This study aims to model genetic, immunologic, metabolomics, and proteomic biomarkers for development of islet autoimmunity (IA) and progression to type 1 diabetes in a prospective high-risk cohort. We studied 67 children: 42 who developed IA (20 of 42 progressed to diabetes) and 25 control subjects matched for sex and age. Biomarkers were assessed at four time points: earliest available sample, just prior to IA, just after IA, and just prior to diabetes onset. Predictors of IA and progression to diabetes were identified across disparate sources using an integrative machine learning algorithm and optimization-based feature selection. Our integrative approach was predictive of IA (area under the receiver operating characteristic curve [AUC] 0.91) and progression to diabetes (AUC 0.92) based on standard cross-validation (CV). Among the strongest predictors of IA were change in serum ascorbate, 3-methyl-oxobutyrate, and the PTPN22 (rs2476601) polymorphism. Serum glucose, ADP fibrinogen, and mannose were among the strongest predictors of progression to diabetes. This proof-of-principle analysis is the first study to integrate large, diverse biomarker data sets into a limited number of features, highlighting differences in pathways leading to IA from those predicting progression to diabetes. Integrated models, if validated in independent populations, could provide novel clues concerning the pathways leading to IA and type 1 diabetes.

Active maintenance of β-cell identity through fine-tuned regulation of key transcription factors ensures β-cell function. Tacrolimus, a widely used immunosuppressant, accelerates onset of diabetes after organ transplantation, but underlying molecular mechanisms are unclear. Here we show that tacrolimus induces loss of human β-cell maturity and β-cell failure through activation of the BMP/SMAD signaling pathway when administered under mild metabolic stress conditions. Tacrolimus-induced phosphorylated SMAD1/5 acts in synergy with metabolic stress-activated FOXO1 through formation of a complex. This interaction is associated with reduced expression of the key β-cell transcription factor MAFA and abolished insulin secretion, both in vitro in primary human islets and in vivo in human islets transplanted into high-fat diet-fed mice. Pharmacological inhibition of BMP signaling protects human β-cells from tacrolimus-induced β-cell dysfunction in vitro. Furthermore, we confirm that BMP/SMAD signaling is activated in protocol pancreas allograft biopsies from recipients on tacrolimus. To conclude, we propose a novel mechanism underlying the diabetogenicity of tacrolimus in primary human β-cells. This insight could lead to new treatment strategies for new-onset diabetes and may have implications for other forms of diabetes.

The ability to switch fuels for oxidation in response to changes in macronutrient composition of diet (metabolic flexibility) may be informative of individuals' susceptibility to weight gain. Seventy-nine healthy, weight-stable participants underwent 24-h assessments of energy expenditure and respiratory quotient (RQ) in a whole-room calorimeter during energy balance (EBL) (50% carbohydrate, 30% fat) and then during 24-h fasting and three 200% overfeeding diets in a crossover design. Metabolic flexibility was defined as the change in 24-h RQ from EBL during fasting and standard overfeeding (STOF) (50% carbohydrate, 30% fat), high-fat overfeeding (HFOF) (60% fat, 20% carbohydrate), and high-carbohydrate overfeeding (HCOF) (75% carbohydrate, 5% fat) diets. Free-living weight change was assessed after 6 and 12 months. Compared with EBL, RQ decreased on average by 9% during fasting and by 4% during HFOF but increased by 4% during STOF and by 8% during HCOF. A smaller decrease in RQ, reflecting a smaller increase in lipid oxidation rate, during HFOF but not during the other diets predicted greater weight gain at both 6 and 12 months. An impaired metabolic flexibility to acute HFOF can identify individuals prone to weight gain, indicating that an individual's capacity to oxidize dietary fat is a metabolic determinant of weight change.

