Biochemical and Biophysical Research Communications
Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is constitutively released from human hepatocytes
Introduction
In mammals, the rate-limiting step in NAD biosynthesis starting from nicotinamide is catalysed by nicotinamide phosphoribosyltransferase or NAMPT, which transfers a phosphoribosyl group onto nicotinamide yielding nicotinamide mononucleotide (NMN) [1]. NAMPT is a regulator of intracellular NAD levels and consequently influences the activity of NAD consuming enzymes [2].
Also known as PBEF or visfatin, circulating NAMPT exerts pleiotropic actions. It has been described as an extracellular NMN producing enzyme [3], with NMN eliciting protective cellular responses [3], [4]. Dimerisation is essential for NAMPT to be enzymatically active [5] and NAMPT has been shown to occur as dimer in human serum [6].
Additionally, NAMPT has been reported to function as a cytokine. Its serum concentration has been shown to be increased in a number of immunological and metabolical disorders [7]. In most of these studies it remained unclear whether enzymatic activity of NAMPT is necessary for its cytokine-like action. However, two studies recently demonstrated that NAMPT acts independent of its NMN biosynthetic activity [8], [9].
Differentiated adipocytes are a source of circulating NAMPT [3], [10], [11], [12], which is possibly released through a non-classical pathway which is blocked neither by brefeldin A and monensin, inhibitors of the ER–Golgi secretory pathway, nor by glibenclamide [11], an inhibitor of ABC-dependent secretion [13]. NAMPT release was reported to be influenced by insulin, glucose and TNFα[10], [14]. It is not known whether adipose tissue is the major source for NAMPT in humans or if there are other organs or tissues capable of releasing significant amounts of this protein. Interestingly, a recent study found NAMPT serum levels to be lower in patients with non-alcoholic steatohepatitis than in patients with simple steatosis or obese healthy controls [15]. Therefore, we aimed to characterize NAMPT expressed in and released from hepatocytes and asked whether NAMPT from hepatocytes may retain its enzymatic activity outside the cells.
Section snippets
Materials and methods
Materials. Cell culture media, supplements and antibiotics were obtained from PAA (Cölbe, Germany) or Invitrogen (Karlsruhe, Germany). Brefeldin A, monensin, glibenclamide, glucose, insulin and chemicals were purchased from Sigma–Aldrich (München, Germany); TNFα was from CellSystems® (St. Katharinen, Germany).
Cell culture of HepG2 and primary rat/human hepatocytes. HepG2 cells were maintained in MEM or in RPMI1640 for glucose stimulation supplemented with 10% FBS and 2 mmol/l glutamine. For
NAMPT is released from HepG2 cells and hepatocytes
Because of the high content of NAMPT in liver extracts [19] we examined NAMPT expression in and release from HepG2 cells and primary hepatocytes. NAMPT content in lysates and supernatants of HepG2, rat and human primary hepatocytes were examined after 2, 6, 16, 24, 48 and 72 h of serum-free incubation. In all three hepatocellular models, NAMPT content in lysates did not change significantly over time (Figs. 1A, 2A and D), while it significantly increased in cell culture supernatants as
Discussion
In this study we examined expression, release and enzymatic activity of NAMPT in three different hepatocellular models. NAMPT was found to be released from HepG2 cells as well as primary rat and human hepatocytes. Our findings are in line with two recent studies [20], [21] in patients with liver cirrhosis. In these studies, a high abundance of NAMPT protein was found in hepatocytes from normal livers, which decreased in hepatocytes from cirrhotic livers. Likewise, plasma from patients with
Acknowledgments
The work was supported by a grant from the Deutsche ForschungsgemeinschaftKFO 152 “Atherobesity” Project BE 1264/10-1 and by the Obesity Research Network of the German Ministry of Science and Education “Kompetenznetz Adipositas”, BMBF, Berlin (to W.K.) and by the “HepatoSys” network of the German Ministry of Science and Education (FKZ 0313081F) (to R.G. and W.T.) as well as a junior research grant by the Medical Faculty, University of Leipzig (to A.G.). Human tissue for research was made
References (23)
- et al.
The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells
J. Biol. Chem.
(2004) - et al.
Nampt/PBEF/visfatin regulates insulin secretion in beta cells as a systemic NAD biosynthetic enzyme
Cell Metab.
(2007) - et al.
Nampt: linking NAD biology, metabolism and cancer
Trends Endocrinol. Metab.
(2009) - et al.
Extracellular Nampt promotes macrophage survival via a non-enzymatic interleukin-6/STAT3 signaling mechanism
J. Biol. Chem.
(2008) - et al.
Regulation of inflammatory cytokine expression in pulmonary epithelial cells by pre-B-cell colony enhancing factor via a nonenzymatic and AP-1 dependent mechanism
J. Biol. Chem.
(2009) - et al.
Visfatin is released from 3T3-L1 adipocytes via a non-classical pathway
Biochem. Biophys. Res. Commun.
(2007) - et al.
Regulated secretion of macrophage migration inhibitory factor is mediated by a non-classical pathway involving an ABC transporter
FEBS Lett.
(2003) - et al.
Pre-B-cell colony-enhancing factor is a secreted cytokine-like protein from the human amniotic epithelium
Am. J. Obstet. Gynecol.
(2005) - et al.
A rapid procedure for assaying nicotinamide phosphoribosyltransferase
Anal. Biochem.
(1980) - et al.
Therapeutic potential of SIRT1 and NAMPT-mediated NAD biosynthesis in type 2 diabetes
Front. Biosci.
(2009)
Extracellular PBEF/NAMPT/visfatin activates pro-inflammatory signalling in human vascular smooth muscle cells through nicotinamide phosphoribosyltransferase activity
Diabetologia
Cited by (124)
Role of adipokines (omentin and visfatin) in coronary artery disease
2023, Nutrition, Metabolism and Cardiovascular DiseasesAdipokines: Deciphering the cardiovascular signature of adipose tissue
2022, Biochemical PharmacologyNAMPT-targeting PROTAC promotes antitumor immunity via suppressing myeloid-derived suppressor cell expansion
2022, Acta Pharmaceutica Sinica BNAMPT: A critical driver and therapeutic target for cancer
2022, International Journal of Biochemistry and Cell BiologyMolecular insights into the interaction between human nicotinamide phosphoribosyltransferase and Toll-like receptor 4
2022, Journal of Biological Chemistry