α-Mangostin reduced the viability of A594 cells in vitro by provoking ROS production through downregulation of NAMPT/NAD.

α-Mangostin (MAN) is a bioactive compound isolated from the inedible pericarp of a tropical fruit mangosteen (Garcinia mangostana Linn). It exhibits notable therapeutic potentials on lung cancers, but the underlying mechanisms are still largely unknown. This study was designed to further explore the mechanisms involved in cytotoxicity of MAN on A549 cells. Apoptosis and cell cycle distribution were analyzed by flow cytometry methods. The fluorescent probes DCFH-DA and JC-1 were used to assess the intracellular reactive oxidative species (ROS) and mitochondrial membrane potential statuses, respectively. The regulation of MAN on relevant pathways was investigated by immunoblotting assays. The results obtained indicated that MAN caused significant apoptosis and cell cycle arrest in A549 cells, which eventually resulted in inhibition on cell proliferation in vitro. All these phenomena were synchronized with escalated oxidative stress and downregulation of nicotinamide phosphoribosyltransferase/nicotinamide adenine dinucleotide (NAMPT/NAD). Supplementation with nicotinamide mononucleotide (NMN) and N-acetylcysteine (NAC) efficiently eased MAN-induced ROS accumulation, and potently antagonized MAN-elicited apoptosis and cell cycle arrest. The pro-apoptotic effect of MAN was further confirmed by increased expressions of cleaved caspase 3, 6, 7, and 9, and its effect on cell cycle progression was validated by the altered expressions of p-p38, p-p53, CDK4, and cyclin D1. The immunoblotting assays also demonstrated that NAC/NMN effectively restored these molecular changes elicited by MAN treatment. Collectively, this study revealed a unique anti-tumor mechanism of MAN by provoking ROS production through downregulation of NAMPT/NAD signaling and further validated MAN as a potential therapeutic reagent for lung cancer treatment.

Nicotinamide phosphoribosyltransferase­related signaling pathway in early Alzheimer's disease mouse models.

Alzheimer's disease (AD) is a neurodegenerative disease of the central nervous system that is characterized by progressive cognitive dysfunction and which ultimately leads to dementia. Studies have shown that energy dysmetabolism contributes significantly to the pathogenesis of a variety of aging­associated diseases and degenerative diseases of the nervous system, including AD. One focus of research thus has been how to regulate the expression of nicotinamide phosphoribosyltransferase (NAMPT) to prevent against neurodegenerative diseases. Therefore, the present study used 6­month­old APPswe/PS1ΔE9 (APP/PS1) transgenic mice as early AD mouse models and sought to evaluate nicotinamide adenine dinucleotide (NAD+) and FK866 (a NAMPT inhibitor) treatment in APP/PS1 mice to study NAMPT dysmetabolism in the process of AD and elucidate the underlying mechanisms. As a result of this treatment, the expression of NAMPT decreased, the synthesis of ATP and NAD+ became insufficient and the NAD+/NADH ratio was reduced. The administration of NAD+ alleviated the spatial learning and memory of APP/PS1 mice and reduced senile plaques. Administration of NAD+ may also increase the expression of the key protein NAMPT and its related protein sirtuin 1 as well as the synthesis of NAD+. Therefore, increasing NAMPT expression levels may promote NAD+ production. Their regulation could form the basis for a new therapeutic strategy.

Nicotinamide phosphoribosyltransferase is a molecular target of potent anticancer agents identified from phenotype-based drug screening.

