The aim of this study was to determine whether antecedent stimulation of γ-aminobutyric acid (GABA) A receptors with the benzodiazepine alprazolam can blunt physiologic responses during next-day moderate (90 min) exercise in healthy man. Thirty-one healthy individuals (16 male/15 female aged 28 ± 1 year, BMI 23 ± 3 kg/m(2)) were studied during separate, 2-day protocols. Day 1 consisted of morning and afternoon 2-h hyperinsulinemic-euglycemic or hypoglycemic clamps with or without 1 mg alprazolam given 30 min before a clamp. Day 2 consisted of 90-min euglycemic cycling exercise at 50% VO2max. Despite similar euglycemia (5.3 ± 0.1 mmol/L) and insulinemia (46 ± 6 pmol/L) during day 2 exercise studies, GABA A activation with alprazolam during day 1 euglycemia resulted in significant blunting of plasma epinephrine, norepinephrine, glucagon, cortisol, and growth hormone responses. Lipolysis (glycerol, nonesterified fatty acids) and endogenous glucose production during exercise were also reduced, and glucose infusion rates were increased following prior euglycemia with alprazolam. Prior hypoglycemia with alprazolam resulted in further reduction of glucagon and cortisol responses during exercise. We conclude that prior activation of GABA A pathways can play a significant role in blunting key autonomous nervous system, neuroendocrine, and metabolic physiologic responses during next-day exercise in healthy man.

Staphylococcus aureus is an opportunistic bacterium capable of causing a wide range of severe diseases when it gains access to underlying tissues. Paradoxically, S. aureus is a common inhabitant of the skin microflora and colonizes the nares and other human mucosa. The purpose of this study was to determine the genetic basis for the differences in the pathogenic versus colonizing potential of S. aureus isolated from diabetic foot ulcers (DFUs). By performing optical map comparisons of a collection of S. aureus strains isolated from DFUs, we brought to light a prophage present in noninfecting bacteria. The phage, namely ROSA-like, was localized in a hotspot region ΦNM2 near the locus isd, the iron surface determinant system. The integrated phage significantly reduces the virulence of the strain and increases the biofilm formation. DFUs seem to be a specific niche of this colonizing strain. The ROSA-like phage represents the first description of a mobile element present mainly in S. aureus isolated from DFUs, which modulates the relationship of the bacteria with its human host. This phage appears to attenuate bacterial virulence and promote colonization.

Aberrant Wnt signaling appears to play an important role in the onset of diabetes. Moreover, the insulin signaling pathway is defective in the nucleus tractus solitarii (NTS) of spontaneously hypertensive rats (SHRs) and fructose-fed rats. Nevertheless, the relationships between Wnt signaling and the insulin pathway and the related modulation of blood pressure (BP) in the central nervous system have yet to be established. The aim of this study was to investigate the potential signaling pathways involved in Wnt-mediated BP regulation in the NTS. Pretreatment with the LDL receptor-related protein (LRP) antagonist Dickkopf-1 (DKK1) significantly attenuated the Wnt3a-induced depressor effect and nitric oxide production. Additionally, the inhibition of LRP6 activity using DKK1 significantly abolished Wnt3a-induced glycogen synthase kinase 3β (GSK-3β)(S9), extracellular signal-regulated kinases 1/2(T202/Y204), ribosomal protein S6 kinase(T359/S363), and Akt(S473) phosphorylation; and increased insulin receptor substrate 1 (IRS1)(S332) phosphorylation. GSK-3β was also found to bind directly to IRS1 and to induce the phosphorylation of IRS1 at serine 332 in the NTS. By contrast, administration of the GSK-3β inhibitor TWS119 into the brain decreased the BP of hypertensive rats by enhancing IRS1 activity. Taken together, these results suggest that the GSK-3β-IRS1 pathway may play a significant role in Wnt-mediated central BP regulation.

