Visfatin, also named nicotinamide phosphoribosyl transferase (NAMPT), is a cytokine secreted from adipose tissue. Visfatin can regulate immune action and is involved in the NAD+ salvage pathway. In addition, recent researches have shown that visfatin helps the regulation of glucose and lipid metabolism, especially in exercise-induced weight reduction for obesity. The aim of this review is to provide an overview of the contribution of visfatin gene polymorphisms to glucose and lipid metabolism and exercise-induced weight reduction in obesity.
Exercise ; physiology ; Glycolipids ; metabolism ; Humans ; Nicotinamide Phosphoribosyltransferase ; genetics ; physiology ; Obesity ; genetics ; metabolism ; Polymorphism, Genetic ; Weight Loss ; genetics

Escherichia coli strain NZN111 is a promising candidate for the fermentative production of succinate. However, because lactate dehydrogenase and pyruvate formate lyase were inactivated in NZN111, this strain had an unbalanced NADH/NAD+ ratio and could not use glucose under anaerobic conditions. In this study, a recombinant strain E. coli NZN111/pTrc99a-pncB was constructed to overexpress the nicotinic acid phosphoribosyl transferase gene (pncB). Under anaerobic conditions with the addition of 0.5 mmol/L nicotinic acid and 0.3 mmol/L isopropyl beta-D-thiogalactopyranoside (IPTG), the specific nicotinic acid phosphoribosyl transferase (NAPRTase, EC 2.4.2.11) activity in the recombinant strain was 11-fold higher than that in E. coli NZN111, the concentration of NAD(H) was increased by 3.85-fold, especially the concentration of NAD+ was increased by 5.17-fold and NADH/NAD+ was decreased from 0.640 to 0.125. The recombinant strain regained the capability of growth and glucose utilization under anaerobic conditions.
Acetyltransferases ; genetics ; metabolism ; Anaerobiosis ; Escherichia coli ; classification ; genetics ; metabolism ; Fermentation ; Genetic Enhancement ; methods ; Glucose ; metabolism ; L-Lactate Dehydrogenase ; genetics ; metabolism ; NAD ; metabolism ; Nicotinamide Phosphoribosyltransferase ; biosynthesis ; genetics ; Succinic Acid ; metabolism

OBJECTIVETo prepare and purify recombinant human NAMPT and NAMPT (H247A) proteins and to detect their enzymatic activity.

METHODSUsing pcDNA3.1-hnampt as template, full-length hnampt was sub-cloned into pET-11a(+) plasmid. The hnampt (H247A) mutant was obtained by site-directed mutagenesis. The plasmids were introduced in Escherichia coli BL21star for protein expression. The recombined NAMPT and NAMPT (H247A) proteins were purified by flowing through nickel column and size-exclusion column. The target proteins were confirmed by SDS-PAGE and mass spectrometry detection. The enzymatic activities of recombinant proteins were assessed by solution NMR.

RESULTThe DNA sequences showed that hnampt (wild type) and hnampt (H247A) (mutation) were cloned into pET-11a(+). The recombinant proteins were expressed in Escherichia coli BL21star in soluble form. The purified protein was confirmed to be NAMPT with a molecular weight of 56 KD. The enzyme activity of NAMPT (H247A) was dramatically decreased compared to wild-type NAMPT.

CONCLUSIONThe recombinant hNAMPT and hNAMPT (H247A) proteins have been successful prepared and purified. The H247A mutation dramatically decreases the enzymatic activity of NAMPT.