Phenotypic screening in drug discovery has been revived with the expectation of providing promising lead compounds and drug targets and improving the success rate of drug approval. However, target identification remains a major bottleneck in phenotype-based drug discovery. We identified the lead compounds K542 and K405 with a selective inhibition of cell viability against sphingosine-1-phosphate lyase 1 (SGPL1)-transduced ES-2 cells by phenotypic screening. We therefore performed an in vivo pharmacological examination and observed the antitumor activity of K542 in an HT-1080 tumor-bearing mouse xenograft model. SGPL1 was expected to be a therapeutic target in some cancers, suggesting that these lead molecules might be promising candidates; however, their mechanisms of action still remain unexplained. We therefore synthesized the affinity probe Ind-tag derived from K542 and identified the proteins binding to Ind-tag via a pull-down experiment. Proteomics and biochemical analyses revealed that the target molecule of these lead compounds was Nicotinamide phosphoribosyltransferase (NAMPT). We established K542-resistant DLD-1 and HT-1080 cells, and genetic analyses of these cells identified a missense mutation in the NAMPT-encoding gene. This enzymatic experiment clearly showed that K393 exerts enzymatic inhibition against NAMPT. These proteomics, genetics and biochemical analyses clarified that compounds K542 and K405 were NAMPT inhibitors.

Metabolomics Analysis Reveals Therapeutic Effects of α-Mangostin on Collagen-Induced Arthritis in Rats by Down-regulating Nicotinamide Phosphoribosyltransferase.

α-Mangostin (MAN) is a bioactive compound isolated from pericarp of mangosteen (Garcinia mangostana Linn.) with significant anti-rheumatic potentials. The purpose of this study was to explore the mechanisms underlying its therapeutic effects on collagen-induced arthritis (CIA) in rats with metabolomics approaches. Therapeutic effects of MAN on CIA were assessed by radiographic, histological, and immunohistochemical methods. Metabolic profiles of rats were characterized based on UPLC-MS/MS analysis of urine samples, followed by verification in HFLS-RA cells using a variety of toxicological and biochemical assays. We found that MAN treatment protected joint structures in CIA rats and caused a decrease of nicotinamide mononucleotide (NMN) in urine. The levels of nicotinamide phosphoribosyltransferase (NAMPT) were reduced in fibroblast-like synoviocytes by MAN both in vivo and in vitro, which was accompanied with a decline in nicotinamide adenine dinucleotide (NAD) production. Secretion of extracellular NAMPT (eNAMPT) in HFLS-RA cells was also decreased upon MAN treatment, which lagged behind the changes of its intracellular counterpart (iNAMPT). Co-treatment with NMN raised the secretion of eNAMPT and restored the decline of p-p65 and TNF-α induced by MAN in vitro. Sirt1 expression was down-regulated under MAN treatments too. These results suggest that MAN treatment suppressed NAD production by inhibiting iNAMPT expression, which in turn decreased eNAMPT secretion and alleviated NF-κB-mediated inflammations in CIA rats.

Dexamethasone mediated downregulation of PGC-1α and visfatin regulates testosterone synthesis and antioxidant system in mouse testis.

Dexamethasone, a synthetic glucocorticoid has been used as an immunosuppressive and anti-inflammatory and affects reproduction. It has been suggested that testicular steroidogenesis involves PGC-1α and visfatin as key regulators. Previous studies have shown that dexamethasone down-regulates PGC-1α and visfatin expression in muscle and mammary epithelial cells respectively. However, the effect of dexamethasone on testicular visfatin and PGC-1α expressions has not been investigated. The aims of the present study were to investigate the effect of dexamethasone, on the expression of PGC-1α, visfatin and antioxidant enzymes activities in mouse testis. The results of the present study showed that dexamethasone treatment significantly decreased the expression of visfatin and PGC-1α in mice testis, along with significant decreased in testicular antioxidant enzymes activates. Further, dexamethasone treatment also significantly increased the testicular lipid peroxidation and decreased testosterone synthesis. The dexamethasone induced changes in PGC-1α and visfatin in the testis were significantly correlated with changes in serum testosterone concentrations and antioxidant enzymes activities. Thus, dexamethasone induced testicular toxicity may involve the PGC-1α and visfatin as important molecules to exhibit its effects.

P7C3-A20 neuroprotection is independent of Wallerian degeneration in primary neuronal culture.

