Obesity-related insulin resistance is closely associated with macrophage accumulation and subsequent cytokine release in local tissues. Sirtuin 6 (Sirt6) is known to exert an anti-inflammatory function, but its role in macrophages in the context of obesity has not been investigated. We generated myeloid-specific Sirt6 knockout (mS6KO) mice and investigated the metabolic characteristics after high-fat diet (HFD) feeding for 16 weeks. Compared with their wild-type littermates, HFD-fed mS6KO mice exhibited greater increases in body weight, fasting blood glucose and insulin levels, hepatic steatosis, glucose intolerance, and insulin resistance. Gene expression, histology, and flow cytometric analyses demonstrated that liver and adipose tissue inflammation were elevated in HFD-fed mS6KO mice relative to wild type, with a greater accumulation of F4/80+CD11b+CD11c+ adipose tissue macrophages. Myeloid Sirt6 deletion facilitated proinflammatory M1 polarization of bone marrow macrophages and augmented the migration potential of macrophages toward adipose-derived chemoattractants. Mechanistically, Sirt6 deletion in macrophages promoted the activation of nuclear factor-κB (NF-κB) and endogenous production of interleukin-6, which led to STAT3 activation and the positive feedback circuits for NF-κB stimulation; this cross talk expedited an M1 polarization. We conclude that Sirt6 in macrophages is required for the prevention of obesity-associated tissue inflammation and insulin resistance.

Although over 40 type 1 diabetes (T1D) risk loci have been mapped in humans, the causative genes and variants for T1D are largely unknown. Here, we investigated a candidate gene in the 21q22.3 risk locus-UBASH3A, which is primarily expressed in T cells where it is thought to play a largely redundant role. Genetic variants in UBASH3A have been shown to be associated with several autoimmune diseases in addition to T1D. However, the molecular mechanism underlying these genetic associations is unresolved. Our study reveals a previously unrecognized role of UBASH3A in human T cells: UBASH3A attenuates the NF-κB signal transduction upon T-cell receptor (TCR) stimulation by specifically suppressing the activation of the IκB kinase complex. We identify novel interactions of UBASH3A with nondegradative polyubiquitin chains, TAK1 and NEMO, suggesting that UBASH3A regulates the NF-κB signaling pathway by an ubiquitin-dependent mechanism. Finally, we show that risk alleles at rs11203203 and rs80054410, two T1D-associated variants in UBASH3A, increase UBASH3A expression in human primary CD4+ T cells upon TCR stimulation, inhibiting NF-κB signaling via its effects on the IκB kinase complex and resulting in reduced IL2 gene expression.

Hepatic acyl-CoA thioesterase 1 (ACOT1) catalyzes the conversion of acyl-CoAs to fatty acids (FAs) and CoA. We sought to determine the role of ACOT1 in hepatic lipid metabolism in C57Bl/6J male mice 1 week after adenovirus-mediated Acot1 knockdown. Acot1 knockdown reduced liver triglyceride (TG) as a result of enhanced TG hydrolysis and subsequent FA oxidation. In vitro experiments demonstrated that Acot1 knockdown led to greater TG turnover and FA oxidation, suggesting that ACOT1 is important for controlling the rate of FA oxidation. Despite increased FA oxidation, Acot1 knockdown reduced the expression of peroxisome proliferator-activated receptor α (PPARα) target genes, whereas overexpression increased PPARα reporter activity, suggesting ACOT1 regulates PPARα by producing FA ligands. Moreover, ACOT1 exhibited partial nuclear localization during fasting and cAMP/cAMP-dependent protein kinase signaling, suggesting local regulation of PPARα. As a consequence of increased FA oxidation and reduced PPARα activity, Acot1 knockdown enhanced hepatic oxidative stress and inflammation. The effects of Acot1 knockdown on PPARα activity, oxidative stress, and inflammation were rescued by supplementation with Wy-14643, a synthetic PPARα ligand. We demonstrate through these results that ACOT1 regulates fasting hepatic FA metabolism by balancing oxidative flux and capacity.