Base Sequence ; Cytokines ; genetics ; isolation & purification ; metabolism ; Escherichia coli ; genetics ; Genetic Vectors ; Humans ; Molecular Sequence Data ; Mutagenesis, Site-Directed ; Mutation ; Nicotinamide Phosphoribosyltransferase ; genetics ; isolation & purification ; metabolism ; Plasmids ; genetics ; Recombinant Proteins ; genetics ; metabolism ; Transformation, Bacterial

BACKGROUNDVisfatin, a visceral fat-derived adipocytokine, plays a significant physiological function in lipid metabolism. However, the precise function of visfatin and its regulation by thyroid hormones are still unknown. This study observed the plasma visfatin concentrations in subjects with hyperthyroidism and hypothyroidism in vivo, and investigated the possible regulation mechanism between visfatin and tri-iodothyronine (T3) in vitro as a further interpretation.

METHODSThe experiment in vivo included clinical subjects (57 patients with thyroid dysfunction and 29 euthyroid healthy volunteers) and an animal model (24 Wistar rats). All subjects were divided into hyperthyroidism, hypothyroidism and euthyroidism groups, with plasma thyroid hormones, thyrotropin, visfatin and triglyceride concentrations determined. Visfatin mRNA expression in visceral fat and liver of rats was detected by quantitative real-time reverse transcription-polymerase chain reaction (RT-PCR). The experiment in vitro studied 3T3-L1 cells and visfatin mRNA expression under nine different T3 concentrations (0, 0.1, 0.25, 0.5, 1, 5, 10, 20, 100 nmol/L) using quantitative real-time RT-PCR.

RESULTSClinical subjects and animal models showed elevated plasma visfatin concentrations in the hyperthyroidism group (20.466 ng/ml (15.263, 26.795 ng/ml) and (1209.164±165.292) ng/L) and hypothyroidism group (12.457 ng/ml (11.115, 15.454 ng/ml) and (1205.425±109.200) ng/L) compared to euthyroidism group (6.891 ng/ml (5.888, 8.803 ng/ml) and (926.650±54.002) ng/L, P<0.001). For animal models, visfatin mRNA expression in visceral fat in the hyperthyroidism and hypothyroidism groups increased about 3.33-fold and 1.98-fold compared to the euthyroidism group (P<0.001), which was positively correlated with plasma visfatin concentrations (r=0.713, P<0.001). However, no significant group difference (P>0.05) and correlation (r=0.121, P=0.572) was found in the liver. T3 induced a remarkable increase of visfatin mRNA expression in 3T3-L1 cells at low concentrations (0-0.5 nmol/L T3) followed by a sharp decrease at higher concentrations (0.5-100 nmol/L T3), with an inflection point at 0.5 nmol/L T3.

CONCLUSIONElevated circulating visfatin levels in subjects with hyperthyroidism and hypothyroidism are possibly due to an increase of visfatin mRNA expression in visceral fat, and a nonlinear regulation mechanism on visfatin mRNA expression under various T3 concentrations might be involved.

3T3-L1 Cells ; Adult ; Animals ; Female ; Humans ; Hyperthyroidism ; blood ; genetics ; metabolism ; Hypothyroidism ; blood ; genetics ; metabolism ; Male ; Mice ; Middle Aged ; Nicotinamide Phosphoribosyltransferase ; genetics ; metabolism ; Reverse Transcriptase Polymerase Chain Reaction ; Triiodothyronine ; blood

BACKGROUNDThe levels of long-term elevated serum or intracellular free fatty acid (FFA) induce insulin resistance associated with central obesity. The insulin-mimetic protein visfatin is preferentially produced by visceral adipose tissues and has been implicated in obesity and insulin resistance. To identify that FFA is capable of inducing insulin resistance and to clarify the role of FFA on visfatin, we examined the effect of monounsaturated FFA oleate (C18:1) and saturated FFA palmitate (C16:0) on glucose transport and visfatin gene expression in cultured 3T3-L1 adipocytes or preadipocytes.

METHODSFFA-free DMEM/F12, 0.125 mmol/L, 0.5 mmol/l and 1.0 mmol/L oleate or palmitate was added to cultured 3T3-L1 adipocytes or preadipocytes and incubated overnight. Glucose transport was assessed as (3)H-2-deoxy-glucose uptake. Total RNA was extracted and subjected to RT-PCR for the measurement of visfatin mRNA levels. Statistical comparisons between control group and other groups were performed with the two-tailed paired t test, and one-way ANOVA was used to compare the mean values among the groups.

