Diabetes impairs the ability to heal cutaneous wounds, leading to hospitalization, amputations, and death. Patients with diabetes experience elevated levels of plasminogen activator inhibitor 1 (PAI-1), regardless of their glycemic control. It has been demonstrated that PAI-1-deficient mice exhibit improved cutaneous wound healing, and that PAI-1 inhibition improves skeletal muscle repair in mice with type 1 diabetes mellitus, leading us to hypothesize that pharmacologically mediated reductions in PAI-1 using PAI-039 would normalize cutaneous wound healing in streptozotocin (STZ)-induced diabetic (STZ-diabetic) mice. To simulate the human condition of variations in wound care, wounds were aggravated or minimally handled postinjury. Following cutaneous injury, PAI-039 was orally administered twice daily for 10 days. Compared with nondiabetic mice, wounds in STZ-diabetic mice healed more slowly. Wound site aggravation exacerbated this deficit. PAI-1 inhibition had no effect on dermal collagen levels or wound bed size. PAI-039 treatment failed to improve angiogenesis in the wounds of STZ-diabetic mice and blunted angiogenesis in the wounds of nondiabetic mice. Importantly, PAI-039 treatment significantly improved epidermal cellular migration and wound re-epithelialization compared with vehicle-treated STZ-diabetic mice. These findings support the use of PAI-039 as a novel therapeutic agent to improve diabetic wound closure and demonstrate the primary mechanism of its action to be related to epidermal closure.

High-carbohydrate diets have been associated with β-cell strain, dyslipidemia, and endothelial dysfunction. We examined how β-cell and endothelial function adapt to carbohydrate overloading and the influence of insulin resistance. On sequential days in randomized order, nondiabetic subjects (classified as insulin-sensitive [IS] [n = 64] or insulin-resistant [IR] [n = 79] by euglycemic clamp) received four mixed meals over 14 h with either standard (300 kcal) or double carbohydrate content. β-Cell function was reconstructed by mathematical modeling; brachial artery flow-mediated dilation (FMD) was measured before and after each meal. Compared with IS, IR subjects showed higher glycemia and insulin hypersecretion due to greater β-cell glucose and rate sensitivity; potentiation of insulin secretion, however, was impaired. Circulating free fatty acids (FFAs) were less suppressed in IR than IS subjects. Baseline FMD was reduced in IR, and postprandial FMD attenuation occurred after each meal, particularly with high carbohydrate, similarly in IR and IS. Throughout the two study days, higher FFA levels were significantly associated with lower (incretin-induced) potentiation and impaired FMD. In nondiabetic individuals, enhanced glucose sensitivity and potentiation upregulate the insulin secretory response to carbohydrate overloading. With insulin resistance, this adaptation is impaired. Defective suppression of endogenous FFA is one common link between impaired potentiation and vascular endothelial dysfunction.

Obesity and diabetes are characterized by increased inflammation reflecting disordered control of innate immunity. We reveal a local intestinal intraepithelial lymphocyte (IEL)-GLP-1 receptor (GLP-1R) signaling network that controls mucosal immune responses. Glp1r expression was enriched in intestinal IEL preparations and copurified with markers of Tαβ and Tγδ IELs, the two main subsets of intestinal IELs. Exendin-4 increased cAMP accumulation in purified IELs and reduced the production of cytokines from activated IELs but not from splenocytes ex vivo. These actions were mimicked by forskolin, absent in IELs from Glp1r(-/-) mice, and attenuated by the GLP-1R agonist exendin (9-39) consistent with a GLP-1R-dependent mechanism of action. Furthermore, Glp1r(-/-) mice exhibited dysregulated intestinal gene expression, an abnormal representation of microbial species in feces, and enhanced sensitivity to intestinal injury following administration of dextran sodium sulfate. Bone marrow transplantation using wild-type C57BL/6 donors normalized expression of multiple genes regulating immune function and epithelial integrity in Glp1r(-/-) recipient mice, whereas acute exendin-4 administration robustly induced the expression of genes encoding cytokines and chemokines in normal and injured intestine. Taken together, these findings define a local enteroendocrine-IEL axis linking energy availability, host microbial responses, and mucosal integrity to the control of innate immunity.