The aim of this study was 1) to clarify β-cell regenerative capacity in the face of glucocorticoid (GC)-induced insulin resistance and 2) to clarify the change in β- and α-cell mass in GC-induced diabetes in humans. We obtained the pancreases from 100 Japanese autopsy case subjects. The case subjects were classified according to whether or not they had received GC therapy before death and the presence or absence of diabetes. Fractional β-cell area (%BCA) and α-cell area (%ACA) were quantified, and the relationship with GC therapy was evaluated. As a result, in case subjects without diabetes, there was no significant difference in %BCA between case subjects with and without GC therapy (1.66 ± 1.05% vs. 1.21 ± 0.59%, P = 0.13). %ACA was also not significantly different between the two groups. In case subjects with type 2 diabetes, %BCA and %ACA were both significantly reduced compared with control subjects without diabetes; however, neither %BCA nor %ACA was significantly decreased in case subjects with GC-induced diabetes. There was a significant negative correlation between %BCA and HbA1c measured before death; however, this relationship was attenuated in case subjects with GC therapy. In conclusion, the current study suggests that β- and α-cell mass remain largely unchanged in the face of GC-induced insulin resistance in Japanese individuals, implying limited capacity of β-cell regeneration in adult humans. The absence of apparent β-cell deficit in case subjects with GC-induced diabetes suggests that GC-induced diabetes is mainly caused by insulin resistance and/or β-cell dysfunction, but not necessarily a deficit of β-cell mass.

Glucagon-like peptide-1 (GLP-1) and cholecystokinin (CCK) exert important complementary beneficial metabolic effects. This study assessed the biological actions and therapeutic utility of a novel (pGlu-Gln)-CCK-8/exendin-4 hybrid peptide compared with the stable GLP-1 and CCK mimetics exendin-4 and (pGlu-Gln)-CCK-8, respectively. All peptides significantly enhanced in vitro insulin secretion. Administration of the peptides, except (pGlu-Gln)-CCK-8 alone, in combination with glucose significantly lowered plasma glucose and increased plasma insulin in mice. All treatments elicited appetite-suppressive effects. Twice-daily administration of the novel (pGlu-Gln)-CCK-8/exendin-4 hybrid, (pGlu-Gln)-CCK-8 alone, or (pGlu-Gln)-CCK-8 in combination with exendin-4 for 21 days to high-fat-fed mice significantly decreased energy intake, body weight, and circulating plasma glucose. HbA1c was reduced in the (pGlu-Gln)-CCK-8/exendin-4 hybrid and combined parent peptide treatment groups. Glucose tolerance and insulin sensitivity also were improved by all treatment modalities. Interestingly, locomotor activity was decreased in the hybrid peptide group, and these mice also exhibited reductions in circulating triglyceride and cholesterol levels. Pancreatic islet number and area, as well β-cell area and insulinotropic responsiveness, were dramatically improved by all treatments. These studies highlight the clear potential of dual activation of GLP-1 and CCK1 receptors for the treatment of type 2 diabetes.

Insulin stimulates the translocation fatty acid transport protein 1 (FATP1) to plasma membrane, and thus greater free fatty acid (FFA) uptake, in adipocyte cell models. Whether insulin stimulates greater FFA clearance into adipose tissue in vivo is unknown. We tested this hypothesis by comparing direct FFA storage in subcutaneous adipose tissue during insulin versus niacin-medicated suppression of lipolysis. We measured direct FFA storage in abdominal and femoral subcutaneous fat in 10 and 11 adults, respectively, during euglycemic hyperinsulinemia or after oral niacin to suppress FFA compared with 11 saline control experiments. Direct palmitate storage was assessed using a [U-(13)C]palmitate infusion to measure palmitate kinetics and an intravenous palmitate radiotracer bolus/timed biopsy. Plasma palmitate concentrations and flux were suppressed to 23 ± 3 and 26 ± 5 µmol ⋅ L(-1) (P = 0.91) and 44 ± 4 and 39 ± 5 µmol ⋅ min(-1) (P = 0.41) in the insulin and niacin groups, respectively, much less (P < 0.001) than the saline control group (102 ± 8 and 104 ± 12 µmol ⋅ min(-1), respectively). In the insulin, niacin, and saline groups, abdominal palmitate storage rates were 0.25 ± 0.05 vs. 0.25 ± 0.07 vs. 0.32 ± 0.05 µmol ⋅ kg adipose lipid(-1) ⋅ min(-1), respectively (P = NS), and femoral adipose storage rates were 0.19 ± 0.06 vs. 0.20 ± 0.05 vs. 0.31 ± 0.05 µmol ⋅ kg adipose lipid(-1) ⋅ min(-1), respectively (P = NS). In conclusion, insulin does not increase FFA storage in adipose tissue compared with niacin, which suppresses lipolysis via a different pathway.

