Characterization of NAD Uptake in Mammalian Cells

Abstract

Recent evidence has shown that NAD(P) plays a variety of roles in cell-signaling processes. Surprisingly, the presence of NAD(P) utilizing ectoenzymes suggests that NAD(P) is present extracellularly. Indeed, nanomolar concentrations of NAD have been found in plasma and other body fluids. Although very high concentrations of NAD have been shown to enter cells, it is not known whether lower, more physiological concentrations are able to be taken up. Here we show that two mammalian cell types are able to transport low NAD concentrations effectively. Furthermore, extracellular application of NAD was able to counteract FK866-induced cell death and restore intracellular NAD(P) levels. We propose that NAD uptake may play a role in physiological NAD homeostasis.Pyridine nucleotides, although often considered as simple redox cofactors in the cell, are in fact multifunctional molecules involved in a wide range of cellular processes (1). It is becoming clear that NAD(P) is involved in more pharmacologically attractive cellular processes such as cell signaling, transcriptional regulation, and post-translational protein modification (2, 3). For example, NAD(P) has been shown to be the precursor of molecules involved in calcium signaling (e.g. cADPR, 2 NAADP, and ADPR), to be involved in the regulation of epigenetic changes via sirtuins and to be a substrate for both monoand poly-ADP-ribosylation (3-5).The general perception is that cellular NAD is synthesized either de novo from tryptophan or via one of two possible recycling pathways: from nicotinic acid or nicotinamide (together referred to as vitamin PP, or niacin) (6). Recently, a third biosynthesis pathway has been described, which uses nicotinamide riboside as a precursor (7).The presence of membrane-bound ectoenzymes that use NAD(P) (8) has led to the investigation and description of several mechanisms for the export of NAD across the plasma membrane, including transport through connexins and stimulus-induced exocytotic release (9 -11). Surprisingly, though, the possibility that low NAD concentrations can be imported across the membrane to directly replenish the cellular NAD(P) pools bypassing biosynthetic pathways has not been conclusively addressed. However, circumstantial evidence suggests that this may occur: (i) High concentrations of extracellularly applied NAD(H) have been shown to increase intracellular NAD levels (10, 12-14): (ii) extracellular NAD counteracts PARP-induced intracellular NAD depletion (12, 15); (iii) CD38 knock-out mice, which are impaired in their ability to degrade extracellular NAD, display higher endogenous SIRT1 activity (16); (iv) uptake processes have been shown for the Ca 2ϩ -mobilizing NAD(P) metabolites cADPR and NAADP in a variety of diverse mammalian cell types (17-21); (v) NAD and at least one of the enzymes involved in biosynthesis are present extracellularly (13,22,23); and (vi) Wallerian degeneration can be slowed by the addition of extracellular NAD (24).Here we show that various mammalian cell types are able to transport pico...

Extracellular PBEF/NAMPT/visfatin activates pro-inflammatory signalling in human vascular smooth muscle cells through nicotinamide phosphoribosyltransferase activity

“…Such a mechanism of action also seems to account for the growth factor-like properties of ePBEF/NAMPT/visfatin in vascular smooth muscle cells [18]. NMN has been recently proposed to permeate mammalian cell membranes, possibly through NAD uptake mechanisms [11,18,32]. Therefore, the extracellular NMN generated trough NAMPT activity may enter the cell and then trigger the intracellular proinflammatory signals explored herein through mechanisms that remain to be elucidated.…”

“…A commercial oligonucleotide (Promega, Madison, WI, USA) encoding the NF-κB consensus sequence (5′-AGTTGAGGGGACTTTC CCAGGC-3′) was 5′-end labelled using [γ- 32 . After addition of the labelled oligonucleotide (~50,000 cpm) the reaction mix was further incubated for 20 min at room temperature.…”

NAMPT (visfatin) in the chicken testis: influence of sexual maturation on cellular localization, plasma levels and gene and protein expression

“…While the NAMPT protein lacks a cleavable signal peptide and is thought to be released from cells by a nonclassical pathway (Tanaka et al 2007), the source and function of circulating NAMPT are still unknown. Nonetheless, the NAMPT activity of NAMPT may catalyze NAD synthesis from nicotinamide in the blood circulation, and produce precursors that are then available for cellular uptake and homeostasis of intracellular NAD levels (Billington et al 2008). Indeed, one such precursor, NMN, has been detected in the blood circulation and extracellular fluid of mice (Revollo et al 2007).…”

Alteration of Neuron-Glia Interactions in Neurodegeneration: Molecular Biomarkers and Therapeutic Strategy

“…This mechanism is very important, because it helps to prevent NAD + loss from cells, depletion of intracellular NAD + due to elevated CD38 activity, and cADPR overproduction leading to calcium accumulation to toxic levels (Salmina et al, 2009a). CD38 and Cx43 also may serve as transporters of nucleotides (NAD(H), cADPR, and NAADP + ) into the cell from the extracellular space (Billington, et al, 2008), thus contributing to maintaining NAD + homeostasis within the cell. NAD + itself can enter astrocytes via gap junction-mediated pathway .…”

“…In amyloid-treated cells, NMNAT-sensitive program is uniquely involved in axonal, but not cell body, degeneration (Vohra et al, 2010). Axonal degeneration can be slowed by the addition of extracellular NAD + (Billington et al, 2008). Nicotinamide N-methyltransferase (NNMT) methylates pyridines, in particular nicotinamide, to N-methyl nicotinamide which is further used for synthesis of NAD(P) and NAD(P)H. Increased activity of NNMT leads to cellular nicotinamide deficiency.…”