Nicotinamide phosphoribosyltransferase (NAMPT/PBEF/visfatin) is constitutively released from human hepatocytes

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Abstract

Circulating NAMPT (PBEF/visfatin) has pleiotropic functions and is secreted from adipocytes. Since it is doubtful whether serum levels can be explained by secretion from adipocytes alone, we asked whether hepatocytes are also able to liberate NAMPT. Using HepG2 cells and primary rat and human hepatocytes, release of NAMPT into the cell culture supernatant was found to occur constitutively in a time-dependent manner. In primary human hepatocytes, secretion within 24 h was far higher than the cellular content, but was neither influenced by inhibitors of secretion nor by glucose, insulin or TNFα. As determined by size exclusion chromatography, HepG2 lysates and supernatants primarily contained the dimeric form of NAMPT which exhibited similar in vitro specific enzymatic activity. In primary human hepatocytes, secreted NAMPT was less active. Our results demonstrate that human hepatocytes are a potential source of circulating NAMPT.

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

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