26RFa is a hypothalamic neuropeptide that promotes food intake. 26RFa is upregulated in obese animal models, and its orexigenic activity is accentuated in rodents fed a high-fat diet, suggesting that this neuropeptide might play a role in the development and maintenance of the obese status. As obesity is frequently associated with type 2 diabetes, we investigated whether 26RFa may be involved in the regulation of glucose homeostasis. In the current study, we show a moderate positive correlation between plasma 26RFa levels and plasma insulin in patients with diabetes. Plasma 26RFa concentration also increases in response to an oral glucose tolerance test. In addition, we found that 26RFa and its receptor GPR103 are present in human pancreatic β-cells as well as in the gut. In mice, 26RFa attenuates the hyperglycemia induced by a glucose load, potentiates insulin sensitivity, and increases plasma insulin concentrations. Consistent with these data, 26RFa stimulates insulin production by MIN6 insulinoma cells. Finally, we show, using in vivo and in vitro approaches, that a glucose load induces a massive secretion of 26RFa by the small intestine. Altogether, the present data indicate that 26RFa acts as an incretin to regulate glucose homeostasis.

The prevalence of obesity-related diabetes is increasing worldwide. Here we report the identification of a pentapeptide, GLP-1(32-36)amide (LVKGRamide), derived from the glucoincretin hormone GLP-1, that increases basal energy expenditure and curtails the development of obesity, insulin resistance, diabetes, and hepatic steatosis in diet-induced obese mice. The pentapeptide inhibited weight gain, reduced fat mass without change in energy intake, and increased basal energy expenditure independent of physical activity. Analyses of tissues from peptide-treated mice reveal increased expression of UCP-1 and UCP-3 in brown adipose tissue and increased UCP-3 and inhibition of acetyl-CoA carboxylase in skeletal muscle, findings consistent with increased fatty acid oxidation and thermogenesis. In palmitate-treated C2C12 skeletal myotubes, GLP-1(32-36)amide activated AMPK and inhibited acetyl-CoA carboxylase, suggesting activation of fat metabolism in response to energy depletion. By mass spectroscopy, the pentapeptide is rapidly formed from GLP-1(9-36)amide, the major form of GLP-1 in the circulation of mice. These findings suggest that the reported insulin-like actions of GLP-1 receptor agonists that occur independently of the GLP-1 receptor might be mediated by the pentapeptide, and the previously reported nonapeptide (FIAWLVKGRamide). We propose that by increasing basal energy expenditure, GLP-1(32-36)amide might be a useful treatment for human obesity and associated metabolic disorders.

Elevated cardiac triacylglycerol (TAG) content is traditionally equated with cardiolipotoxicity and suggested to be a culprit in cardiac dysfunction. However, previous work demonstrated that myosin heavy-chain-mediated cardiac-specific overexpression of diacylglycerol transferase 1 (MHC-DGAT1), the primary enzyme for TAG synthesis, preserved cardiac function in two lipotoxic mouse models despite maintaining high TAG content. Therefore, we examined whether increased cardiomyocyte TAG levels due to DGAT1 overexpression led to changes in cardiac TAG turnover rates under normoxia and ischemia-reperfusion conditions. MHC-DGAT1 mice had elevated TAG content and synthesis rates, which did not alter cardiac function, substrate oxidation, or myocardial energetics. MHC-DGAT1 hearts had ischemia-induced lipolysis; however, when a physiologic mixture of long-chain fatty acids was provided, enhanced TAG turnover rates were associated with improved functional recovery from low-flow ischemia. Conversely, exogenous supply of palmitate during reperfusion suppressed elevated TAG turnover rates and impaired recovery from ischemia in MHC-DGAT1 hearts. Collectively, this study shows that elevated TAG content, accompanied by enhanced turnover, does not adversely affect cardiac function and, in fact, provides cardioprotection from ischemic stress. In addition, the results highlight the importance of exogenous supply of fatty acids when assessing cardiac lipid metabolism and its relationship with cardiac function.