Current pharmacological options for type 2 diabetes do not cure the disease. Despite the availability of multiple drug classes that modulate glycemia effectively and minimize long-term complications, these agents do not reverse pathogenesis, and in practice they are not selected to correct the molecular profile specific to the patient. Pharmaceutical companies find drug development programs increasingly costly and burdensome, and many promising compounds fail before launch to market. Human genetics can help advance the therapeutic enterprise. Genomic discovery that is agnostic to preexisting knowledge has uncovered dozens of loci that influence glycemic dysregulation. Physiological investigation has begun to define disease subtypes, clarifying heterogeneity and suggesting molecular pathways for intervention. Convincing genetic associations have paved the way for the identification of effector transcripts that underlie the phenotype, and genetic or experimental proof of gain or loss of function in select cases has clarified the direction of effect to guide therapeutic development. Genetic studies can also examine off-target effects and furnish causal inference. As this information is curated and made widely available to all stakeholders, it is hoped that it will enhance therapeutic development pipelines by accelerating efficiency, maximizing cost-effectiveness, and raising ultimate success rates.

Exocytosis of the hormone glucagon-like peptide 1 (GLP-1) by the intestinal L cell is essential for the incretin effect after nutrient ingestion and is critical for the actions of dipeptidyl peptidase 4 inhibitors that enhance GLP-1 levels in patients with type 2 diabetes. Two-photon microscopy revealed that exocytosis of GLP-1 is biphasic, with a first peak at 1-6 min and a second peak at 7-12 min after stimulation with forskolin. Approximately 75% of the exocytotic events were represented by compound granule fusion, and the remainder were accounted for by full fusion of single granules under basal and stimulated conditions. The core SNARE protein syntaxin-1a (syn1a) was expressed by murine ileal L cells. At the single L-cell level, first-phase forskolin-induced exocytosis was reduced to basal (P < 0.05) and second-phase exocytosis abolished (P < 0.05) by syn1a knockout. L cells from intestinal-epithelial syn1a-deficient mice demonstrated a 63% reduction in forskolin-induced GLP-1 release in vitro (P < 0.001) and a 23% reduction in oral glucose-stimulated GLP-1 secretion (P < 0.05) in association with impairments in glucose-stimulated insulin release (by 60%; P < 0.01) and glucose tolerance (by 20%; P < 0.01). The findings identify an exquisite mechanism of metered secretory output that precisely regulates release of the incretin hormone GLP-1 and hence insulin secretion after a meal.

Hypoglycemia is the leading limiting factor in glycemic management of insulin-treated diabetes. Skeletal muscle is the predominant site of insulin-mediated glucose disposal. Our study used a crossover design to test to what extent insulin-induced hypoglycemia affects glucose uptake in skeletal muscle and whether hypoglycemia counterregulation modulates insulin and catecholamine signaling and glycogen synthase activity in skeletal muscle. Nine healthy volunteers were examined on three randomized study days: 1) hyperinsulinemic hypoglycemia (bolus insulin), 2) hyperinsulinemic euglycemia (bolus insulin and glucose infusion), and 3) saline control with skeletal muscle biopsies taken just before, 30 min after, and 75 min after insulin/saline injection. During hypoglycemia, glucose levels reached a nadir of ∼2.0 mmol/L, and epinephrine rose to ∼900 pg/mL. Hypoglycemia impaired insulin-stimulated glucose disposal and glucose clearance in skeletal muscle, whereas insulin signaling in glucose transport was unaffected by hypoglycemia. Insulin-stimulated glycogen synthase activity was completely ablated during hyperinsulinemic hypoglycemia, and catecholamine signaling via cAMP-dependent protein kinase and phosphorylation of inhibiting sites on glycogen synthase all increased.

Diabetic keratopathy decreases corneal sensation and tear secretion and delays wound healing after injury. In the current study, we tested the effect of treatment with pigment epithelium-derived factor (PEDF) in combination with docosahexaenoic acid (DHA) on corneal nerve regeneration in a mouse model of diabetes with or without corneal injury. The study was performed in streptozotocin-induced diabetic mice (C57BL/6). Ten weeks after streptozotocin injection, diabetic mice showed significant decreases of corneal sensitivity, tear production, and epithelial subbasal nerve density when compared with age-matched normal mice. After diabetic mice were wounded in the right eye and treated in both eyes with PEDF+DHA for 2 weeks, there was a significant increase in corneal epithelial nerve regeneration and substance P-positive nerve density in both wounded and unwounded eyes compared with vehicle-treated corneas. There also was elevated corneal sensitivity and tear production in the treated corneas compared with vehicle. In addition, PEDF+DHA accelerated corneal wound healing, selectively recruited type 2 macrophages, and prevented neutrophil infiltration in diabetic wounded corneas. These results suggest that topical treatment with PEDF+DHA promotes corneal nerve regeneration and wound healing in diabetic mice and could potentially be exploited as a therapeutic option for the treatment of diabetic keratopathy.

