mitigated MASLD, reducing liver steatosis and inflammation as well as systemic fat accumulation, while improving insulin sensitivity. Further studies identified a dramatic decrease in the lipid-associated macrophages marker glycoprotein NMB (Gpnmb) by Activin A, and Gpnmb knockdown in the same model produced similar benefits and transcriptional changes to Activin A expression. These studies reveal a surprising protective role for Activin A in MASLD and the potential for SASP proteins to have context-specific beneficial effects. Moreover, they implicate both Activin A and Gpnmb as potential therapeutic targets for this condition.