Optimal immune-based therapies for type 1 diabetes (T1D) should restore self-tolerance without inducing chronic immunosuppression. CD4+Foxp3+ regulatory T cells (Tregs) are a key cell population capable of facilitating durable immune tolerance. However, clinical trials with expanded Tregs in T1D and solid-organ transplant recipients are limited by poor Treg engraftment without host manipulation. We showed that Treg engraftment and therapeutic benefit in nonautoimmune models required ablative host conditioning. Here, we evaluated Treg engraftment and therapeutic efficacy in the nonobese diabetic (NOD) mouse model of autoimmune diabetes using nonablative, combinatorial regimens involving the anti-CD3 (αCD3), cyclophosphamide (CyP), and IAC (IL-2/JES6-1) antibody complex. We demonstrate that αCD3 alone induced substantial T-cell depletion, impacting both conventional T cells (Tconv) and Tregs, subsequently followed by more rapid rebound of Tregs Despite robust depletion of host Tconv and host Tregs, donor Tregs failed to engraft even with interleukin-2 (IL-2) support. A single dose of CyP after αCD3 depleted rebounding host Tregs and resulted in a 43-fold increase in donor Treg engraftment, yet polyclonal donor Tregs failed to reverse diabetes. However, infusion of autoantigen-specific Tregs after αCD3 alone resulted in robust Treg engraftment within the islets and induced remission in all mice. This novel combinatorial therapy promotes engraftment of autoantigen-specific donor Tregs and controls islet autoimmunity without long-term immunosuppression.

The whitening and loss of brown adipose tissue (BAT) during obesity and aging promote metabolic disorders and related diseases. The imbalance of Ca2+ homeostasis accounts for the dysfunction and clearance of mitochondria during BAT whitening. Capsaicin, a dietary factor activating TRPV1, can inhibit obesity induced by high-fat diet (HFD), but whether capsaicin inhibits BAT loss and the underlying mechanism remain unclear. In this study, we determined that the inhibitory effects of capsaicin on HFD-induced obesity and BAT whitening were dependent on the participation of SIRT3, a critical mitochondrial deacetylase. SIRT3 also mediated all of the beneficial effects of capsaicin on alleviating reactive oxygen species generation, elevating mitochondrial activity, and restricting mitochondrial calcium overload induced by HFD. Mechanistically, SIRT3 inhibits mitochondrial calcium uniporter (MCU)-mediated mitochondrial calcium overload by reducing the H3K27ac level on the MCU promoter in an AMPK-dependent manner. In addition, HFD also inhibits AMPK activity to reduce SIRT3 expression, which could be reversed by capsaicin. Capsaicin intervention also inhibited aging-induced BAT whitening through this mechanism. In conclusion, this study emphasizes a critical role of the AMPK/SIRT3 pathway in the maintenance of BAT morphology and function and suggests that intervention in this pathway may be an effective target for preventing obesity- or age-related metabolic diseases.

Adipose tissue macrophages (ATMs) are involved in the development of insulin resistance in obesity. We have recently shown that myeloid cell-specific reduction of HMG-CoA reductase (Hmgcrm-/m- ), which is the rate-limiting enzyme in cholesterol biosynthesis, protects against atherosclerosis by inhibiting macrophage migration in mice. We hypothesized that ATMs are harder to accumulate in Hmgcrm-/m- mice than in control Hmgcrfl/fl mice in the setting of obesity. To test this hypothesis, we fed Hmgcrm-/m- and Hmgcrfl/fl mice a high-fat diet (HFD) for 24 weeks and compared plasma glucose metabolism as well as insulin signaling and histology between the two groups. Myeloid cell-specific reduction of Hmgcr improved glucose tolerance and insulin sensitivity without altering body weight in the HFD-induced obese mice. The improvement was due to a decrease in the number of ATMs. The ATMs were reduced by decreased recruitment of macrophages as a result of their impaired chemotactic activity. These changes were associated with decreased expression of proinflammatory cytokines in adipose tissues. Myeloid cell-specific reduction of Hmgcr also attenuated hepatic steatosis. In conclusion, reducing myeloid HMGCR may be a promising strategy to improve insulin resistance and hepatic steatosis in obesity.