Methylglyoxal (MGO), a major precursor for advanced glycation end products, is increased in diabetes. In diabetic rodents, inhibition of MGO prevents cardiovascular disease (CVD). Whether plasma MGO levels are associated with incident CVD in people with type 1 diabetes is unknown. We included 159 individuals with persistent normoalbuminuria and 162 individuals with diabetic nephropathy (DN) from the outpatient clinic at Steno Diabetes Center. We measured MGO at baseline and recorded fatal and nonfatal CVD over a median follow-up of 12.3 years (interquartile range 7.6-12.5 years). Data were analyzed by Cox regression, with adjustment for sex, age, HbA1c, DN, diabetes duration, smoking, systolic blood pressure, antihypertensive medication, and BMI. During follow-up, 73 individuals suffered at least one CVD event (36 fatal and 53 nonfatal). Higher MGO levels were associated with total, fatal, and nonfatal incident CVD (hazard ratios [HRs] 1.47 [95% CI 1.13-1.91], 1.42 [1.01-1.99], and 1.46 [1.08-1.98], respectively). We observed a similar trend for total mortality (HR 1.24 [0.99-1.56]). This study shows for the first time in our knowledge that plasma MGO levels are associated with cardiovascular events in individuals with type 1 diabetes. MGO may explain, at least in part, the increased risk for CVD in type 1 diabetes.

Aging is associated with increased risk for type 2 diabetes, resulting from reduced insulin sensitivity and secretion. Reduced insulin secretion can result from reduced proliferative capacity and reduced islet function. Mechanisms underlying altered β-cell function in aging are poorly understood in mouse and human islets, and the impact of aging on intraislet communication has not been characterized. Here, we examine how β-cell [Ca2+] and electrical communication are impacted during aging in mouse and human islets. Islets from human donors and from mice were studied using [Ca2+] imaging, static and perifusion insulin secretion assays, and gap junction permeability measurements. In human islets, [Ca2+] dynamics were coordinated within distinct subregions of the islet, invariant with islet size. There was a marked decline in the coordination of [Ca2+] dynamics, gap junction coupling, and insulin secretion dynamics with age. These age-dependent declines were reversed by pharmacological gap junction activation. These results show that human islet function declines with aging, which can reduce insulin action and may contribute to increased risk of type 2 diabetes.

Obesity is associated with hypothalamic inflammation (HI) in animal models. In the current study, we examined the mediobasal hypothalamus (MBH) of 57 obese human subjects and 54 age- and sex- matched nonobese control subjects by MRI and analyzed the T2 hyperintensity as a measure of HI. Obese subjects exhibited T2 hyperintensity in the left but not the right MBH, which was strongly associated with systemic low-grade inflammation. MRS revealed the number of neurons in the left hypothalamic region to be similar in obese versus control subjects, suggesting functional but not structural impairment due to the inflammatory process. To gain mechanistic insights, we performed nutritional analysis and 16S rDNA microbiome sequencing, which showed that high-fat diet induces reduction of Parasutterella sp. in the gut, which is significantly correlated with MBH T2 hyperintensity. In addition to these environmental factors, we found subjects carrying common polymorphisms in the JNK or the MC4R gene to be more susceptible to HI. Finally, in a subgroup analysis, bariatric surgery had no effect on MBH T2 hyperintensity despite inducing significant weight loss and improvement of peripheral insulin sensitivity. In conclusion, obesity in humans is associated with HI and disturbances in the gut-brain axis, which are influenced by both environmental and genetic factors.

Poor maternal diet can lead to metabolic disease in offspring, whereas maternal exercise may have beneficial effects on offspring health. In this study, we determined ifmaternal exercise could reverse the detrimental effects of maternal high-fat feeding on offspring metabolism of female mice. C57BL/6 female mice were fed a chow (21%) or high-fat (60%) diet and further divided by housing in static cages or cages with running wheels for 2 weeks prior to breeding and throughout gestation. Females were bred with chow-fed sedentary C57BL/6 males. High fat-fed sedentary dams produced female offspring with impaired glucose tolerance compared with offspring of chow-fed dams throughout their first year of life, an effect not present in the offspring from high fat-fed dams that had trained. Offspring from high fat-fed trained dams had normalized glucose tolerance, decreased fasting insulin, and decreased adiposity. Liver metabolic function, measured by hepatic glucose production in isolated hepatocytes, hyperinsulinemic-euglycemic clamps, liver triglyceride content, and liver enzyme expression, was enhanced in offspring from trained dams. In conclusion, maternal exercise negates the detrimental effects of a maternal high-fat diet on glucose tolerance and hepatocyte glucose metabolism in female offspring. The ability of maternal exercise to improve the metabolic health of female offspring is important, as this intervention could combat the transmission of obesity and diabetes to subsequent generations.