RESULTSInsulin increased specific membrane glucose transport in 3T3-L1 preadipocytes. Upregulation was evident from 15 minutes to 1 hour exposure to insulin. However, after 6-hour exposure to insulin, there was a downregulation in the response to insulin. Dose response studies demonstrated that 2-deoxy glucose transport was increased by 336% at 50 nmol/L insulin (P < 0.01), and reached a maximal effect at 100 nmol/L insulin (P < 0.01). Oleate and palmitate treatment did not influence basal glucose transport (without insulin stimulation), whereas insulin-stimulated glucose transport was inhibited after overnight oleate and palmitate treatment in preadipocytes and adipocytes. In 3T3-L1 preadipocytes, insulin resistance could be achieved at 0.125 mmol/L oleate or palmitate (P < 0.05, respectively), and the inhibition was dose dependent. In adipocytes, the inhibition was noted at 0.5 mmol/L oleate or 1.0 mmol/L palmitate. Visfatin mRNA expression increased during differentiation more than 1.5-fold. Bovine serum albumin (BSA) did not influence visfatin mRNA expression compared with the control group. Dose-response studies demonstrated that addition of 0.125 mmol/L oleate and palmitate to 3T3-L1 adipocytes decreased visfatin mRNA expression significantly (78%, 77%, respectively, relative to untreated control, P < 0.05), and further to 65% (relative to untreated control, P < 0.05) and 55% (relative to untreated control, P < 0.01) at 1.0 mmol/L FFA. Furthermore, the suppression on preadipocytes was similar to that of adipocytes, which reached a maximal reduction of 44% (oleate, P < 0.05) and 47% (palmitate, P < 0.05) at 1.0 mmol/L FFA.

CONCLUSIONSOleic acid and palmitic acid may induce insulin resistance in 3T3-L1 adipocytes and preadipocytes. Downregulation of visfatin mRNA may contribute to impair insulin sensitivity caused by oleate and palmitate.

3T3-L1 Cells ; Adipocytes ; cytology ; metabolism ; Animals ; Cell Differentiation ; Cytokines ; genetics ; Dose-Response Relationship, Drug ; Gene Expression Regulation ; drug effects ; Insulin Resistance ; Mice ; Nicotinamide Phosphoribosyltransferase ; Oleic Acid ; pharmacology ; Palmitic Acid ; pharmacology ; RNA, Messenger ; analysis ; Stem Cells ; metabolism

BACKGROUND/AIMS: Adipose tissue is an active endocrine organ that secretes various metabolically important substances including adipokines, which represent a link between insulin resistance and nonalcoholic steatohepatitis (NASH). The factors responsible for the progression from simple steatosis to steatohepatitis remain elusive, but adipokine imbalance may play a pivotal role. We evaluated the expressions of adipokines such as visfatin, adipocyte-fatty-acid-binding protein (A-FABP), and retinol-binding protein-4 (RBP-4) in serum and tissue. The aim was to discover whether these adipokines are potential predictors of NASH. METHODS: Polymerase chain reaction, quantification of mRNA, and Western blots encoding A-FABP, RBP-4, and visfatin were used to study tissue samples from the liver, and visceral and subcutaneous adipose tissue. The tissue samples were from biopsy specimens obtained from patients with proven NASH who were undergoing laparoscopic cholecystectomy due to gallbladder polyps. RESULTS: Patients were classified into two groups: NASH, n=10 and non-NASH, n=20 according to their nonalcoholic fatty liver disease Activity Score. Although serum A-FABP levels did not differ between the two groups, the expressions of A-FABP mRNA and protein in the visceral adipose tissue were significantly higher in NASH group than in non-NASH group (104.34 vs. 97.05, P<0.05, and 190.01 vs. 95.15, P<0.01, respectively). Furthermore, the A-FABP protein expression ratio between visceral adipose tissue and liver was higher in NASH group than in non-NASH group (4.38 vs. 1.64, P<0.05). CONCLUSIONS: NASH patients had higher levels of A-FABP expression in their visceral fat compared to non-NASH patients. This differential A-FABP expression may predispose patients to the progressive form of NASH.
Adipose Tissue/metabolism/pathology ; Adult ; Aged ; Fatty Acid-Binding Proteins/genetics/*metabolism ; Fatty Liver/metabolism/*pathology ; *Gene Expression Regulation ; Humans ; Intra-Abdominal Fat/*metabolism ; Liver/metabolism/pathology ; Middle Aged ; Nicotinamide Phosphoribosyltransferase/genetics/metabolism ; RNA, Messenger/metabolism ; Retinol-Binding Proteins, Plasma/genetics/metabolism

