Metrnl deficiency decreases blood HDL cholesterol and increases blood triglyceride.

Dyslipidemia is a risk factor for cardiovascular diseases and type 2 diabetes. Several adipokines play important roles in modulation of blood lipids. Metrnl is a recently identified adipokine, and adipose Metrnl participates in regulation of blood triglyceride (TG). In this study, we generated Metrnl global, intestine-specific and liver-specific knockout mice, and explored the effects of Metrnl on serum lipid parameters. Global knockout of Metrnl had no effects on serum lipid parameters under normal chow diet, but increased blood TG by 14%, and decreased total cholesterol (TC) by 16% and high density lipoprotein cholesterol (HDL-C) by 24% under high fat diet. Nevertheless, intestine-specific knockout of Metrnl did not alter the serum lipids parameters under normal chow diet or high fat diet. Notably, liver-specific knockout of Metrnl decreased HDL-C by 24%, TC by 20% and low density lipoprotein cholesterol (LDL-C) by 16% without alterations of blood TG and nonesterified fatty acids (NEFA) under high fat diet. But deficiency of Metrnl in liver did not change VLDL secretion and expression of lipid synthetic and metabolic genes. We conclude that tissue-specific Metrnl controls different components of blood lipids. In addition to modulation of blood TG by adipose Metrnl, blood HDL-C is regulated by liver Metrnl.

Antishock effect of anisodamine involves a novel pathway for activating alpha7 nicotinic acetylcholine receptor.

OBJECTIVE: Vagus nerve stimulation inhibits proinflammatory cytokine production by signaling through the alpha7 nicotinic acetylcholine receptor (alpha7nAChR). Anisodamine, a muscarinic acetylcholine receptor antagonist, has been used clinically in China for treatment of various shocks, but the mechanism was poorly understood. Here, we tested the hypothesis whether anisodamine attained its antishock effect through activation of alpha7nAChR. DESIGN: : Randomized and controlled in vitro and in vivo study. SETTINGS: Research laboratory and animal facility rooms. SUBJECTS: Sprague-Dawley rats, Kunming mice, alpha7nAChR-deficient mice, and RAW264.7 cells. INTERVENTIONS: Sprague-Dawley rats were injected with lipopolysaccharide (LPS) (15 mg/kg, intravenous) to induce septic shock. Methyllycaconitine, a selective alpha7nAChR antagonist, was administered (10 mg/kg, intraperitoneal) 10 minutes before anisodamine (10 mg/kg, intravenous). Mean arterial pressure was monitored and cytokines were analyzed 2 hours after the onset of LPS. In vagotomized mice and alpha7nAChR-deficient mice, the antishock effect of anisodamine was appraised, respectively. RAW264.7 cells were stained by fluorescein isothiocyanate- labeled-alpha-bungarotoxin and the fluorescence intensity was observed. Mice peritoneal macrophages were pretreated and stimulated with LPS, and tumor necrosis factor (TNF)-alpha in the supernatant was measured by enzyme-linked immunosorbent assay. MEASUREMENTS AND MAIN RESULTS: Methyllycaconitine significantly antagonized the beneficial effect of anisodamine on mean arterial pressure and TNF-alpha, interleukin-1beta expression in response to LPS. The antishock effects of anisodamine were markedly attenuated in vagotomized mice and alpha7nAChR-deficient mice. In vitro, anisodamine significantly augmented the effect of acetylcholine on fluorescence intensity stained with fluorescein isothiocyanate-labeled-alpha-bungarotoxin and TNF-alpha production stimulated with LPS. CONCLUSION: These findings demonstrate that the antishock effect of anisodamine is intimately linked to alpha7nAChR-dependent anti-inflammatory pathway.

Exogenous porcine surfactants increase the infiltration of leukocytes in the lung of rats.