The antiapoptotic, neuroprotective compound P7C3-A20 reduces neurological deficits when administered to murine in-vivo models of traumatic brain injury. P7C3-A20 is thought to exert its activity through small-molecule activation of the enzyme nicotinamide phosphoribosyltransferase. This enzyme converts nicotinamide to nicotinamide mononucleotide, the precursor to nicotinamide adenine dinucleotide synthesis. Alterations to this bioenergetic pathway have been shown to induce Wallerian degeneration (WD) of the distal neurite following injury. This study aimed to establish whether P7C3-A20, through induction of nicotinamide phosphoribosyltransferase activity, would affect the rate of WD. The model systems used were dissociated primary cortical neurons, dissociated superior cervical ganglion neurons and superior cervical ganglion explants. P7C3-A20 failed to show any protection against WD induced by neurite transection or vincristine administration. Furthermore, there was a concentration-dependent neurotoxicity. These findings are important in understanding the mechanism by which P7C3-A20 mediates its effects - a key step before moving to human clinical trials.

NAMPT inhibitor protects ischemic neuronal injury in rat brain via anti-neuroinflammation.

Nicotinamide phosphoribosyltransferase (NAMPT) is an important neuroprotective factor in cerebral ischemia, and it has been reported that NAMPT inhibitors can aggravate neuronal injury in the acute phase. However, because it is a cytokine, NAMPT participates in many inflammatory diseases in the peripheral system, and its inhibitors have therapeutic effects. Following cerebral ischemia, the peripheral and resident inflammatory and immune cells produce many pro-inflammatory mediators in the ischemic area, which induce neuroinflammation and impair the brain. However, the effects of NAMPT inhibitors in the neuroinflammation after ischemic brain injury remain unknown. Here, we found that FK866, a potent NAMPT inhibitor, decreased the level of TNF-α, NAMPT and IL-6 in the ischemic brain tissue one day after middle-cerebral-artery occlusion and reperfusion (MCAO/R), improved neurological dysfunction, decreased infarct volume and neuronal loss, and inhibited microgliosis and astrogliosis 14days after MCAO/R. The expression of NAMPT protein was induced in Iba1-positive microglia/macrophages in the ischemia core 14days after MCAO/R. In vitro studies show that oxygen-glucose deprivation and recovery (OGD/R) activate microglia. Activated microglia increased the activity of NF-κB, increased the mRNA synthesis of TNF-α, NAMPT and IL-6, and increased the secretion of TNF-α, NAMPT and IL-6. On the other hand, NAMPT can act synergistically with other cytokines and activate microglia. FK866 strongly inhibited these changes and alleviated OGD/R-induced activation of microglia. As such, NAMPT is a crucial determinant of cellular inflammation after cerebral ischemia. NAMPT inhibitors are novel compounds to protect neuronal injury from ischemia via anti-inflammatory effects.

Effects of antihypertensive treatment on plasma apelin, resistin, and visfatin concentrations.

INTRODUCTION Adipose tissue has been recently recognized as an endocrine organ secreting a number of adipokines contributing to the development of atherosclerosis, hypertension, chronic kidney disease, endothelial dysfunction, insulin resistance, and vascular remodeling. OBJECTIVES The aim of this study was to determine whether treatment with a ß-blocker, calcium antagonist, thiazide-like diuretic, or angiotensin II receptor type 1 influences plasma concentrations of apelin, resistin, and visfatin in obese hypertensive patients. PATIENTS AND METHODS The study included 84 obese patients with essential hypertension. One control group included obese subjects without hypertension, and the other, lean subjects without hypertension. Patients with hypertension were randomized into 4 groups treated for 6 weeks with bisoprolol, amlodipine, indapamide, or candesartan, respectively. RESULTS Mean daily plasma apelin concentrations in patients treated with amlodipine was significantly higher than the baseline values, whereas the difference in plasma apelin concentrations in other treatment groups was not significant. Mean daily plasma resistin concentrations were significantly lower after 6-week treatment with amlodipine, bisoprolol, or indapamide compared with the baseline values. In patients treated with candesartan, no significant differences in resistin concentrations were shown. After 6-week treatment with bisoprolol, mean daily plasma concentrations of visfatin were significantly lower compared with the baseline values. Treatment with amlodipine, candesartan, or indapamide did not significantly affect plasma visfatin levels. CONCLUSIONS Antihypertensive treatment exerts significant and varied effects on adipokine secretion in obese hypertensive patients. Changes in apelin secretion, caused by the use of different antihypertensive drugs, may protect the cardiovascular system and kidneys. The involvement of adipokins in the mechanism of diverse protective effects of antihypertensive drugs, independently of the effect on blood pressure, requires further research.

