Tumor Necrosis Factor-α Antagonism Improves Vasodilation During Hyperinsulinemia in Metabolic Syndrome

OBJECTIVE—Obesity is associated with chronic inflammation due to overproduction of proinflammatory cytokines, including tumor necrosis factor (TNF)-α. We assessed the effects of TNF-α neutralization by infliximab on vascular reactivity during hyperinsulinemia in obesity-related metabolic syndrome

Non-linear Analysis of Heart rate Variability Improves Differential Diagnosis Between Parkinson Diseases and Multiple System Atrophy

Aims: Parkinson's disease (PD) and multiple system atrophy (MSA) are neurodegenerative disorders characterized by motor "parkinsonian" symptoms and non-motor symptoms related to autonomic nervous system (ANS) dysfunction. The latter can be quantified with the analysis of Heart Rate Variability (HRVa) and of its complexity. In this study nonlinear (NL) HRV complexity parameters were calculated to assess their predictive accuracy as markers of “disease” useful for early differentiation between PD and MSA in parkinsonian syndromes of uncertain diagnosis. Study Design: Observational study. Place and Duration of Study: Clinical Physiology-Biomagnetism Center, Policlinico A. Gemelli, Rome Italy. Patients enrolled from January 2010 to October 2013. Methodology: 51 patients [25 with “certain” diagnosis of PD, 9 with a “highly probable” diagnosis of MSA and 17 with parkinsonian syndromes of uncertain neurological definition (6 with “undefined parkinsonism” and 11 with “suspected MSA”)] and 40 age-matched healthy control subjects were studied. Short-term NL HRVa was performed during daily activity and during REM/NREM sleep from 24 h ECG recordings. Discriminant analysis (DA) was used to identify which NL HRV parameters (or their combination) were efficient to differentiate between PD and MSA in cases of uncertain diagnosis. Results: Compared with healthy controls, most NL HRV parameters were significantly altered in patients (p<0.05), during both active and passive awakeness and during sleep. Most evident HRV abnormalities were found during active awakeness in MSA. DA of recurrence plot parameters provided the best predictive accuracy (76.5%) for the classification of parkinsonian patients with uncertain diagnosis. Conclusion: NL HRVa is efficient in differentiating MSA from PD and may improve earlier diagnosis in patients with parkinsonian symptoms of uncertain nature, useful to address second level diagnostic steps and to guide more individualized drug treatment

Glucocorticoid-Treated Mice Are an Inappropriate Positive Control for Long-Term Preclinical Studies in the mdx Mouse

Dmd(mdx) (mdx) mice are used as a genetic and biochemical model of dystrophin deficiency. The long-term consequences of glucocorticoid (GC) treatment on dystrophin-deficient skeletal and heart muscle are not yet known. Here we used systematic phenotyping to assess the long-term consequences of GC treatment in mdx mice. Our investigation addressed not only the effects of GC on the disease phenotype but also the question of whether GCs can be used as a positive control for preclinical drug evaluations.We performed nine pre-clinical efficacy trials (treated N = 129, untreated N = 106) of different durations in 9-to-50-week-old dystrophic mdx mice over a 3-year time period using standardized methods. In all these trials, we used either 1 mg/kg body weight of prednisone or 5 mg/kg body weight of prednisolone as positive controls to compare the efficacy of various test drugs. Data from untreated controls and GC-treated mice in the various trials have been pooled and analyzed to assess the effects of GCs on dystrophin-deficient skeletal and cardiac muscles of mdx mice. Our results indicate that continuous GC treatment results in early (e.g., at 50 days) improvements in normalized parameters such as grip strength, motor coordination and maximal in vitro force contractions on isolated EDL muscle, but these initial benefits are followed by a progressive loss of muscle strength after 100 days. We also found a significant increase in heart fibrosis that is reflected in a significant deterioration in cardiac systolic function after 100 days of treatment.Continuous administration of prednisone to mdx mice initially improves skeletal muscle strength, but further therapy result in deterioration of muscle strength and cardiac function associated with enhanced cardiac fibrosis. These results suggest that GCs may not serve as an appropriate positive control for long-term mdx mouse preclinical trials

A Concerted Kinase Interplay Identifies PPARγ as a Molecular Target of Ghrelin Signaling in Macrophages