BACKGROUND: Several studies have investigated the influence of exogenous surfactants on inflammatory response in the lung, however results reported about effects of surfactants on the lung infiltration of leukocytes are controversial. Our previous study noticed that treatment of porcine surfactant (PS) significantly increased the lung infiltration of leukocytes in rats with acute lung injury (ALI). The objective of this study was to verify the effect of exogenous PS on the lung infiltration of leukocytes in vivo and investigate the possible mechanisms involved in vitro. METHODS: The number of leukocytes in bronchoalveolar lavage fluid (BALF) of rats with or without lipopolysaccharide (LPS)-induced ALI was determined after treatment with different concentrations of PS, dexamethasone (Dex) or PS + Dex. The effect of PS and Curosurf, a commercially available porcine surfactant, on human peripheral neutrophil migration was determined by the Boyden Chamber Assay. RESULTS: Instillation of PS significantly increased the number of leukocytes in BALF of normal rats and rats with LPS-induced ALI. Most of the increased leukocytes were neutrophils. Dex significantly decreased the number of leukocytes and TNF-alpha concentration in BALF caused by LPS, but did not significantly reduce the number of leukocytes increased by PS. In vitro experiments further demonstrated that both PS and Curosurf had direct chemotactic effects on neutrophils. CONCLUSIONS: These results suggest that PS contain chemoattractant(s) which induce the infiltration of leukocytes, especially neutrophils, into lung.

Androgen alleviates neurotoxicity of ß-amyloid peptide (Aß) by promoting microglial clearance of Aß and inhibiting microglial inflammatory response to Aß.

AIMS: Lower androgen level in elderly men is a risk factor of Alzheimer's disease (AD). It has been reported that androgen reduces amyloid peptides (Aß) production and increases Aß degradation by neurons. Activated microglia are involved in AD by either clearing Aß deposits through uptake of Aß or releasing cytotoxic substances and pro-inflammatory cytokines. Here, we investigated the effect of androgen on Aß uptake and clearance and Aß-induced inflammatory response in microglia, on neuronal death induced by Aß-activated microglia, and explored underlying mechanisms. METHODS: Intracellular and extracellular Aß were examined by immunofluorescence staining and Western blot. Amyloid peptides (Aß) receptors, Aß degrading enzymes, and pro-inflammatory cytokines were detected by RT-PCR, real-time PCR, and ELISA. Phosphorylation of MAP kinases and NF-κB was examined by Western blot. RESULTS: We found that physiological concentrations of androgen enhanced Aß42 uptake and clearance, suppressed Aß42 -induced IL-1ß and TNFα expression by murine microglia cell line N9 and primary microglia, and alleviated neuronal death induced by Aß42 -activated microglia. Androgen administration also reduced Aß42 -induced IL-1ß expression and neuronal death in murine hippocampus. Mechanistic studies revealed that androgen promoted microglia to phagocytose and degrade Aß42 through upregulating formyl peptide receptor 2 and endothelin-converting enzyme 1c expression, and inhibited Aß42 -induced pro-inflammatory cytokines expression via suppressing MAPK p38 and NF-κB activation by Aß42 , in an androgen receptor independent manner. CONCLUSION: Our study demonstrates that androgen promotes microglia to phagocytose and clear Aß42 and inhibits Aß42 -induced inflammatory response, which may play an important role in reducing the neurotoxicity of Aß.

A novel monoclonal antibody against the N-terminus of Aß1-42 reduces plaques and improves cognition in a mouse model of Alzheimer's disease.