Insulin-induced hypoglycemia leads to far-ranging negative consequences in patients with diabetes. Components of the counterregulatory response (CRR) system that help minimize and reverse hypoglycemia and coordination between those components are well studied but not yet fully characterized. Here, we tested the hypothesis that acyl-ghrelin, a hormone that defends against hypoglycemia in a preclinical starvation model, is permissive for the normal CRR to insulin-induced hypoglycemia. Ghrelin knockout (KO) mice and wild-type (WT) littermates underwent an insulin bolus-induced hypoglycemia test and a low-dose hyperinsulinemic-hypoglycemic clamp procedure. Clamps also were performed in ghrelin-KO mice and C57BL/6N mice administered the growth hormone secretagogue receptor agonist HM01 or vehicle. Results show that hypoglycemia, as induced by an insulin bolus, was more pronounced and prolonged in ghrelin-KO mice, supporting previous studies suggesting increased insulin sensitivity upon ghrelin deletion. Furthermore, during hyperinsulinemic-hypoglycemic clamps, ghrelin-KO mice required a 10-fold higher glucose infusion rate (GIR) and exhibited less robust corticosterone and growth hormone responses. Conversely, HM01 administration, which reduced the GIR required by ghrelin-KO mice during the clamps, increased plasma corticosterone and growth hormone. Thus, our data suggest that endogenously produced acyl-ghrelin not only influences insulin sensitivity but also is permissive for the normal CRR to insulin-induced hypoglycemia.

Endothelial dysfunction plays a crucial role in the progress of diabetic vasculopathy. C1q/tumor necrosis factor-related protein 13 (CTRP13) is a secreted adipokine that can ameliorate atherosclerosis and vascular calcification. However, the role of CTRP13 in regulating endothelial function in diabetes has yet to be explored. In this study, CTRP13 treatment improved endothelium-dependent relaxation in the aortae and mesenteric arteries of both db/db mice and streptozotocin-injected mice. CTRP13 supplement also rescued the impaired endothelium-dependent relaxation ex vivo in the db/db mouse aortae and in high glucose (HG)-treated mouse aortae. Additionally, CTRP13 treatment reduced reactive oxygen species overproduction and improved nitric oxide (NO) production and endothelial NO synthase (eNOS) coupling in the aortae of diabetic mice and in HG-treated human umbilical vein endothelial cells. Mechanistically, CTRP13 could increase GTP cyclohydrolase 1 (GCH1) expression and tetrahydrobiopterin (BH4) levels to ameliorate eNOS coupling. More importantly, CTRP13 rescued HG-induced inhibition of protein kinase A (PKA) activity. Increased PKA activity enhanced phosphorylation of the peroxisome proliferator-activated receptor α and its recruitment to the GCH1 promoter, thus activating GCH1 transcription and, ultimately, endothelial relaxation. Together, these results suggest that CTRP13 preserves endothelial function in diabetic mice by regulating GCH1/BH4 axis-dependent eNOS coupling, suggesting the therapeutic potential of CTRP13 against diabetic vasculopathy.

It is estimated that ∼1% of European ancestry patients clinically diagnosed with type 1 diabetes (T1D) actually have monogenic forms of the disease. Because of the much lower incidence of true T1D in East Asians, we hypothesized that the percentage would be much higher. To test this, we sequenced the exome of 82 Chinese Han patients clinically diagnosed with T1D but negative for three autoantibodies. Analysis focused on established or proposed monogenic diabetes genes. We found credible mutations in 18 of the 82 autoantibody-negative patients (22%). All mutations had consensus pathogenicity support by five algorithms. As in Europeans, the most common gene was HNF1A (MODY3), in 6 of 18 cases. Surprisingly, almost as frequent were diallelic mutations in WFS1, known to cause Wolfram syndrome but also described in nonsyndromic cases. Fasting C-peptide varied widely and was not predictive. Given the 27.4% autoantibody negativity in Chinese and 22% mutation rate, we estimate that ∼6% of Chinese with a clinical T1D diagnosis have monogenic diabetes. Our findings support universal sequencing of autoantibody-negative cases as standard of care in East Asian patients with a clinical T1D diagnosis. Nonsyndromic diabetes with WSF1 mutations is not rare in Chinese. Its response to alternative treatments should be investigated.