The peroxisome proliferator-activator receptor PPARγ plays an essential role in vascular biology, modulating macrophage function and atherosclerosis progression. Recently, we have described the beneficial effect of combined activation of the ghrelin/GHS-R1a receptor and the scavenger receptor CD36 to induce macrophage cholesterol release through transcriptional activation of PPARγ. Although the interplay between CD36 and PPARγ in atherogenesis is well recognized, the contribution of the ghrelin receptor to regulate PPARγ remains unknown. Here, we demonstrate that ghrelin triggers PPARγ activation through a concerted signaling cascade involving Erk1/2 and Akt kinases, resulting in enhanced expression of downstream effectors LXRα and ABC sterol transporters in human macrophages. These effects were associated with enhanced PPARγ phosphorylation independently of the inhibitory conserved serine-84. Src tyrosine kinase Fyn was identified as being recruited to GHS-R1a in response to ghrelin, but failure of activated Fyn to enhance PPARγ Ser-84 specific phosphorylation relied on the concomitant recruitment of docking protein Dok-1, which prevented optimal activation of the Erk1/2 pathway. Also, substitution of Ser-84 preserved the ghrelin-induced PPARγ activity and responsiveness to Src inhibition, supporting a mechanism independent of Ser-84 in PPARγ response to ghrelin. Consistent with this, we found that ghrelin promoted the PI3-K/Akt pathway in a Gαq-dependent manner, resulting in Akt recruitment to PPARγ, enhanced PPARγ phosphorylation and activation independently of Ser-84, and increased expression of LXRα and ABCA1/G1. Collectively, these results illustrate a complex interplay involving Fyn/Dok-1/Erk and Gαq/PI3-K/Akt pathways to transduce in a concerted manner responsiveness of PPARγ to ghrelin in macrophages

Ghrelin improves endothelial function in patients with metabolic syndrome

Background-Metabolic syndrome importantly accelerates the atherosclerotic process, the earliest event of which is endothelial dysfunction. Ghrelin, a gastric peptide with cardiovascular actions, has been shown to inhibit proatherogenic changes in experimental models. This study therefore investigated whether ghrelin administration might beneficially affect endothelial function in metabolic syndrome. Methods and Results-Endothelium-dependent and -independent vasodilator responses to intra-arterial infusion of increasing doses of acetylcholine and sodium nitroprusside ( SNP), respectively, were assessed by strain-gauge plethysmography before and after local administration of human ghrelin (200 mu g/min). During saline, the vasodilator response to acetylcholine was significantly blunted (P = 0.008) in patients with metabolic syndrome (n = 12, 5 female) compared with controls (n = 12, 7 female), whereas the vasodilator response to SNP was not different between groups (P = 0.68). In patients with metabolic syndrome, basal plasma ghrelin was significantly lower than in controls (P = 0.02). In these patients, ghrelin infusion markedly increased intravascular concentrations of the peptide (P = 0.001) and resulted in a potentiation of the vasodilator response to acetylcholine (P = 0.001 versus saline) but not to SNP (P = 0.22). This effect was likely related to enhanced nitric oxide bioavailability because, in a group of patients with metabolic syndrome (n = 6, 2 female), ghrelin had no effect on the vasodilator response to acetylcholine (P = 0.78 versus saline) after nitric oxide inhibition by N-G-monomethyl-L-arginine. Conclusions-These findings indicate that ghrelin reverses endothelial dysfunction in patients with metabolic syndrome by increasing nitric oxide bioactivity, thereby suggesting that decreased circulating levels of the peptide, such as those found in these patients, might play a role in the pathobiology of atherosclerosis

Ghrelin has novel vascular actions that mimic PI 3-kinase-dependent actions of insulin to stimulate production of NO from endothelial cells

Ghrelin is an orexigenic peptide hormone secreted by the stomach. In patients with metabolic syndrome and low ghrelin levels, intra-arterial ghrelin administration acutely improves their endothelial dysfunction. Therefore, we hypothesized that ghrelin activates endothelial nitric oxide synthase (eNOS) in vascular endothelium, resulting in increased production of nitric oxide (NO) using signaling pathways shared in common with the insulin receptor. Similar to insulin, ghrelin acutely stimulated increased production of NO in bovine aortic endothelial cells (BAEC) in primary culture (assessed using NO-specific fluorescent dye 4,5-diaminofluorescein) in a time- and dose-dependent manner. Production of NO in response to ghrelin (100 nM, 10 min) in human aortic endothelial cells was blocked by pretreatment of cells with NG-nitro-L-arginine methyl ester (nitric oxide synthase inhibitor), wortmannin [phosphatidylinositol (PI) 3-kinase inhibitor], or (D-Lys3)-GHRP-6 (selective antagonist of ghrelin receptor GHSR-1a), as well as by knockdown of GHSR-1a using small-interfering (si) RNA (but not by mitogen/extracellular signal-regulated kinase inhibitor PD-98059). Moreover, ghrelin stimulated increased phosphorylation of Akt (Ser473) and eNOS (Akt phosphorylation site Ser1179) that was inhibitable by knockdown of GHSR-1a using siRNA or by pretreatment of cells with wortmannin but not with PD-98059. Ghrelin also stimulated phosphorylation of mitogen-activated protein (MAP) kinase in BAEC. However, unlike insulin, ghrelin did not stimulate MAP kinase-dependent secretion of the vasoconstrictor endothelin-1 from BAEC. We conclude that ghrelin has novel vascular actions to acutely stimulate production of NO in endothelium using a signaling pathway that involves GHSR-1a, PI 3-kinase, Akt, and eNOS. Our findings may be relevant to developing novel therapeutic strategies to treat diabetes and related diseases characterized by reciprocal relationships between endothelial dysfunction and insulin resistance