Characterization of NAD Uptake in Mammalian Cells

Richard A Billington,Cristina Travelli,Emanuela Ercolano,Ubaldina Galli,Cintia Blasi Roman,Ambra A Grolla,Pier Luigi Canonico,Fabrizio Condorelli,Armando A Genazzani

doi:10.1074/jbc.m706204200

Richard A Billington, Cristina Travelli + Show 7 more

Open Access

https://doi.org/10.1074/jbc.m706204200

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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.

Highlights

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 [6]
Circumstantial evidence suggests that this may occur: (i) High concentrations of extracellularly applied NAD(H) have been shown to increase intracellular NAD levels [10, 12,13,14]: (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 Ca2ϩ-mobilizing NAD(P) metabolites cADPR and NAADP in a variety of diverse mammalian cell types [17,18,19,20,21]; (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]
Cells could be rescued from FK866-induced NAD depletion, autophagy, and cell death by addition of NAD to the culture medium

Summary

EXPERIMENTAL PROCEDURES

Cell Culture—NIH-3T3 murine epithelial cells were cultured in RPMI 1640 (Sigma) supplemented with 5% FBS (fetal bovine serum), 2 mM glutamine, 10 units/ml penicillin, and 100 ␮g/ml streptomycin. For HPLC analysis of transported NAD, cells were scraped after transport experiments in ddH2O, extracted by adding an equal volume of chloroform and run on HPLC as described previously [27]. In a black 96-well plate (OptiPlateTM-96 F: PerkinElmer Life Sciences) 60 ␮l of cycling mix was added to 100 ␮l of the extracted NAD(P). Apoptotic Nuclei Staining—For confocal-microscopy analysis of DNA integrity and AIF nuclear localization counterstaining, neuroblastoma cells were plated on 12-mm glass coverslips and maintained for appropriate length of time with different compounds. The dye was added to cultured cells at 10 ␮M for 30 min followed by fixing in a 3.7% PBS/paraform-. Cells were washed twice with PBS, stained for AIF protein (as described above), and mounted onto coverslips for visualization with a confocal microscopy (HeNe laser UV, 633 nm)

RESULTS

DISCUSSION

Genazzani