Adenylyl cyclase type 5 knockout (AC5KO) mice have increased longevity and share a similar phenotype with calorie-restricted wild-type (WT) mice. To determine the in vivo metabolic properties of AC5 deficiency, we compared the effects of standard diet (SD) and high-fat diet (HFD) on obesity, energy balance, glucose regulation, and insulin sensitivity. AC5KO mice on SD had reduced body weight and adiposity compared with WT mice. Blood cholesterol and triglyceride levels were also significantly reduced in AC5KO mice. Indirect calorimetry demonstrated increased oxygen consumption, respiratory exchange ratio, and energy expenditure in AC5KO compared with WT mice on both SD and HFD. AC5KO mice also displayed improved glucose tolerance and increased whole-body insulin sensitivity, accompanied by decreased liver glycogen stores. Euglycemic-hyperinsulinemic clamp studies confirmed the marked improvement of glucose homeostasis and insulin sensitivity in AC5KO mice primarily through increased insulin sensitivity in skeletal muscle. Moreover, the genes involved in mitochondrial biogenesis and function were significantly increased in AC5KO skeletal muscle. These data demonstrate that deficiency of AC5 protects against obesity, glucose intolerance, and insulin resistance, supporting AC5 as a potential novel therapeutic target for treatment of obesity and diabetes.

Type 1 diabetes (T1D) is the result of an autoimmune assault against the insulin-producing pancreatic β-cells, where chronic local inflammation (insulitis) leads to β-cell destruction. T cells and macrophages infiltrate into islets early in T1D pathogenesis. These immune cells secrete cytokines that lead to the production of reactive oxygen species (ROS) and T-cell invasion and activation. Cytokine-signaling pathways are very tightly regulated by protein tyrosine phosphatases (PTPs) to prevent excessive activation. Here, we demonstrate that pancreata from NOD mice with islet infiltration have enhanced oxidation/inactivation of PTPs and STAT1 signaling compared with NOD mice that do not have insulitis. Inactivation of PTPs with sodium orthovanadate in human and rodent islets and β-cells leads to increased activation of interferon signaling and chemokine production mediated by STAT1 phosphorylation. Furthermore, this exacerbated STAT1 activation-induced cell death in islets was prevented by overexpression of the suppressor of cytokine signaling-1 or inactivation of the BH3-only protein Bim. Together our data provide a mechanism by which PTP inactivation induces signaling in pancreatic islets that results in increased expression of inflammatory genes and exacerbated insulitis.

The paucity of animal models exhibiting full pathology of diabetic retinopathy (DR) has impeded understanding of the pathogenesis of DR and the development of therapeutic interventions. Here, we investigated whether hyperhexosemic marmosets (Callithrix jacchus) develop characteristic retinal vascular lesions including macular edema (ME), a leading cause of vision loss in DR. Marmosets maintained on 30% galactose (gal)-rich diet for 2 years were monitored for retinal vascular permeability, development of ME, and morphological characteristics including acellular capillaries (AC) and pericyte loss (PL), vessel tortuosity, and capillary basement membrane (BM) thickness. Excess vascular permeability, increased number of AC and PL, vascular BM thickening, and increased vessel tortuosity were observed in the retinas of gal-fed marmosets. Optical coherence tomography (OCT) images revealed significant thickening of the retinal foveal and the juxtafoveal area, and histological analysis showed incipient microaneurysms in retinas of gal-fed marmosets. Findings from this study indicate that hyperhexosemia can trigger retinal vascular changes similar to those seen in human DR including ME and microaneurysms. The striking similarities between the marmoset retina and the human retina, and the exceptionally small size of the monkey, offer significant advantages to this primate model of DR.

Increasing prevalence of type 2 diabetes in women of childbearing age has led to a higher incidence of diabetes-associated birth defects. We established a model of type 2 diabetic embryopathy by feeding 4-week-old female mice a high-fat diet (HFD) (60% fat). After 15 weeks on HFD, the mice showed characteristics of type 2 diabetes mellitus (DM) and were mated with lean male mice. During pregnancy, control dams fed a normal diet (10% fat) were maintained on either normal diet or HFD, serving as a control group with elevated circulating free fatty acids. DM dams produced offspring at a rate of 11.3% for neural tube defect (NTD) formation, whereas no embryos in the control groups developed NTDs. Elevated markers of oxidative stress, endoplasmic reticulum stress, caspase activation, and neuroepithelial cell apoptosis (causal events in type 1 diabetic embryopathy) were observed in embryos of DM dams. DM dams treated with 200 mg/kg metformin in drinking water ameliorated fasting hyperglycemia, glucose intolerance, and insulin resistance with consequent reduction of cellular stress, apoptosis, and NTDs in their embryos. We conclude that cellular stress and apoptosis occur and that metformin effectively reduces type 2 diabetic embryopathy in a useful rodent model.