Nicotine may affect the secretion of adipokines leptin, resistin, and visfatin through activation of KATP channel.

OBJECTIVE: It has been confirmed that adipokines are associated with atherosclerosis. Cigarette smoking was found to possibly influence adipokine secretion. However, the precise role of smoking in adipokine secretion and the underlying mechanisms are largely unknown. The aim of this study was to determine whether nicotine, the principal active ingredient of cigarettes, can influence adipokine secretion and its potential mechanism. METHODS: The present study consecutively enrolled 96 men, including 50 smokers with early atherosclerosis and 46 nonsmokers. Serum adipokines, including leptin, resistin, and visfatin, were determined with enzyme-linked immunosorbent assay in all participants. Furthermore, the effect of nicotine on secretion of these adipokines was examined in differentiated 3T3-L1 preadipocytes under the conditions of ATP-dependent potassium (KATP) channel blocked or unblocked. RESULTS: Compared with the control group, serum levels of leptin, resistin, and visfatin in smokers were significantly higher. In 3T3-L1 adipocytes, nicotine treatment significantly increased the levels of these adipokines (P = 0.014, 0.001, and 0.029, respectively). When the KATP channel was blocked, secretion of resistin and visfatin was reduced (P < 0.001), but no change was found in the leptin secretion (P = 0.522). CONCLUSIONS: Nicotine may affect the secretion of adipokines leptin, resistin, and visfatin through activation of KATP channel.

Effects of 1-year orlistat treatment compared to placebo on insulin resistance parameters in patients with type 2 diabetes.

WHAT IS KNOWN AND OBJECTIVE: The behavioural approach is usually slow and not always sufficient to achieve optimal targets in weight and metabolic control in obese diabetic patients, and a pharmacological treatment is often necessary. The aim of this study was to compare the effects of orlistat and placebo on body weight, glycaemic and lipid profile and insulin resistance in patients with type 2 diabetes. METHODS: Two hundred and fifty-four obese, diabetic patients were enrolled in this study and randomized to take orlistat 360mg or placebo for 1year. We evaluated at baseline and after 3, 6, 9 and 12months body weight, waist circumference (WC), body mass index (BMI), glycated haemoglobin (HbA(1c) ), fasting plasma glucose (FPG), post-prandial plasma glucose (PPG), fasting plasma insulin (FPI), homeostasis model assessment insulin resistance index (HOMA-IR), lipid profile, retinol-binding protein-4 (RBP-4), resistin, visfatin and high-sensitivity C-reactive protein (Hs-CRP). RESULTS AND DISCUSSION: We observed a significant reduction in body weight, WC, BMI, lipid profile, RBP-4 and visfatin in the orlistat group but not in control group. Faster improvements in HbA(1c) , PPG, FPI, HOMA-IR, resistin and Hs-CRP were recorded with orlistat than with placebo. A similar decrease in FPG was seen in the two groups. Significant predictors of change in insulin resistance (HOMA-IR) were RBP-4 and resistin concentration in the orlistat group (r=-0·53, P<0·05, and r=-0·59, P<0·01, respectively). WHAT IS NEW AND CONCLUSION: To the best of our knowledge, this is the first study investigating the effect of orlistat on insulin resistance and markers of inflammation. Orlistat improved lipid profile and led to faster glycaemic control and insulin resistance parameters than the control, without any serious adverse event. Orlistat also improved RBP-4 and visfatin, effects not observed with placebo.