Berberine, an isoquinoline alkaloid isolated from some Chinese medicinal herbs such as Coptidis rhizoma, has been used for the treatment of diarrhea and other gastrointestinal infections as an antibacterial drug in Chinese medicine. In recent years, it was reported to have beneficial effects on the metabolism disorders states of diabetes. The mechanisms involve many aspects of the diabetes, including regulating the blood cholesterol and triglyceride, lowering blood glucose, ameliorating the insulin resistant state and influencing the function of the pancreatic beta cell.
Animals ; Berberine ; isolation & purification ; pharmacology ; Blood Glucose ; metabolism ; Coptis ; chemistry ; Diabetes Mellitus ; metabolism ; Drugs, Chinese Herbal ; isolation & purification ; pharmacology ; Humans ; Insulin ; metabolism ; secretion ; Insulin-Secreting Cells ; drug effects ; Metabolic Diseases ; metabolism ; Nicotinamide Phosphoribosyltransferase ; biosynthesis ; genetics ; Plants, Medicinal ; chemistry ; Protein Kinases ; metabolism ; RNA, Messenger ; metabolism ; Receptors, LDL ; genetics ; metabolism ; Signal Transduction

Nicotinamide phosphoribosyltransferase (NAMPT) catalyzes the first rate-limiting step in converting nicotinamide to NAD(+), essential for a number of enzymes and regulatory proteins involved in a variety of cellular processes, including deacetylation enzyme SIRT1 which modulates several tumor suppressors such as p53 and FOXO. Herein we report that NQO1 substrates Tanshione IIA (TSA) and β-lapachone (β-lap) induced a rapid depletion of NAD(+) pool but adaptively a significant upregulation of NAMPT. NAMPT inhibition by FK866 at a nontoxic dose significantly enhanced NQO1-targeting agent-induced apoptotic cell death. Compared with TSA or β-lap treatment alone, co-treatment with FK866 induced a more dramatic depletion of NAD(+), repression of SIRT1 activity, and thereby the increased accumulation of acetylated FOXO1 and the activation of apoptotic pathway. In conclusion, the results from the present study support that NAMPT inhibition can synergize with NQO1 activation to induce apoptotic cell death, thereby providing a new rationale for the development of combinative therapeutic drugs in combating non-small lung cancer.
Abietanes ; pharmacology ; Apoptosis ; drug effects ; Carcinoma, Non-Small-Cell Lung ; drug therapy ; enzymology ; genetics ; physiopathology ; Cell Line, Tumor ; Cytokines ; antagonists & inhibitors ; genetics ; metabolism ; Enzyme Inhibitors ; pharmacology ; Humans ; NAD ; metabolism ; NAD(P)H Dehydrogenase (Quinone) ; genetics ; metabolism ; Naphthoquinones ; pharmacology ; Nicotinamide Phosphoribosyltransferase ; antagonists & inhibitors ; genetics ; metabolism