To characterize type 2 diabetes (T2D)-associated variation across the allele frequency spectrum, we conducted a meta-analysis of genome-wide association data from 26,676 T2D case and 132,532 control subjects of European ancestry after imputation using the 1000 Genomes multiethnic reference panel. Promising association signals were followed up in additional data sets (of 14,545 or 7,397 T2D case and 38,994 or 71,604 control subjects). We identified 13 novel T2D-associated loci (P < 5 × 10-8), including variants near the GLP2R, GIP, and HLA-DQA1 genes. Our analysis brought the total number of independent T2D associations to 128 distinct signals at 113 loci. Despite substantially increased sample size and more complete coverage of low-frequency variation, all novel associations were driven by common single nucleotide variants. Credible sets of potentially causal variants were generally larger than those based on imputation with earlier reference panels, consistent with resolution of causal signals to common risk haplotypes. Stratification of T2D-associated loci based on T2D-related quantitative trait associations revealed tissue-specific enrichment of regulatory annotations in pancreatic islet enhancers for loci influencing insulin secretion and in adipocytes, monocytes, and hepatocytes for insulin action-associated loci. These findings highlight the predominant role played by common variants of modest effect and the diversity of biological mechanisms influencing T2D pathophysiology.

Recent work has renewed interest in therapies targeting the renin-angiotensin system (RAS) to improve β-cell function in type 2 diabetes. Studies show that generation of angiotensin-(1-7) by ACE2 and its binding to the Mas receptor (MasR) improves glucose homeostasis, partly by enhancing glucose-stimulated insulin secretion (GSIS). Thus, islet ACE2 upregulation is viewed as a desirable therapeutic goal. Here, we show that, although endogenous islet ACE2 expression is sparse, its inhibition abrogates angiotensin-(1-7)-mediated GSIS. However, a more widely expressed islet peptidase, neprilysin, degrades angiotensin-(1-7) into several peptides. In neprilysin-deficient mouse islets, angiotensin-(1-7) and neprilysin-derived degradation products angiotensin-(1-4), angiotensin-(5-7), and angiotensin-(3-4) failed to enhance GSIS. Conversely, angiotensin-(1-2) enhanced GSIS in both neprilysin-deficient and wild-type islets. Rather than mediating this effect via activation of the G-protein-coupled receptor (GPCR) MasR, angiotensin-(1-2) was found to signal via another GPCR, namely GPCR family C group 6 member A (GPRC6A). In conclusion, in islets, intact angiotensin-(1-7) is not the primary mediator of beneficial effects ascribed to the ACE2/angiotensin-(1-7)/MasR axis. Our findings warrant caution for the concurrent use of angiotensin-(1-7) compounds and neprilysin inhibitors as therapies for diabetes.

Progressive reduction in β-cell mass and function comprise the core of the pathogenesis mechanism of type 2 diabetes. The process of deteriorating pancreatic islets, in which a complex network of molecular events is involved, is not yet fully characterized. We used RNA sequencing and tandem mass tag-based quantitative proteomics technology to measure the temporal mRNA and protein expression changes of pancreatic islets in Goto-Kakizaki (GK) rats from 4 to 24 weeks of age. Our omics data set outlines the dynamics of the molecular network during the deterioration of GK islets as two stages: The early stage (4-6 weeks) is characterized by anaerobic glycolysis, inflammation priming, and compensation for insulin synthesis, and the late stage (8-24 weeks) is characterized by inflammation amplification and compensation failure. Further time course analysis allowed us to reveal 5,551 differentially expressed genes, a large portion of which have not been reported before. Our comprehensive and temporal transcriptome and proteome data offer a valuable resource for the diabetes research community and for quantitative biology.

We used mice lacking Abcc8, a key component of the β-cell KATP-channel, to analyze the effects of a sustained elevation in the intracellular Ca2+ concentration ([Ca2+]i) on β-cell identity and gene expression. Lineage tracing analysis revealed the conversion of β-cells lacking Abcc8 into pancreatic polypeptide cells but not to α- or δ-cells. RNA-sequencing analysis of FACS-purified Abcc8-/- β-cells confirmed an increase in Ppy gene expression and revealed altered expression of more than 4,200 genes, many of which are involved in Ca2+ signaling, the maintenance of β-cell identity, and cell adhesion. The expression of S100a6 and S100a4, two highly upregulated genes, is closely correlated with membrane depolarization, suggesting their use as markers for an increase in [Ca2+]i Moreover, a bioinformatics analysis predicts that many of the dysregulated genes are regulated by common transcription factors, one of which, Ascl1, was confirmed to be directly controlled by Ca2+ influx in β-cells. Interestingly, among the upregulated genes is Aldh1a3, a putative marker of β-cell dedifferentiation, and other genes associated with β-cell failure. Taken together, our results suggest that chronically elevated β-cell [Ca2+]i in Abcc8-/- islets contributes to the alteration of β-cell identity, islet cell numbers and morphology, and gene expression by disrupting a network of Ca2+-regulated genes.

