Insulin plays pivotal role in cellular fuel metabolism in skeletal muscle. Despite being the primary site of energy metabolism, the underlying mechanism on how insulin deficiency deranges skeletal muscle mitochondrial physiology remains to be fully understood. Here we report an important link between altered skeletal muscle proteome homeostasis and mitochondrial physiology during insulin deficiency. Deprivation of insulin in streptozotocin-induced diabetic mice decreased mitochondrial ATP production, reduced coupling and phosphorylation efficiency, and increased oxidant emission in skeletal muscle. Proteomic survey revealed that the mitochondrial derangements during insulin deficiency were related to increased mitochondrial protein degradation and decreased protein synthesis, resulting in reduced abundance of proteins involved in mitochondrial respiration and β-oxidation. However, a paradoxical upregulation of proteins involved in cellular uptake of fatty acids triggered an accumulation of incomplete fatty acid oxidation products in skeletal muscle. These data implicate a mismatch of β-oxidation and fatty acid uptake as a mechanism leading to increased oxidative stress in diabetes. This notion was supported by elevated oxidative stress in cultured myotubes exposed to palmitate in the presence of a β-oxidation inhibitor. Together, these results indicate that insulin deficiency alters the balance of proteins involved in fatty acid transport and oxidation in skeletal muscle, leading to impaired mitochondrial function and increased oxidative stress.

The high mortality and disability of diabetic nonhealing skin ulcers create an urgent need for the development of more efficacious strategies targeting diabetic wound healing. In the current study, using human clinical specimens, we show that perilesional skin tissues from patients with diabetes are under more severe oxidative stress and display higher activation of the nuclear factor-E2-related factor 2 (NRF2)-mediated antioxidant response than perilesional skin tissues from normoglycemic patients. In a streptozotocin-induced diabetes mouse model, Nrf2(-/-) mice have delayed wound closure rates compared with Nrf2(+/+) mice, which is, at least partially, due to greater oxidative DNA damage, low transforming growth factor-β1 (TGF-β1) and high matrix metalloproteinase 9 (MMP9) expression, and increased apoptosis. More importantly, pharmacological activation of the NRF2 pathway significantly improves diabetic wound healing. In vitro experiments in human immortalized keratinocyte cells confirm that NRF2 contributes to wound healing by alleviating oxidative stress, increasing proliferation and migration, decreasing apoptosis, and increasing the expression of TGF-β1 and lowering MMP9 under high-glucose conditions. This study indicates an essential role for NRF2 in diabetic wound healing and the therapeutic benefits of activating NRF2 in this disease, laying the foundation for future clinical trials using NRF2 activators in treating diabetic skin ulcers.

The risk of long-term diabetes complications is not fully explained by diabetes duration or long-term glycemic exposure, suggesting the involvement of genetic factors. Because thiamine regulates intracellular glucose metabolism and corrects for multiple damaging effects of high glucose, we hypothesized that variants in specific thiamine transporters are associated with risk of severe retinopathy and/or severe nephropathy because they modify an individual's ability to achieve sufficiently high intracellular thiamine levels. We tested 134 single nucleotide polymorphisms (SNPs) in two thiamine transporters (SLC19A2/3) and their transcription factors (SP1/2) for an association with severe retinopathy or nephropathy or their combination in the FinnDiane cohort. Subsequently, the results were examined for replication in the DCCT/EDIC and Wisconsin Epidemiologic Study of Diabetic Retinopathy (WESDR) cohorts. We found two SNPs in strong linkage disequilibrium in the SLC19A3 locus associated with a reduced rate of severe retinopathy and the combined phenotype of severe retinopathy and end-stage renal disease. The association for the combined phenotype reached genome-wide significance in a meta-analysis that included the WESDR cohort. These findings suggest that genetic variations in SLC19A3 play an important role in the pathogenesis of severe diabetic retinopathy and nephropathy and may explain why some individuals with type 1 diabetes are less prone than others to develop microvascular complications.