Effects of visfatin on proliferation and collagen synthesis in rat cardiac fibroblasts.

The proliferation of cardiac fibroblasts (CFs) and excessive extracellular matrix protein accumulation are the basic pathological processes of myocardial fibrosis. Visfatin is a novel adipokine involved in the regulation of inflammatory cytokines, however, the effects of visfatin on proliferation and collagen synthesis of CFs are unclear. The aim of this study was to determine whether visfatin has any effect on the proliferation and collagen synthesis in rat CFs. Incorporation of [ (3)H]-thymidine and [ (3)H]-proline were used for evaluating DNA and collagen synthesis. Flow cytometry techniques were adopted to analyze cell cycle. Enzyme-linked immunosorbent assay was used for measuring collagen type I and III production. RT-PCR and Western blot analysis were used for determining procollagen I and III mRNA expression and protein production. The inhibitors used for pathway determination were: wortmannin [phosphatiylinositol 3-kinase (PI3K)], SB203580 [p38 mitogen-activated protein kinase (MAPK)], PD98059 [extracellular signal-regulated kinase (ERK)1/2)], and JNK inhibitor II [c-Jun NH 2-terminal kinase (JNK)]. We demonstrated that visfatin significantly increased DNA and collagen synthesis in a dose- and time-dependent manner. Cell cycle analysis showed that visfatin increased S-stage percentage and proliferation index in a dose- and time-dependent manner. It was also found that visfatin upregulated collagen I and III production, procollagen I and III mRNA expression and protein production. These effects were diminished by SB203580, wortmannin, and PD98059, but not by JNK inhibitor II. These results suggest that visfatin promote CFs proliferation and collagen synthesis via p38MAPK, PI3K, and ERK 1/2 pathways rather than JNK pathways, which also indicate that visfatin might play a role in myocardial fibrosis.

Angiotensin inhibition stimulates PPARgamma and the release of visfatin.

BACKGROUND: Angiotensin converting enzyme inhibitors (ACE-I) and angiotensin receptor blockers (ARB) exhibit beneficial antidiabetic effects in patients with type 2 diabetes independent of their blood pressure-lowering effects. Some antidiabetic properties of ARB and ACE-I might by exerted by activation of peroxisome proliferator-activated receptor gamma (PPARgamma). However, it is not clear whether this action is drug specific. MATERIALS AND METHODS: The binding affinity of telmisartan, valsartan, lisinopril, rosiglitazone and angiotensin II to PPARgamma was assessed in a cell-free assay system. PPARgamma signalling was studied in isolated skeletal muscle cells using Western blot analysis of phosphorylated protein kinase B (pAKT) and phosphorylated insulin like growth factor-1 receptor (pILGF-1R). Further, the ability of the drugs under study to stimulate the release of the adipocytokine visfatin was investigated in isolated human adipocytes, skeletal muscle cells, and umbilical vein endothelial cells (HUVEC). RESULTS: The binding affinity to PPARgamma was highest for telmisartan with a half-maximal effective concentration of 463 nM, followed by lisinopril (2.9 microM) and valsartan (6.2 microM). In skeletal muscle cells phosphorylation of ILGF-1R was 2-fold increased after incubation with telmisartan or valsartan and 1.7-fold with lisinopril. pAKT expression was enhanced after incubation with telmisartan, valsartan and with lisinopril. The release of visfatin from adipocytes was 1.6-fold increased after treatment with lisinopril and about 2.0-fold increased with telmisartan and valsartan. Similar results were obtained in skeletal muscle cells and HUVEC. CONCLUSIONS: Our data confirm agonism of telmisartan, valsartan and lisinopril on PPARgamma. Pharmacokinetic differences may explain different potencies of PPARgamma stimulation by drugs acting on the renin-angiotensin system in clinical settings.