Glucagon and its partner insulin are dually linked in both their secretion from islet cells and their action in the liver. Glucagon signaling increases hepatic glucose output, and hyperglucagonemia is partly responsible for the hyperglycemia in diabetes, making glucagon an attractive target for therapeutic intervention. Interrupting glucagon signaling lowers blood glucose but also results in hyperglucagonemia and α-cell hyperplasia. Investigation of the mechanism for α-cell proliferation led to the description of a conserved liver-α-cell axis where glucagon is a critical regulator of amino acid homeostasis. In return, amino acids regulate α-cell function and proliferation. New evidence suggests that dysfunction of the axis in humans may result in the hyperglucagonemia observed in diabetes. This discussion outlines important but often overlooked roles for glucagon that extend beyond glycemia and supports a new role for α-cells as amino acid sensors.

Statins are cholesterol-lowering agents that increase the incidence of diabetes and impair glucose tolerance via their detrimental effects on nonhepatic tissues, such as pancreatic islets, but the underlying mechanism has not been determined. In atorvastatin (ator)-treated high-fat diet-fed mice, we found reduced pancreatic β-cell size and β-cell mass, fewer mature insulin granules, and reduced insulin secretion and glucose tolerance. Transcriptome profiling of primary pancreatic islets showed that ator inhibited the expression of pancreatic transcription factor, mechanistic target of rapamycin (mTOR) signaling, and small G protein (sGP) genes. Supplementation of the mevalonate pathway intermediate geranylgeranyl pyrophosphate (GGPP), which is produced by 3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) reductase, significantly restored the attenuated mTOR activity, v-maf musculoaponeurotic fibrosarcoma oncogene homolog A (MafA) expression, and β-cell function after ator, lovastatin, rosuvastatin, and fluvastatin treatment; this effect was potentially mediated by sGP prenylation. Rab5a, the sGP in pancreatic islets most affected by ator treatment, was found to positively regulate mTOR signaling and β-cell function. Rab5a knockdown mimicked the effect of ator treatment on β-cells. Thus, ator impairs β-cell function by regulating sGPs, for example, Rab5a, which subsequently attenuates islet mTOR signaling and reduces functional β-cell mass. GGPP supplementation could constitute a new approach for preventing statin-induced hyperglycemia.

Coronary artery disease (CAD) is more frequent among individuals with dysglycemia. Preventive interventions for diabetes can improve cardiometabolic risk factors (CRFs), but it is unclear whether the benefits on CRFs are similar for individuals at different genetic risk for CAD. We built a 201-variant polygenic risk score (PRS) for CAD and tested for interaction with diabetes prevention strategies on 1-year changes in CRFs in 2,658 Diabetes Prevention Program (DPP) participants. We also examined whether separate lifestyle behaviors interact with PRS and affect changes in CRFs in each intervention group. Participants in both the lifestyle and metformin interventions had greater improvement in the majority of recognized CRFs compared with placebo (P < 0.001) irrespective of CAD genetic risk (Pinteraction > 0.05). We detected nominal significant interactions between PRS and dietary quality and physical activity on 1-year change in BMI, fasting glucose, triglycerides, and HDL cholesterol in individuals randomized to metformin or placebo, but none of them achieved the multiple-testing correction for significance. This study confirms that diabetes preventive interventions improve CRFs regardless of CAD genetic risk and delivers hypothesis-generating data on the varying benefit of increasing physical activity and improving diet on intermediate cardiovascular risk factors depending on individual CAD genetic risk profile.

Insulin secretion is tightly regulated by membrane trafficking. RILP (Rab7 interacting lysosomal protein) regulates the endocytic trafficking, but its role in insulin secretion has not been investigated. In this study, we found that overexpression of RILP inhibited insulin secretion in both the β-cell lines and freshly isolated islets. Consequently, the expression of RILP in islets suppressed the ability to recover the glucose homeostasis in type 1 diabetes mice upon transplantation. Of physiological relevance is that RILP expression was upregulated in the diabetic mouse islets. Mechanistically, overexpression of RILP induced insulin granule clustering, decreased the number of proinsulin-containing granules in β-cells, and significantly promoted proinsulin degradation. Conversely, RILP depletion sustained proinsulin and increased insulin secretion. The proinsulin degradation induced by RILP expression was inhibited by lysosomal inhibitors and was Rab7-dependent. Finally, we showed that RILP interacts with insulin granule-associated Rab26 to restrict insulin secretion. This study presents a new pathway regulating insulin secretion and mechanically demonstrates a novel function of RILP in modulating insulin secretion through mediating the lysosomal degradation of proinsulin.