There is an ongoing need to develop strategic combinations of therapeutic agents to prevent type 1 diabetes (T1D) or to preserve islet β-cell mass in new-onset disease. Although clinical trials using candidate therapeutics are commonly based on preclinical studies, concern is growing regarding the reproducibility as well as the potential clinical translation of reported results using animal models of human disorders. In response, the National Institutes of Health Immune Tolerance Network and JDRF established a multicenter consortium of academic institutions designed to assess the efficacy and intergroup reproducibility of clinically applicable immunotherapies for reversing new-onset disease in the NOD mouse model of T1D. Predicated on prior studies, this consortium conducted coordinated, prospective studies, using joint standard operating procedures, fixed criteria for study entry, and common reagents, to optimize combined anti-CD3 treatment plus interleukin-1 (IL-1) blockade to reverse new-onset disease in NOD mice. We did not find that IL-1 blockade with anti-IL-1β monoclonal antibody or IL-1trap provided additional benefit for reversing new-onset disease compared with anti-CD3 treatment alone. These results demonstrate the value of larger, multicenter preclinical studies for vetting and prioritizing therapeutics for future clinical use.

The innate immune cell sensor leucine-rich-containing family, pyrin domain containing 3 (NLRP3) inflammasome controls the activation of caspase-1, and the release of proinflammatory cytokines interleukin (IL)-1β and IL-18. The NLRP3 inflammasome is implicated in adipose tissue inflammation and the pathogenesis of insulin resistance. Herein, we tested the hypothesis that adipose tissue inflammation and NLRP3 inflammasome are linked to the downregulation of subcutaneous adipose tissue (SAT) adipogenesis/lipogenesis in obese adolescents with altered abdominal fat partitioning. We performed abdominal SAT biopsies on 58 obese adolescents and grouped them by MRI-derived visceral fat to visceral adipose tissue (VAT) plus SAT (VAT/VAT+SAT) ratio (cutoff 0.11). Adolescents with a high VAT/VAT+SAT ratio showed higher SAT macrophage infiltration and higher expression of the NLRP3 inflammasome-related genes (i.e., TLR4, NLRP3, IL1B, and CASP1). The increase in inflammation markers was paralleled by a decrease in genes related to insulin sensitivity (ADIPOQ, GLUT4, PPARG2, and SIRT1) and lipogenesis (SREBP1c, ACC, LPL, and FASN). Furthermore, SAT ceramide concentrations correlated with the expression of CASP1 and IL1B. Infiltration of macrophages and upregulation of the NLRP3 inflammasome together with the associated high ceramide content in the plasma and SAT of obese adolescents with a high VAT/VAT+SAT may contribute to the limited expansion of the subcutaneous abdominal adipose depot and the development of insulin resistance.

Human proinsulin with C-peptide-bearing Superfolder Green Fluorescent Protein (CpepSfGFP) has been expressed in transgenic mice, driven by the Ins1 promoter. The protein, expressed exclusively in β-cells, is processed and stored as CpepSfGFP and human insulin comprising only ∼0.04% of total islet proinsulin plus insulin, exerting no metabolic impact. The kinetics of the release of insulin and CpepSfGFP from isolated islets appear identical. Upon a single acute stimulatory challenge in vitro, fractional release of insulin does not detectably deplete islet fluorescence. In vivo, fluorescence imaging of the pancreatic surface allows, for the first time, visual assessment of pancreatic islet insulin content, and we demonstrate that CpepSfGFP visibly declines upon diabetes progression in live lepR(db/db) mice. In anesthetized mice, after intragastric or intravenous saline delivery, pancreatic CpepSfGFP (insulin) content remains undiminished. Remarkably, however, within 20 min after acute intragastric or intravenous glucose delivery (with blood glucose concentrations reaching >15 mmol/L), a small subset of islets shows rapid dispossession of a major fraction of their stored CpepSfGFP (insulin) content, whereas most islets exhibit no demonstrable loss of CpepSfGFP (insulin). These studies strongly suggest that there are "first responder" islets to an in vivo glycemic challenge, which cannot be replicated by islets in vitro.

Chronic hyperglycemia impairs intracellular redox homeostasis and contributes to impaired diabetic tissue regeneration. The Keap1/Nrf2 pathway is a critical regulator of the endogenous antioxidant response system, and its dysfunction has been implicated in numerous pathologies. Here we characterize the effect of chronic hyperglycemia on Nrf2 signaling within a diabetic cutaneous regeneration model. We characterized the effects of chronic hyperglycemia on the Keap1/Nrf2 pathway within models of diabetic cutaneous wound regeneration. We assessed reactive oxygen species (ROS) production and antioxidant gene expression following alterations in the Nrf2 suppressor Keap1 and the subsequent changes in Nrf2 signaling. We also developed a topical small interfering RNA (siRNA)-based therapy to restore redox homeostasis within diabetic wounds. Western blotting demonstrated that chronic hyperglycemia-associated oxidative stress inhibits nuclear translocation of Nrf2 and impairs activation of antioxidant genes, thus contributing to ROS accumulation. Keap1 inhibition increased Nrf2 nuclear translocation, increased antioxidant gene expression, and reduced ROS production to normoglycemic levels, both in vitro and in vivo. Topical siKeap1 therapy resulted in improved regenerative capacity of diabetic wounds and accelerated closure. We report that chronic hyperglycemia weakens the endogenous antioxidant response, and the consequences of this defect are manifested by intracellular redox dysregulation, which can be restored by Keap1 inhibition. Targeted siRNA-based therapy represents a novel, efficacious strategy to reestablish redox homeostasis and accelerate diabetic cutaneous tissue regeneration.