Effects of ezetimibe, either alone or in combination with atorvastatin, on serum visfatin levels: a pilot study.

INTRODUCTION: Ezetimibe inhibits intestinal absorption of cholesterol and lowers circulating low-density lipoprotein cholesterol levels. Visfatin is a novel adipokine, which may be implicated in the atherosclerotic process. OBJECTIVE: The aim of this study was to explore the possible association between ezetimibe administration and serum visfatin concentrations. METHODS: Patients (n = 30) with primary dyslipidemia and another 30 who failed to reach their assigned low-density lipoprotein cholesterol target on atorvastatin therapy (20 mg/day) were included in the study. All participants were given ezetimibe at 10 mg/day for 12 weeks. RESULTS: At baseline the visfatin levels correlated significantly with the total cholesterol (r = 0.61 and p < 0.01) and low-density lipoprotein cholesterol (r = 0.51 and p < 0.01) levels in the statin pretreatment group. Furthermore, in the statin group the post-treatment levels of visfatin and low-density lipoprotein cholesterol were significantly correlated (r = 0.57 and p < 0.01). The serum visfatin concentrations did not change significantly in either the monotherapy or statin pretreatment groups or in subgroups divided according to the baseline lipid variables. In both the ezetimibe monotherapy and ezetimibe plus atorvastatin groups the effect of ezetimibe on the lipid variables depended on the baseline lipid values. The low-density lipoprotein cholesterol:high density lipoprotein cholesterol ratio was consistently improved by ezetimibe in all groups or subgroups. CONCLUSIONS: Ezetimibe did not alter serum visfatin concentrations, either when administered as monotherapy or combined with a statin. Future studies investigating the effect of ezetimibe on visfatin levels need to include groups of patients with distinct lipid characteristics.

Effects of sibutramine plus verapamil sustained release/trandolapril combination on blood pressure and metabolic variables in obese hypertensive patients.

OBJECTIVE: The management of obese hypertensive subjects may require the administration of anti-obesity and antihypertensive drugs. Sibutramine use has raised concerns regarding a potential increase in subjects' blood pressure and heart rate. The primary end-points of this study were an evaluation of the effect of sibutramine together with a verapamil sustained release/trandolapril combination tablet versus verapamil sustained release/trandolapril alone on the blood pressure and heart rate in obese hypertensive patients. RESEARCH DESIGN/METHODS: Patients received a low-fat low-calorie diet and were randomly allocated to open-label verapamil sustained release/trandolapril 180/2 mg (n = 26) or sibutramine 10 mg together with verapamil sustained release/trandolapril 180/2 mg (n = 28) daily for 6 months. RESULTS: Significant reductions in the subjects' systolic blood pressure and diastolic blood pressure were observed in both groups (p < 0.01 versus baseline). At 6 months a greater fall in blood pressure was observed in the sibutramine/verapamil sustained release/trandolapril group compared with the verapamil sustained release/trandolapril group (systolic blood pressure 21.9 +/- 8.1 versus 15.9 +/- 12.3 mmHg and diastolic blood pressure 15.7 +/- 8.1 versus 9.1 +/- 9.9 mmHg) but this was only significant (p = 0.03) for diastolic blood pressure. The subjects' heart rate did not change significantly in any group. No significant sibutramine-associated attenuation of blood pressure reduction was observed during the study. The sibutramine/verapamil sustained release/trandolapril treatment resulted in significantly greater improvement in the subjects' anthropometric variables, homeostasis model assessment and lipid profiles compared with verapamil sustained release/trandolapril administration. The subjects' small dense low-density lipoprotein cholesterol, high-sensitivity C-reactive protein and visfatin plasma levels were only measured in the sibutramine/verapamil sustained release/trandolapril group (all decreased by p < 0.05 versus baseline). CONCLUSIONS: The sibutramine/verapamil sustained release/trandolapril combination in obese hypertensive patients significantly reduced their blood pressure and improved their anthropometric and metabolic variables without affecting the heart rate.