Activation of AMPK suppresses inflammation, but the underlying mechanisms remain poorly understood. This study was designed to characterize the molecular mechanisms by which AMPK suppresses vascular inflammation. In cultured human aortic smooth muscle cells, pharmacologic or genetic activation of AMPK inhibited the signal transducer and activator of transcription-1 (STAT1), while inhibition of AMPK had opposite effects. Deletion of AMPKα1 or AMPKα2 resulted in activation of STAT1 and in increases in proinflammatory mediators, both of which were attenuated by administration of STAT1 small interfering RNA or fludarabine, a selective STAT1 inhibitor. Moreover, AMPK activation attenuated the proinflammatory actions induced by STAT1 activators such as interferon-γ and angiotensin II (AngII). Mechanistically, we found that AMPK activation increased, whereas AMPK inhibition decreased, the levels of mitogen-activated protein kinase phosphatase-1 (MKP-1), an inducible nuclear phosphatase, by regulating proteasome-dependent degradation of MKP-1. Gene silencing of MKP-1 increased STAT1 phosphorylation and prevented 5-aminoimidazole-4-carboxyamide ribonucleoside-reduced STAT1 phosphorylation. Finally, we found that infusion of AngII caused a more severe inflammatory response in AMPKα2 knockout mouse aortas, all of which were suppressed by chronic administration of fludarabine. We conclude that AMPK activation suppresses STAT1 signaling and inhibits vascular inflammation through the upregulation of MKP-1.

Understanding the molecular networks controlling ectopic lipid deposition, glucose tolerance, and insulin sensitivity is essential to identifying new pharmacological approaches to treat type 2 diabetes. We recently identified serine/threonine protein kinase 25 (STK25) as a negative regulator of glucose and insulin homeostasis based on observations in myoblasts with acute depletion of STK25 and in STK25-overexpressing transgenic mice. Here, we challenged Stk25 knockout mice and wild-type littermates with a high-fat diet and showed that STK25 deficiency suppressed development of hyperglycemia and hyperinsulinemia, improved systemic glucose tolerance, reduced hepatic gluconeogenesis, and increased insulin sensitivity. Stk25(-/-) mice were protected from diet-induced liver steatosis accompanied by decreased protein levels of acetyl-CoA carboxylase, a key regulator of both lipid oxidation and synthesis. Lipid accumulation in Stk25(-/-) skeletal muscle was reduced, and expression of enzymes controlling the muscle oxidative capacity (Cpt1, Acox1, Cs, Cycs, Ucp3) and glucose metabolism (Glut1, Glut4, Hk2) was increased. These data are consistent with our previous study of STK25 knockdown in myoblasts and reciprocal to the metabolic phenotype of Stk25 transgenic mice, reinforcing the validity of the results. The findings suggest that STK25 deficiency protects against the metabolic consequences of chronic exposure to dietary lipids and highlight the potential of STK25 antagonists for the treatment of type 2 diabetes.

Endothelial nitric oxide (NO) signaling plays a physiological role in limiting obesity-associated insulin resistance and inflammation. This study was undertaken to investigate whether this NO effect involves polarization of macrophages toward an anti-inflammatory M2 phenotype. Mice with transgenic endothelial NO synthase overexpression were protected against high-fat diet (HFD)-induced hepatic inflammation and insulin resistance, and this effect was associated with reduced proinflammatory M1 and increased anti-inflammatory M2 activation of Kupffer cells. In cell culture studies, exposure of macrophages to endothelial NO similarly reduced inflammatory (M1) and increased anti-inflammatory (M2) gene expression. Similar effects were induced by macrophage overexpression of vasodilator-stimulated phosphoprotein (VASP), a key downstream mediator of intracellular NO signaling. Conversely, VASP deficiency induced proinflammatory M1 macrophage activation, and the transplantation of bone marrow from VASP-deficient donor mice into normal recipients caused hepatic inflammation and insulin resistance resembling that induced in normal mice by consumption of an HFD. These data suggest that proinflammatory macrophage M1 activation and macrophage-mediated inflammation are tonically inhibited by NO → VASP signal transduction, and that reduced NO → VASP signaling is involved in the effect of HFD feeding to induce M1 activation of Kupffer cells and associated hepatic inflammation. Our data implicate endothelial NO → VASP signaling as a physiological determinant of macrophage polarization and show that signaling via this pathway is required to prevent hepatic inflammation and insulin resistance.