The sequelae of diabetes include microvascular complications such as diabetic kidney disease (DKD), which involves glucose-mediated renal injury associated with a disruption in mitochondrial metabolic agility, inflammation, and fibrosis. We explored the role of the innate immune complement component C5a, a potent mediator of inflammation, in the pathogenesis of DKD in clinical and experimental diabetes. Marked systemic elevation in C5a activity was demonstrated in patients with diabetes; conventional renoprotective agents did not therapeutically target this elevation. C5a and its receptor (C5aR1) were upregulated early in the disease process and prior to manifest kidney injury in several diverse rodent models of diabetes. Genetic deletion of C5aR1 in mice conferred protection against diabetes-induced renal injury. Transcriptomic profiling of kidney revealed diabetes-induced downregulation of pathways involved in mitochondrial fatty acid metabolism. Interrogation of the lipidomics signature revealed abnormal cardiolipin remodeling in diabetic kidneys, a cardinal sign of disrupted mitochondrial architecture and bioenergetics. In vivo delivery of an orally active inhibitor of C5aR1 (PMX53) reversed the phenotypic changes and normalized the renal mitochondrial fatty acid profile, cardiolipin remodeling, and citric acid cycle intermediates. In vitro exposure of human renal proximal tubular epithelial cells to C5a led to altered mitochondrial respiratory function and reactive oxygen species generation. These experiments provide evidence for a pivotal role of the C5a/C5aR1 axis in propagating renal injury in the development of DKD by disrupting mitochondrial agility, thereby establishing a new immunometabolic signaling pathway in DKD.

Adipose tissue is the key organ coordinating whole-body energy homeostasis. Although it has been reported that ring finger protein 20 (RNF20) regulates lipid metabolism in the liver and kidney, the roles of RNF20 in adipose tissue have not been explored. Here, we demonstrate that RNF20 promotes adipogenesis by potentiating the transcriptional activity of peroxisome proliferator-activated receptor-γ (PPARγ). Under normal chow diet feeding, Rnf20 defective (Rnf20+/- ) mice exhibited reduced fat mass with smaller adipocytes compared with wild-type littermates. In addition, high-fat diet-fed Rnf20+/- mice alleviated systemic insulin resistance accompanied by a reduced expansion of fat tissue. Quantitative proteomic analyses revealed significantly decreased levels of PPARγ target proteins in adipose tissue of Rnf20+/- mice. Mechanistically, RNF20 promoted proteasomal degradation of nuclear corepressor 1 (NCoR1), which led to stimulation of the transcriptional activity of PPARγ. Collectively, these data suggest that RNF20-NCoR1 is a novel axis in adipocyte biology through fine-tuning the transcriptional activity of PPARγ.

Abnormalities of methyl-CpG binding protein 2 (Mecp2) cause neurological disorders with metabolic dysfunction; however, its role in adipose tissues remains unclear. Here, we report upregulated Mecp2 in white adipose tissues (WAT) of obese humans, as well as in obese mice and during in vitro adipogenesis. Normal chow-fed adipocyte-specific Mecp2 knockout mice (Mecp2Adi KO mice) showed a lean phenotype, with downregulated lipogenic genes and upregulated thermogenic genes that were identified using RNA sequencing. Consistently, the deficiency of Mecp2 in adipocytes protected mice from high-fat diet (HFD)-induced obesity and inhibited in vitro adipogenesis. Furthermore, Mecp2Adi KO mice showed increased browning under different stimuli, including cold treatment. Mechanistically, Mecp2 bound to the promoter of secretory leukocyte protease inhibitor (Slpi) and negatively regulated its expression. Knockdown of Slpi in inguinal WAT of Mecp2Adi KO mice prevented cold-induced browning. Moreover, recombinant SLPI treatment reduced the HFD-induced obesity via enhancing browning. Together, our results suggest a novel non-central nervous system function of Mecp2 in obesity by suppressing browning, at least partially, through regulating adipokine Slpi.