Senile plaques consisting of Amyloid-beta (Aß) peptides, in particular Aß1-42, are the hallmark of Alzheimer's disease (AD) and have been the primary therapeutic targets. Passive immunotherapy with monoclonal antibodies (mAbs) has shown initial success in mouse models of AD. However, the existing Aß-directed mAbs mostly were tested on animal models or patients with advanced disease. The effects and mechanisms of mAbs on animals or human trial participants in the prodromal phase of AD are not fully clarified. In the current study, a novel mAb (3F5) directed against the 1-11 amino acids of Aß1-42 was generated by immunizing mice with an emulsion of full length human Aß1-42. The mAb (3F5) showed the ability to disrupt Aß1-42 aggregation and prevent Aß-mediated neurotoxicity in vitro. In a mouse model of AD, administration with 3F5 for 3 months in 6 months-old mice demonstrated that the mAb specifically bound with Aß1-42 to promote the depolymerization of Aß fibrils, facilitated endocytosis of Aß1-42 by microglia, and attenuated the death and apoptosis of neuronal cells, accompanied by neurite outgrowth. APP/PS1 double-transgenic mice treated with 3F5 mAb showed reduced memory loss, cognitive decline, and decreased levels of amyloid deposits in the brain. Aß1-42 levels in cerebral tissues were also significantly reduced, whereas serum Aß1-42 was markedly increased. Interestingly, the concentration of 3F5 in peripheral circulation is much higher than that in the brain. These results indicate that 3F5 is able to cross the blood-brain barrier (BBB) to bind Aß and initiates the phagocytosis of antibody/Aß complexes by microglia in the amyloid depositing mice. 3F5 also promotes Aß efflux from the brain. As a consequence, the antibody reduces plaques in the AD mouse brain, in association with reduction in the pathology of AD.

Role of resistin in inflammation and inflammation-related diseases.

Resistin is a newly identified adipocyte secreted hormone belonging to a cysteine-rich protein family. It is expressed in white adipose tissues in rodents and has also been found in several other tissues in human. Insulin, glucose, many cytokines and anti-diabetic thiazolidinediones are regulators of resistin gene expression. Resistin was firstly proposed to be involved in insulin resistance and type 2 diabetes. Recently, it was found to be relevant to inflammation and inflammation-related diseases like atherosclerosis and arthritis.

Discovery of Novel Inhibitors and Fluorescent Probe Targeting NAMPT.

Nicotinamide phosphoribosyltransferase (NAMPT) is a promising antitumor target. Novel NAMPT inhibitors with diverse chemotypes are highly desirable for development of antitumor agents. Using high throughput screening system targeting NAMPT on a chemical library of 30000 small-molecules, we found a non-fluorescent compound F671-0003 and a fluorescent compound M049-0244 with excellent in vitro activity (IC50: 85 nM and 170 nM respectively) and anti-proliferative activity against HepG2 cells. These two compounds significantly depleted cellular NAD levels. Exogenous NMN rescued their anti-proliferative activity against HepG2 cells. Structure-activity relationship study proposed a binding mode for NAMPT inhibitor F671-0003 and highlighted the importance of hydrogen bonding, hydrophobic and π-π interactions in inhibitor binding. Imaging study provided the evidence that fluorescent compound M049-0244 (3 µM) significantly stained living HepG2 cells. Cellular fluorescence was further verified to be NAMPT dependent by using RNA interference and NAMPT over expression transgenic mice. Our findings provide novel antitumor lead compounds and a "first-in-class" fluorescent probe for imaging NAMPT.

The grape component resveratrol interferes with the function of chemoattractant receptors on phagocytic leukocytes.

Resveratrol (3,5,4'-trihydroxystilbene) (RV) is a constituent of grape seeds with anti-inflammatory and anti-oxidant activities. In this study, we examined the capacity of RV to modulate the function of G protein-coupled chemoattractant receptors, which play important roles in inflammation and immune responses. RV, over a non-cytotoxic concentration range, inhibited chemotactic and calcium mobilization responses of phagocytic cells to selected chemoattractants. At low micromolar concentrations, RV potently reduced superoxide anion production by phagocytic leukocytes in response to the bacterial chemotactic peptide fMLF, a high affinity ligand for formylpeptide receptor FPR, and A beta42, an Alzheimer's disease-associated peptide and a ligand for the FPR variant FPRL1. In addition, RV reduced phosphorylation of extracellular signal-regulated kinase (ERK1/2) and the activation of nuclear factor NF-kappaB induced by formylpeptide receptor agonists. These results suggest that the inhibition of the function of chemoattractant receptors may contribute to the anti-inflammatory properties of RV. Thus, RV may be therapeutically promising for diseases in which activation of formylpeptide receptors contributes to the pathogenic processes.