Metabolic syndrome (MetS) doubles the risk of adverse cardiovascular events. Glucagon-like peptide 1 (GLP-1) receptor agonists induce weight loss, increase insulin secretion, and improve glucose tolerance. Studies in healthy animals suggest cardioprotective properties of GLP-1 receptor agonists, perhaps partially mediated by improved sarco-endoplasmic reticulum Ca(2+) ATPase (SERCA) activity. We examined the acute effect of GLP-1 receptor agonists on coronary smooth muscle cells (CSM) enzymatically isolated from lean, healthy Ossabaw miniature swine. Intracellular Ca(2+) handling was interrogated with fura-2. The GLP-1 receptor agonist exenatide activated SERCA but did not alter other Ca(2+) transporters. Further, we tested the hypothesis that chronic, in vivo treatment with GLP-1 receptor agonist AC3174 would attenuate coronary artery disease (CAD) in swine with MetS. MetS was induced in 20 swine by 6 months' feeding of a hypercaloric, atherogenic diet. Swine were then randomized (n = 10/group) into placebo or AC3174 treatment groups and continued the diet for an additional 6 months. AC3174 treatment attenuated weight gain, increased insulin secretion, and improved glucose tolerance. Intravascular ultrasound and histology showed no effect of AC3174 on CAD. MetS abolished SERCA activation by GLP-1 receptor agonists. We conclude that MetS confers vascular resistance to GLP-1 receptor agonists, partially through impaired cellular signaling steps involving SERCA.

Mitochondrial ADP transport may represent a convergence point unifying two prominent working models for the development of insulin resistance, as reactive lipids (specifically palmitoyl-CoA [P-CoA]) can inhibit ADP transport and subsequently increase mitochondrial reactive oxygen species emissions. In the current study, we aimed to determine if exercise training in humans diminished P-CoA attenuation of mitochondrial ADP respiratory sensitivity. Six weeks of exercise training increased whole-body glucose homeostasis and skeletal muscle Akt signaling and reduced markers of oxidative stress without reducing maximal mitochondrial H2O2 emissions. To ascertain if enhanced mitochondrial ADP transport contributed to the improvement in the in vivo oxidative state, we determined mitochondrial ADP sensitivity in the presence and absence of P-CoA. In the absence of P-CoA, exercise training reduced mitochondrial ADP sensitivity. In contrast, exercise training increased mitochondrial ADP sensitivity with P-CoA present. We further show that P-CoA noncompetitively inhibits mitochondrial ADP transport and the ability of ADP to attenuate mitochondrial H2O2 emission. Altogether, the current data provide a potential mechanism for how P-CoA contributes to insulin resistance and highlight the ability of exercise training to diminish P-CoA attenuation in mitochondrial ADP transport.

Dietary polyphenols protect against metabolic syndrome, despite limited absorption and digestion, raising questions about their mechanism of action. We hypothesized that one mechanism may involve the gut microbiota. To test this hypothesis, C57BL/6J mice were fed a high-fat diet (HFD) containing 1% Concord grape polyphenols (GP). Relative to vehicle controls, GP attenuated several effects of HFD feeding, including weight gain, adiposity, serum inflammatory markers (tumor necrosis factor [TNF]α, interleukin [IL]-6, and lipopolysaccharide), and glucose intolerance. GP lowered intestinal expression of inflammatory markers (TNFα, IL-6, inducible nitric oxide synthase) and a gene for glucose absorption (Glut2). GP increased intestinal expression of genes involved in barrier function (occludin) and limiting triglyceride storage (fasting-induced adipocyte factor). GP also increased intestinal gene expression of proglucagon, a precursor of proteins that promote insulin production and gut barrier integrity. 16S rRNA gene sequencing and quantitative PCR of cecal and fecal samples demonstrated that GP dramatically increased the growth of Akkermansia muciniphila and decreased the proportion of Firmicutes to Bacteroidetes, consistent with prior reports that similar changes in microbial community structure can protect from diet-induced obesity and metabolic disease. These data suggest that GP act in the intestine to modify gut microbial community structure, resulting in lower intestinal and systemic inflammation and improved metabolic outcomes. The gut microbiota may thus provide the missing link in the mechanism of action of poorly absorbed dietary polyphenols.