Despite finding more than 40 risk loci for type 1 diabetes (T1D), the causative variants and genes remain largely unknown. Here, we sought to identify rare deleterious variants of moderate-to-large effects contributing to T1D. We deeply sequenced 301 protein-coding genes located in 49 previously reported T1D risk loci in 70 T1D cases of European ancestry. These cases were selected from putatively high-risk families that had three or more siblings diagnosed with T1D at early ages. A cluster of rare deleterious variants in PTPN22 was identified, including two novel frameshift mutations (ss538819444 and rs371865329) and two missense variants (rs74163663 and rs56048322). Genotyping in 3,609 T1D families showed that rs56048322 was significantly associated with T1D and that this association was independent of the T1D-associated common variant rs2476601. The risk allele at rs56048322 affects splicing of PTPN22, resulting in the production of two alternative PTPN22 transcripts and a novel isoform of LYP (the protein encoded by PTPN22). This isoform competes with the wild-type LYP for binding to CSK and results in hyporesponsiveness of CD4(+) T cells to antigen stimulation in T1D subjects. These findings demonstrate that in addition to common variants, rare deleterious variants in PTPN22 exist and can affect T1D risk.

Transcription factor expression fluctuates during β-cell ontogeny, and disruptions in this pattern can affect the development or function of those cells. Here we uncovered that murine endocrine pancreatic progenitors express high levels of the homeodomain transcription factor Prox1, whereas both immature and mature β-cells scarcely express this protein. We also investigated if sustained Prox1 expression is incompatible with β-cell development or maintenance using transgenic mouse approaches. We discovered that Prox1 upregulation in mature β-cells has no functional consequences; in contrast, Prox1 overexpression in immature β-cells promotes acute fasting hyperglycemia. Using a combination of immunostaining and quantitative and comparative gene expression analyses, we determined that Prox1 upregulation reduces proliferation, impairs maturation, and enables apoptosis in postnatal β-cells. Also, we uncovered substantial deficiency in β-cells that overexpress Prox1 of the key regulator of β-cell maturation MafA, several MafA downstream targets required for glucose-stimulated insulin secretion, and genes encoding important components of FGF signaling. Moreover, knocking down PROX1 in human EndoC-βH1 β-cells caused increased expression of many of these same gene products. These and other results in our study indicate that reducing the expression of Prox1 is beneficial for the expansion and maturation of postnatal β-cells.

Elevated concentrations of albumin in the urine, albuminuria, are a hallmark of diabetic kidney disease and are associated with an increased risk for end-stage renal disease and cardiovascular events. To gain insight into the pathophysiological mechanisms underlying albuminuria, we conducted meta-analyses of genome-wide association studies and independent replication in up to 5,825 individuals of European ancestry with diabetes and up to 46,061 without diabetes, followed by functional studies. Known associations of variants in CUBN, encoding cubilin, with the urinary albumin-to-creatinine ratio (UACR) were confirmed in the overall sample (P = 2.4 × 10(-10)). Gene-by-diabetes interactions were detected and confirmed for variants in HS6ST1 and near RAB38/CTSC. Single nucleotide polymorphisms at these loci demonstrated a genetic effect on UACR in individuals with but not without diabetes. The change in the average UACR per minor allele was 21% for HS6ST1 (P = 6.3 × 10(-7)) and 13% for RAB38/CTSC (P = 5.8 × 10(-7)). Experiments using streptozotocin-induced diabetic Rab38 knockout and control rats showed higher urinary albumin concentrations and reduced amounts of megalin and cubilin at the proximal tubule cell surface in Rab38 knockout versus control rats. Relative expression of RAB38 was higher in tubuli of patients with diabetic kidney disease compared with control subjects. The loci identified here confirm known pathways and highlight novel pathways influencing albuminuria.