Resveratrol protects vascular endothelial cells from high glucose-induced apoptosis through inhibition of NADPH oxidase activation-driven oxidative stress.

INTRODUCTION: Hyperglycemia-induced oxidative stress has been implicated in diabetic vascular complications in which NADPH oxidase is a major source of reactive oxygen species (ROS) generation. Resveratrol is a naturally occurring polyphenol, which has vasoprotective effects in diabetic animal models and inhibits high glucose (HG)-induced oxidative stress in endothelial cells. AIMS: We aimed to examine whether HG-induced NADPH oxidase activation and ROS production contribute to glucotoxicity to endothelial cells and the effect of resveratrol on glucotoxicity. RESULTS: Using a murine brain microvascular endothelial cell line bEnd3, we found that NADPH oxidase inhibitor (apocynin) and resveratrol both inhibited HG-induced endothelial cell apoptosis. HG-induced elevation of NADPH oxidase activity and production of ROS were inhibited by apocynin, suggesting that HG induces endothelial cell apoptosis through NADPH oxidase-mediated ROS production. Mechanistic studies revealed that HG upregulated NADPH oxidase subunit Nox1 but not Nox2, Nox4, and p22(phox) expression through NF-κB activation, which resulted in elevation of NADPH oxidase activity and consequent ROS production. Resveratrol prevented HG-induced endothelial cell apoptosis through inhibiting HG-induced NF-κB activation, NADPH oxidase activity elevation, and ROS production. CONCLUSIONS: HG induces endothelial cell apoptosis through NF-κB/NADPH oxidase/ROS pathway, which was inhibited by resveratrol. Our findings provide new potential therapeutic targets against brain vascular complications of diabetes.

Chronic exposure to nicotine enhances insulin sensitivity through α7 nicotinic acetylcholine receptor-STAT3 pathway.

This study was to investigate the effect of nicotine on insulin sensitivity and explore the underlying mechanisms. Treatment of Sprague-Dawley rats with nicotine (3 mg/kg/day) for 6 weeks reduced 43% body weight gain and 65% blood insulin level, but had no effect on blood glucose level. Both insulin tolerance test and glucose tolerance test demonstrated that nicotine treatment enhanced insulin sensitivity. Pretreatment of rats with hexamethonium (20 mg/kg/day) to antagonize peripheral nicotinic receptors except for α7 nicotinic acetylcholine receptor (α7-nAChR) had no effect on the insulin sensitizing effect of nicotine. However, the insulin sensitizing effect but not the bodyweight reducing effect of nicotine was abrogated in α7-nAChR knockout mice. Further, chronic treatment with PNU-282987 (0.53 mg/kg/day), a selective α7-nAChR agonist, significantly enhanced insulin sensitivity without apparently modifying bodyweight not only in normal mice but also in AMP-activated kinase-α2 knockout mice, an animal model of insulin resistance with no sign of inflammation. Moreover, PNU-282987 treatment enhanced phosphorylation of signal transducer and activator of transcription 3 (STAT3) in skeletal muscle, adipose tissue and liver in normal mice. PNU-282987 treatment also increased glucose uptake by 25% in C2C12 myotubes and this effect was total abrogated by STAT3 inhibitor, S3I-201. All together, these findings demonstrated that nicotine enhanced insulin sensitivity in animals with or without insulin resistance, at least in part via stimulating α7-nAChR-STAT3 pathway independent of inflammation. Our results contribute not only to the understanding of the pharmacological effects of nicotine, but also to the identifying of new therapeutic targets against insulin resistance.