Islet autoantibodies detected at disease onset in patients with type 1 diabetes are signs of an autoimmune destruction of the insulin-producing β-cells. To further investigate the genetic determinants of autoantibody positivity, we performed dense immune-focused genotyping on the Immunochip array and tested for association with seven disease-specific autoantibodies in a large cross-sectional cohort of 6,160 type 1 diabetes-affected siblings. The genetic association with positivity for GAD autoantibodies (GADAs), IA2 antigen (IA-2A), zinc transporter 8, thyroid peroxidase, gastric parietal cells (PCAs), tissue transglutaminase, and 21-hydroxylase was tested using a linear mixed-model regression approach to simultaneously control for population structure and family relatedness. Four loci were associated with autoantibody positivity at genome-wide significance. Positivity for GADA was associated with 3q28/LPP, for IA-2A with 1q23/FCRL3 and 11q13/RELA, and for PCAs with 2q24/IFIH1. The 3q28 locus showed association after only 3 years duration and might therefore be a marker of persistent GADA positivity. The 1q23, 11q13, and 2q24 loci were associated with autoantibodies close to diabetes onset and constitute candidates for early screening. Major susceptibility loci for islet autoantibodies are separate from type 1 diabetes risk, which may have consequences for intervention strategies to reduce autoimmunity.

Perilipin 5 (PLIN5) is a lipid droplet protein and is highly expressed in oxidative tissue. Expression of the PLIN5 gene is regulated by peroxisome proliferator-activated receptor-α, fasting, and exercise. However, the effect of increased muscle PLIN5 expression on whole-body energy homeostasis remains unclear. To examine this, we developed a mouse line with skeletal muscle PLIN5 overexpression (MCK-Plin5). We show that MCK-Plin5 mice have increased energy metabolism and accumulate more intramyocellular triacylglycerol but have normal glucose and insulin tolerance. MCK-Plin5 mice fed high-fat chow manifest lower expression of inflammatory markers in their liver and increased expression of "browning" factors in adipose tissue. This muscle-driven phenotype is, at least in part, mediated by myokines; the MCK-Plin5 mice have 80-fold higher FGF21 gene expression in muscle and increased serum FGF21 concentration. The increase in FGF21 occurs mainly in muscles with a predominance of fast-twitch fibers, suggesting that fiber type-specific lipid storage may be part of the mechanism conferring metabolic protection in MCK-Plin5 mice. In conclusion, upregulating the PLIN5 level in skeletal muscle drives expression of the FGF21 gene in fast-twitch fibers and is metabolically protective. These findings provide insight into the physiology of PLIN5 and the potential contribution of its upregulation during exercise.

Intestine contributes to energy homeostasis through the absorption, metabolism, and transfer of nutrients to the organism. We demonstrated previously that hepatocyte nuclear receptor-4α (HNF-4α) controls intestinal epithelium homeostasis and intestinal absorption of dietary lipids. HNF-4γ, the other HNF-4 form highly expressed in intestine, is much less studied. In HNF-4γ knockout mice, we detect an exaggerated insulin peak and improvement in glucose tolerance during oral but not intraperitoneal glucose tolerance tests, highlighting the involvement of intestine. Moreover, the enteroendocrine L-type cell lineage is modified, as assessed by the increased expression of transcription factors Isl1, Foxa1/2, and Hnf4a, leading to an increase of both GLP-1-positive cell number and basal and stimulated GLP-1 plasma levels potentiating the glucose-stimulated insulin secretion. Using the GLP-1 antagonist exendin (9-39), we demonstrate a direct effect of GLP-1 on improved glucose tolerance. GLP-1 exerts a trophic effect on pancreatic β-cells, and we report an increase of the β-cell fraction correlated with an augmented number of proliferative islet cells and with resistance to streptozotocin-induced diabetes. In conclusion, the loss of HNF-4γ improves glucose homeostasis through a modulation of the enteroendocrine cell lineage.