The Lin28a/Let-7 axis has been studied in peripheral tissues for its role in metabolism regulation. However, its central function remains unclear. Here we found that Lin28a is highly expressed in the hypothalamus compared with peripheral tissues. Its expression is positively correlated with positive energy balance, suggesting a potential central role for Lin28a in metabolism regulation. Thus, we targeted the hypothalamic ventromedial nucleus (VMH) to selectively overexpress (Lin28aKIVMH ) or downregulate (Lin28aKDVMH ) Lin28a expression in mice. With mice on a standard chow diet, body weight and glucose homeostasis were not affected in Lin28aKIVMH or Lin28aKDVMH mice. On a high-fat diet, although no differences in body weight and composition were observed, Lin28aKIVMH mice showed improved glucose tolerance and insulin sensitivity compared with controls. Conversely, Lin28aKDVMH mice displayed glucose intolerance and insulin resistance. Changes in VMH AKT activation of diet-induced obese Lin28aKIVMH or Lin28aKDVMH mice were not associated with alterations in Let-7 levels or insulin receptor activation. Rather, we observed altered expression of TANK-binding kinase-1 (TBK-1), which was found to be a direct Lin28a target mRNA. VMH-specific inhibition of TBK-1 in mice with diet-induced obesity impaired glucose metabolism and AKT activation. Altogether, our data show a TBK-1-dependent role for central Lin28a in glucose homeostasis.

We have previously reported that the topical application of erythropoietin (EPO) to cutaneous wounds in rats and mice with experimentally induced diabetes accelerates their healing by stimulating angiogenesis, reepithelialization, and collagen deposition, and by suppressing the inflammatory response and apoptosis. Aquaporins (AQPs) are integral membrane proteins whose function is to regulate intracellular fluid hemostasis by enabling the transport of water and glycerol. AQP3 is the AQP that is expressed in the skin where it facilitates cell migration and proliferation and re-epithelialization during wound healing. In this report, we provide the results of an investigation that examined the contribution of AQP3 to the mechanism of EPO action on the healing of burn wounds in the skin of pigs with experimentally induced type 1 diabetes. We found that topical EPO treatment of the burns accelerated their healing through an AQP3-dependent mechanism that activates angiogenesis, triggers collagen and hyaluronic acid synthesis and the formation of the extracellular matrix (ECM), and stimulates reepithelialization by keratinocytes. We also found that incorporating fibronectin, a crucial constituent of the ECM, into the topical EPO-containing gel, can potentiate the accelerating action of EPO on the healing of the burn injury.

Regulation of glucose homeostasis by insulin depends on β-cell growth and function. Nutrients and growth factor stimuli converge on the conserved protein kinase mechanistic target of rapamycin (mTOR), existing in two complexes, mTORC1 and mTORC2. To understand the functional relevance of mTOR enzymatic activity in β-cell development and glucose homeostasis, we generated mice overexpressing either one or two copies of a kinase-dead mTOR mutant (KD-mTOR) transgene exclusively in β-cells. We examined glucose homeostasis and β-cell function of these mice fed a control chow or high-fat diet. Mice with two copies of the transgene [RIPCre;KD-mTOR (Homozygous)] develop glucose intolerance due to a defect in β-cell function without alterations in β-cell mass with control chow. Islets from RIPCre;KD-mTOR (Homozygous) mice showed reduced mTORC1 and mTORC2 signaling along with transcripts and protein levels of Pdx-1. Islets with reduced mTORC2 signaling in their β-cells (RIPCre;Rictorfl/fl) also showed reduced Pdx-1. When challenged with a high-fat diet, mice carrying one copy of KD-mTOR mutant transgene developed glucose intolerance and β-cell insulin secretion defect but showed no changes in β-cell mass. These findings suggest that the mTOR-mediated signaling pathway is not essential to β-cell growth but is involved in regulating β-cell function in normal and diabetogenic conditions.