Within the human pancreas, exocrine and endocrine cells control secretion of digestive enzymes and production of hormones to maintain metabolic homeostasis, respectively. While the vast majority of type 1 diabetes research efforts have focused on endocrine function and autoimmunity, recent studies identified a series of unique features (e.g., reduced weight and volume, increased density of leukocytes) within the exocrine pancreas in this disease, but the mechanisms underlying these aberrancies are unknown. Therefore, we histologically assessed amylase, insulin, glucagon, lipase, and/or trypsinogen in 78 organ donor pancreata from birth through adulthood in control subjects and those at various stages of type 1 diabetes. While amylase-positive (AMY+) acinar cells were detectable in pancreata from all study groups, tissues from individuals >2 years of age contained clusters of acinar cells devoid of amylase (AMY-). A majority of these AMY- cell clusters localized proximal to islets (i.e., peri-islet). Additionally, most AMY- clusters were positive for the exocrine enzymes lipase and trypsinogen. Interestingly, type 1 diabetes pancreata displayed significant reductions in the frequency of these AMY- cell clusters. These results support a contribution of the islet-acinar axis in pancreatic development and underscore a potential role for the exocrine pancreas in the pathogenesis of type 1 diabetes.

A declining first-phase insulin response (FPIR) is associated with positivity for multiple islet autoantibodies, irrespective of class II HLA DR-DQ genotype. We examined the associations of FPIR with genetic variants outside the HLA DR-DQ region in the Finnish Type 1 Diabetes Prediction and Prevention (DIPP) study in children with and without multiple autoantibodies. Association between FPIR and class I alleles A*24 and B*39 and eight single nucleotide polymorphisms outside the HLA region were analyzed in 438 children who had one or more FPIR results available after seroconversion. Hierarchical linear mixed models were used to analyze repeated measurements of FPIR. In children with multiple autoantibodies, the change in FPIR over time was significantly different between those with various PTPN2 (rs45450798), FUT2 (rs601338), CTSH (rs3825932), and IKZF4 (rs1701704) genotypes in at least one of the models. In general, children carrying susceptibility alleles for type 1 diabetes experienced a more rapid decline in insulin secretion compared with children without susceptibility alleles. The presence of the class I HLA A*24 allele was also associated with a steeper decline of FPIR over time in children with multiple autoantibodies. Certain genetic variants outside the class II HLA region may have a significant impact on the longitudinal pattern of FPIR.

Reduction of β-cell mass and function is central to the pathogenesis of type 2 diabetes. The terms glucotoxicity, lipotoxicity, and glucolipotoxicity are used to describe potentially responsible processes. The premise is that chronically elevated glucose levels are toxic to β-cells, that elevated lipid levels in the form of circulating free fatty acids (FFA) also have toxic effects, and that the combination of the two, glucolipotoxicity, is particularly harmful. Much work has shown that high concentrations of FFA can be very damaging to β-cells when used for in vitro experiments, and when infused in large amounts in humans and rodents they produce suppression of insulin secretion. The purpose of this Perspective is to raise doubts about whether the FFA levels found in real-life situations are ever high enough to cause problems. Evidence supporting the importance of glucotoxicity is strong because there is such a tight correlation between defective insulin secretion and rising glucose levels. However, there is virtually no convincing evidence that the alterations in FFA levels occurring during progression to diabetes are pathogenic. Thus, the terms lipotoxicity and glucolipotoxicity should be used with great caution, if at all, because evidence supporting their importance has not yet emerged.

Glucagon is historically described as the counterregulatory hormone to insulin, induced by fasting/hypoglycemia to raise blood glucose through action mediated in the liver. However, it is becoming clear that the biology of glucagon is much more complex and extends beyond hepatic actions to exert control on glucose metabolism. We discuss the inconsistencies with the canonical view that glucagon is primarily a hyperglycemic agent driven by fasting/hypoglycemia and highlight the recent advances that have reshaped the metabolic role of glucagon. These concepts are placed within the context of both normal physiology and the pathophysiology of disease and then extended to discuss emerging strategies that incorporate glucagon agonism in the pharmacology of treating diabetes.