Maternal obesity increases the risk for pediatric obesity; however, the molecular mechanisms in human infants remain poorly understood. We hypothesized that mesenchymal stem cells (MSCs) from infants born to obese mothers would demonstrate greater potential for adipogenesis and less potential for myogenesis, driven by differences in β-catenin, a regulator of MSC commitment. MSCs were cultured from the umbilical cords of infants born to normal-weight (prepregnancy [pp] BMI 21.1 ± 0.3 kg/m(2); n = 15; NW-MSCs) and obese mothers (ppBMI 34.6 ± 1.0 kg/m(2); n = 14; Ob-MSCs). Upon differentiation, Ob-MSCs exhibit evidence of greater adipogenesis (+30% Oil Red O stain [ORO], +50% peroxisome proliferator-activated receptor (PPAR)-γ protein; P < 0.05) compared with NW-MSCs. In undifferentiated cells, total β-catenin protein content was 10% lower and phosphorylated Thr41Ser45/total β-catenin was 25% higher (P < 0.05) in Ob-MSCs versus NW-MSCs (P < 0.05). Coupled with 25% lower inhibitory phosphorylation of GSK-3β in Ob-MSCs (P < 0.05), these data suggest greater β-catenin degradation in Ob-MSCs. Lithium chloride inhibition of GSK-3β increased nuclear β-catenin content and normalized nuclear PPAR-γ in Ob-MSCs. Last, ORO in adipogenic differentiating cells was positively correlated with the percent fat mass in infants (r = 0.475; P < 0.05). These results suggest that altered GSK-3β/β-catenin signaling in MSCs of infants exposed to maternal obesity may have important consequences for MSC lineage commitment, fetal fat accrual, and offspring obesity risk.

Gluconeogenesis is a complex metabolic process that involves multiple enzymatic steps regulated by myriad factors, including substrate concentrations, the redox state, activation and inhibition of specific enzyme steps, and hormonal modulation. At present, the most widely accepted technique to determine gluconeogenesis is by measuring the incorporation of deuterium from the body water pool into newly formed glucose. However, several techniques using radioactive and stable-labeled isotopes have been used to quantitate the contribution and regulation of gluconeogenesis in humans. Each method has its advantages, methodological assumptions, and set of propagated errors. In this review, we examine the strengths and weaknesses of the most commonly used stable isotopes methods to measure gluconeogenesis in vivo. We discuss the advantages and limitations of each method and summarize the applicability of these measurements in understanding normal and pathophysiological conditions.

The hypothesis that plasma prekallikrein (PK) is a risk factor for the development of vascular complications was assessed in a study using the Diabetes Control and Complications Trial (DCCT)/Epidemiology and Diabetes Interventions and Complications (EDIC) cohort of subjects with type 1 diabetes. The circulating levels of plasma PK activity were measured in the plasma of 636 subjects with type 1 diabetes (EDIC years 3-5). Common and internal carotid intima-media thickness (IMT) were measured by B-mode ultrasonography in EDIC years 1 and 6. Plasma PK levels were positively and significantly associated with BMI, hemoglobin A1c, systolic blood pressure, total cholesterol, LDL cholesterol, and triglycerides but not with age, sex, duration of diabetes, or HDL cholesterol. Univariate and multivariable statistical models after controlling for other risk factors consistently demonstrated a positive association between plasma PK and progression of internal carotid IMT. Multivariate analysis using a general linear model showed plasma PK to be significantly associated with progression of both internal and combined IMT (Wilks Λ P value of 0.005). In addition, the mean internal carotid IMT levels were higher in subjects with plasma PK levels in the highest 10th percentile compared with subjects with plasma PK levels in the lower 10th percentile (P = 0.048). These novel findings implicate plasma PK as a risk factor for vascular disease in type 1 diabetes.

Pancreatic β-cells modulate insulin secretion through rapid sensing of blood glucose and integration of gut-derived signals. Increased insulin demand during pregnancy and obesity alters islet function and mass and leads to gestational diabetes mellitus and type 2 diabetes in predisposed individuals. However, it is unclear how blood-borne factors dynamically access the islets of Langerhans. Thus, understanding the changes in circulating molecule distribution that accompany compensatory β-cell expansion may be key to developing novel antidiabetic therapies. Here, using two-photon microscopy in vivo in mice, we demonstrate that islets are almost instantly exposed to peaks of circulating molecules, which rapidly pervade the tissue before clearance. In addition, both gestation and short-term high-fat-diet feeding decrease molecule extravasation and uptake rates in vivo in islets, independently of β-cell expansion or islet blood flow velocity. Together, these data support a role for islet vascular permeability in shaping β-cell adaptive responses to metabolic demand by modulating the access and sensing of circulating molecules.