The aim of the present study was to investigate the role of peroxisome proliferator-activated receptor γ (PPARγ) signal transduction pathway in the expression of ATP binding cassette transporter A1 (ABCA1) and acyl-CoA:cholesterol acyltransferase 1 (ACAT1) induced by visfatin and to discuss the mechanism of foam cell formation induced by visfatin. THP-1 monocytes were induced into macrophages by 160 nmol/L phorbol myristate acetate (PMA) for 48 h, and then the macrophages were exposed to visfatin and PPARγ activator rosiglitazone, respectively. The expressions of PPARγ, ABCA1 and ACAT1 mRNA and protein were determined by RT-PCR and Western blot respectively. The contents of total cholesterol (TC) and free cholesterol (FC) were detected by enzyme fluorescence analysis. The content of cholesterol ester (CE) was calculated by the difference between TC and FC. The results showed that visfatin decreased the mRNA and protein expressions of PPARγ and ABCA1, increased the mRNA and protein expressions of ACAT1, and increased the contents of FC and CE in a concentration-dependent manner. These above effects of visfatin were inhibited by rosiglitazone in a concentration-dependent manner. These results suggest that visfatin may down-regulate the ABCA1 expression and up-regulate the ACAT1 expression via PPARγ signal transduction pathway, which decreases the outflow of FC, increases the content of CE, and then induces foam cell formation.
ATP Binding Cassette Transporter 1 ; ATP-Binding Cassette Transporters ; genetics ; metabolism ; Acetyl-CoA C-Acetyltransferase ; genetics ; metabolism ; Cell Differentiation ; Cell Line ; Cholesterol Esters ; metabolism ; Foam Cells ; cytology ; Humans ; Macrophages ; cytology ; Monocytes ; cytology ; Nicotinamide Phosphoribosyltransferase ; pharmacology ; PPAR gamma ; agonists ; physiology ; RNA, Messenger ; genetics ; metabolism ; Signal Transduction ; Thiazolidinediones ; pharmacology

DNA double-strand break (DSB) is the most severe form of DNA damage, which is repaired mainly through high-fidelity homologous recombination (HR) or error-prone non-homologous end joining (NHEJ). Defects in the DNA damage response lead to genomic instability and ultimately predispose organs to cancer. Nicotinamide phosphoribosyltransferase (Nampt), which is involved in nicotinamide adenine dinucleotide metabolism, is overexpressed in a variety of tumors. In this report, we found that Nampt physically associated with CtIP and DNA-PKcs/Ku80, which are key factors in HR and NHEJ, respectively. Depletion of Nampt by small interfering RNA (siRNA) led to defective NHEJ-mediated DSB repair and enhanced HR-mediated repair. Furthermore, the inhibition of Nampt expression promoted proliferation of cancer cells and normal human fibroblasts and decreased β-galactosidase staining, indicating a delay in the onset of cellular senescence in normal human fibroblasts. Taken together, our results suggest that Nampt is a suppressor of HR-mediated DSB repair and an enhancer of NHEJ-mediated DSB repair, contributing to the acceleration of cellular senescence.
Antigen-Antibody Complex ; metabolism ; Antigens, Nuclear ; genetics ; metabolism ; Carrier Proteins ; genetics ; metabolism ; Cell Line ; Cell Proliferation ; Cellular Senescence ; DNA Breaks, Double-Stranded ; DNA End-Joining Repair ; DNA Repair ; DNA-Activated Protein Kinase ; genetics ; metabolism ; DNA-Binding Proteins ; genetics ; metabolism ; Fibroblasts ; cytology ; HeLa Cells ; Homologous Recombination ; genetics ; physiology ; Humans ; Ku Autoantigen ; Nicotinamide Phosphoribosyltransferase ; genetics ; metabolism ; physiology ; Nuclear Proteins ; genetics ; metabolism ; RNA, Small Interfering ; genetics ; beta-Galactosidase ; metabolism