GLP-1 receptor (GLP-1R) agonists may improve endothelial function (EF) via metabolic improvement and direct vascular action. The current study determined the effect of GLP-1R agonist exenatide on postprandial EF in type 2 diabetes and the mechanisms underlying GLP-1R agonist-mediated vasodilation. Two crossover studies were conducted: 36 participants with type 2 diabetes received subcutaneous exenatide or placebo for 11 days and EF, and glucose and lipid responses to breakfast and lunch were determined; and 32 participants with impaired glucose tolerance (IGT) or diet-controlled type 2 diabetes had EF measured before and after intravenous exenatide, with or without the GLP-1R antagonist exendin-9. Mechanisms of GLP-1R agonist action were studied ex vivo on human subcutaneous adipose tissue arterioles and endothelial cells. Subcutaneous exenatide increased postprandial EF independent of reductions in plasma glucose and triglycerides. Intravenous exenatide increased fasting EF, and exendin-9 abolished this effect. Exenatide elicited eNOS activation and NO production in endothelial cells, and induced dose-dependent vasorelaxation and reduced high-glucose or lipid-induced endothelial dysfunction in arterioles ex vivo. These effects were reduced with AMPK inhibition. In conclusion, exenatide augmented postprandial EF in subjects with diabetes and prevented high-glucose and lipid-induced endothelial dysfunction in human arterioles. These effects were largely direct, via GLP-1R and AMPK activation.

Intrauterine exposure to gestational diabetes mellitus (GDM) is linked to development of hypertension, obesity, and type 2 diabetes in children. Our previous studies determined that endothelial colony-forming cells (ECFCs) from neonates exposed to GDM exhibit impaired function. The current goals were to identify aberrantly expressed genes that contribute to impaired function of GDM-exposed ECFCs and to evaluate for evidence of altered epigenetic regulation of gene expression. Genome-wide mRNA expression analysis was conducted on ECFCs from control and GDM pregnancies. Candidate genes were validated by quantitative RT-PCR and Western blotting. Bisulfite sequencing evaluated DNA methylation of placenta-specific 8 (PLAC8). Proliferation and senescence assays of ECFCs transfected with siRNA to knockdown PLAC8 were performed to determine functional impact. Thirty-eight genes were differentially expressed between control and GDM-exposed ECFCs. PLAC8 was highly expressed in GDM-exposed ECFCs, and PLAC8 expression correlated with maternal hyperglycemia. Methylation status of 17 CpG sites in PLAC8 negatively correlated with mRNA expression. Knockdown of PLAC8 in GDM-exposed ECFCs improved proliferation and senescence defects. This study provides strong evidence in neonatal endothelial progenitor cells that GDM exposure in utero leads to altered gene expression and DNA methylation, suggesting the possibility of altered epigenetic regulation.

The FTO gene harbors variation with the strongest effect on adiposity and obesity risk. Previous data support a role for FTO variation in influencing food intake. We conducted a combined analysis of 16,094 boys and girls aged 1-18 years from 14 studies to examine the following: 1) the association between the FTO rs9939609 variant (or a proxy) and total energy and macronutrient intake; and 2) the interaction between the FTO variant and dietary intake, and the effect on BMI. We found that the BMI-increasing allele (minor allele) of the FTO variant was associated with increased total energy intake (effect per allele = 14.3 kcal/day [95% CI 5.9, 22.7 kcal/day], P = 6.5 × 10(-4)), but not with protein, carbohydrate, or fat intake. We also found that protein intake modified the association between the FTO variant and BMI (interactive effect per allele = 0.08 SD [0.03, 0.12 SD], P for interaction = 7.2 × 10(-4)): the association between FTO genotype and BMI was much stronger in individuals with high protein intake (effect per allele = 0.10 SD [0.07, 0.13 SD], P = 8.2 × 10(-10)) than in those with low intake (effect per allele = 0.04 SD [0.01, 0.07 SD], P = 0.02). Our results suggest that the FTO variant that confers a predisposition to higher BMI is associated with higher total energy intake, and that lower dietary protein intake attenuates the association between FTO genotype and adiposity in children and adolescents.

Diabetes is characterized by altered metabolism of key molecules and regulatory pathways. The phenotypic expression of diabetes and associated complications encompasses complex interactions between genetic, environmental, and tissue-specific factors that require an integrated understanding of perturbations in the network of genes, proteins, and metabolites. Metabolomics attempts to systematically identify and quantitate small molecule metabolites from biological systems. The recent rapid development of a variety of analytical platforms based on mass spectrometry and nuclear magnetic resonance have enabled identification of complex metabolic phenotypes. Continued development of bioinformatics and analytical strategies has facilitated the discovery of causal links in understanding the pathophysiology of diabetes and its complications. Here, we summarize the metabolomics workflow, including analytical, statistical, and computational tools, highlight recent applications of metabolomics in diabetes research, and discuss the challenges in the field.