Descriptions of insulitis in human islets throughout the natural history of type 1 diabetes are limited. We determined insulitis frequency (the percent of islets displaying insulitis to total islets), infiltrating leukocyte subtypes, and β-cell and α-cell mass in pancreata recovered from organ donors with type 1 diabetes (n = 80), as well as from donors without diabetes, both with islet autoantibodies (AAb(+), n = 18) and without islet autoantibodies (AAb(-), n = 61). Insulitis was observed in four of four donors (100%) with type 1 diabetes duration of ≤1 year and two AAb(+) donors (2 of 18 donors, 11%). Insulitis frequency showed a significant but limited inverse correlation with diabetes duration (r = -0.58, P = 0.01) but not with age at disease onset. Residual β-cells were observed in all type 1 diabetes donors with insulitis, while β-cell area and mass were significantly higher in type 1 diabetes donors with insulitis compared with those without insulitis. Insulitis affected 33% of insulin(+) islets compared with 2% of insulin(-) islets in donors with type 1 diabetes. A significant correlation was observed between insulitis frequency and CD45(+), CD3(+), CD4(+), CD8(+), and CD20(+) cell numbers within the insulitis (r = 0.53-0.73, P = 0.004-0.04), but not CD68(+) or CD11c(+) cells. The presence of β-cells as well as insulitis several years after diagnosis in children and young adults suggests that the chronicity of islet autoimmunity extends well into the postdiagnosis period. This information should aid considerations of therapeutic strategies seeking type 1 diabetes prevention and reversal.

At the CDKN2A/B locus, three independent signals for type 2 diabetes risk are located in a noncoding region near CDKN2A. The disease-associated alleles have been implicated in reduced β-cell function, but the underlying mechanism remains elusive. In mice, β-cell-specific loss of Cdkn2a causes hyperplasia, while overexpression leads to diabetes, highlighting CDKN2A as a candidate effector transcript. Rare CDKN2A loss-of-function mutations are a cause of familial melanoma and offer the opportunity to determine the impact of CDKN2A haploinsufficiency on glucose homeostasis in humans. To test the hypothesis that such individuals have improved β-cell function, we performed oral and intravenous glucose tolerance tests on mutation carriers and matched control subjects. Compared with control subjects, carriers displayed increased insulin secretion, impaired insulin sensitivity, and reduced hepatic insulin clearance. These results are consistent with a model whereby CDKN2A loss affects a range of different tissues, including pancreatic β-cells and liver. To test for direct effects of CDKN2A-loss on β-cell function, we performed knockdown in a human β-cell line, EndoC-bH1. This revealed increased insulin secretion independent of proliferation. Overall, we demonstrated that CDKN2A is an important regulator of glucose homeostasis in humans, thus supporting its candidacy as an effector transcript for type 2 diabetes-associated alleles in the region.

Our objective was to evaluate the relationship of urine metals including barium, cadmium, cobalt, cesium, molybdenum, lead, antimony, thallium, tungsten, and uranium with diabetes prevalence. Data were from a cross-sectional study of 9,447 participants of the 1999-2010 National Health and Nutrition Examination Survey, a representative sample of the U.S. civilian noninstitutionalized population. Metals were measured in a spot urine sample, and diabetes status was determined based on a previous diagnosis or an A1C ≥6.5% (48 mmol/mol). After multivariable adjustment, the odds ratios of diabetes associated with the highest quartile of metal, compared with the lowest quartile, were 0.86 (95% CI 0.66-1.12) for barium (Ptrend = 0.13), 0.74 (0.51-1.09) for cadmium (Ptrend = 0.35), 1.21 (0.85-1.72) for cobalt (Ptrend = 0.59), 1.31 (0.90-1.91) for cesium (Ptrend = 0.29), 1.76 (1.24-2.50) for molybdenum (Ptrend = 0.01), 0.79 (0.56-1.13) for lead (Ptrend = 0.10), 1.72 (1.27-2.33) for antimony (Ptrend < 0.01), 0.76 (0.51-1.13) for thallium (Ptrend = 0.13), 2.18 (1.51-3.15) for tungsten (Ptrend < 0.01), and 1.46 (1.09-1.96) for uranium (Ptrend = 0.02). Higher quartiles of barium, molybdenum, and antimony were associated with greater HOMA of insulin resistance after adjustment. Molybdenum, antimony, tungsten, and uranium were positively associated with diabetes, even at the relatively low levels seen in the U.S.