Elevation of Cellular NAD Levels by Nicotinic Acid and Involvement of Nicotinic Acid Phosphoribosyltransferase in Human Cells

NAD plays critical roles in various biological processes through the function of SIRT1. Although classical studies in mammals showed that nicotinic acid (NA) is a better precursor than nicotinamide (Nam) in elevating tissue NAD levels, molecular details of NAD synthesis from NA remain largely unknown. We here identified NA phosphoribosyltransferase (NAPRT) in humans and provided direct evidence of tight link between NAPRT and the increase in cellular NAD levels. The enzyme was abundantly expressed in the small intestine, liver, and kidney in mice and mediated 14CNAD synthesis from 14CNA in human cells. In cells expressing endogenous NAPRT, the addition of NA but not Nam almost doubled cellular NAD contents and decreased cytotoxicity by H2O2. Both effects were reversed by knockdown of NAPRT expression. These results indicate that NAPRT is essential for NA to increase cellular NAD levels and, thus, to prevent oxidative stress of the cells. Kinetic analyses revealed that NAPRT, but not Nam phosphoribosyltransferase (NamPRT, also known as pre-B-cell colony-enhancing factor or visfatin), is insensitive to the physiological concentration of NAD. Together, we conclude that NA elevates cellular NAD levels through NAPRT function and, thus, protects the cells against stress, partly due to lack of feedback inhibition of NAPRT but not NamPRT by NAD. The ability of NA to increase cellular NAD contents may account for some of the clinically observed effects of the vitamin and further implies a novel application of the vitamin to treat diseases such as those associated with the depletion of cellular NAD pools. NAD plays critical roles in various biological processes through the function of SIRT1. Although classical studies in mammals showed that nicotinic acid (NA) is a better precursor than nicotinamide (Nam) in elevating tissue NAD levels, molecular details of NAD synthesis from NA remain largely unknown. We here identified NA phosphoribosyltransferase (NAPRT) in humans and provided direct evidence of tight link between NAPRT and the increase in cellular NAD levels. The enzyme was abundantly expressed in the small intestine, liver, and kidney in mice and mediated 14CNAD synthesis from 14CNA in human cells. In cells expressing endogenous NAPRT, the addition of NA but not Nam almost doubled cellular NAD contents and decreased cytotoxicity by H2O2. Both effects were reversed by knockdown of NAPRT expression. These results indicate that NAPRT is essential for NA to increase cellular NAD levels and, thus, to prevent oxidative stress of the cells. Kinetic analyses revealed that NAPRT, but not Nam phosphoribosyltransferase (NamPRT, also known as pre-B-cell colony-enhancing factor or visfatin), is insensitive to the physiological concentration of NAD. Together, we conclude that NA elevates cellular NAD levels through NAPRT function and, thus, protects the cells against stress, partly due to lack of feedback inhibition of NAPRT but not NamPRT by NAD. The ability of NA to increase cellular NAD contents may account for some of the clinically observed effects of the vitamin and further implies a novel application of the vitamin to treat diseases such as those associated with the depletion of cellular NAD pools. NAD serves as a coenzyme in cellular redox reactions and is, thus, an essential component of metabolic pathways in all living cells. Numerous recent studies have demonstrated that NAD plays important roles in a variety of biological processes in mammals, such as cell survival and apoptosis (1Luo J. Nikolaev A.Y. Imai S. Chen D. Su F. Shiloh A. Guarente L. Gu W. Cell. 2001; 107: 137-148Abstract Full Text Full Text PDF PubMed Scopus (1891) Google Scholar, 2Vaziri H. Dessain S.K. Ng-Eaton E. Imai S. Frye R.A. Pandita T.K. 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Nature. 2004; 429: 771-776Crossref PubMed Scopus (1664) Google Scholar) through the activity of a longevity factor NAD-dependent histone/protein deacetylase SIRT1. Changes in the cellular NAD level would, thus, have a significant impact on mammal physiology, including humans, and NAD biosynthesis reactions should be tightly regulated; however, the mechanisms regulating the cellular content of NAD remain to be determined. Mammalian NAD biosynthesis is accomplished through either the de novo pathway from tryptophan or salvage pathway from nicotinamide (Nam) 2The abbreviations used are: NamnicotinamideNAnicotinic acidNAPRTNA phosphoribosyltransferaseNaMNNA mononucleotideNaADNA adenine dinucleotideNamPRTNam phosphoribosyltransferaseNMNNam mononucleotidePRPP5-phosphoribosyl 1-pyrophosphateESI-MSelectrospray ionization mass spectrometryRTreverse transcriptionsiRNAshort interfering RNA and nicotinic acid (NA) (Fig. 1) (14Magni G. Amici A. Emanuelli M. Raffaelli N. Ruggieri S. Adv. Enzymol. Relat. Areas Mol. Biol. 1999; 73: 135-182PubMed Google Scholar). In the salvage pathway Nam is recycled to NAD by two enzymes, Nam phosphoribosyltransferase (NamPRT, also known as pre-B-cell colony-enhancing factor (15Samal B. Sun Y. Stearns G. Xie C. Suggs S. McNiece I. Mol. Cell. Biol. 1994; 14: 1431-1437Crossref PubMed Google Scholar), or visfatin (16Fukuhara A. Matsuda M. Nishizawa M. Segawa K. Tanaka M. Kishimoto K. Matsuki Y. Murakami M. Ichisaka T. Murakami H. Watanabe E. Takagi T. Akiyoshi M. Ohtsubo T. Kihara S. Yamashita S. Makishima M. Funahashi T. Yamanaka S. Hiramatsu R. Matsuzawa Y. Shimomura I. Science. 2005; 307: 426-430Crossref PubMed Scopus (1660) Google Scholar)), and Nam mononucleotide (NMN) adenylyltransferase, which convert Nam to NMN and NMN to NAD, respectively. Although Nam has been thought to represent the main precursor of the salvage synthesis to keep cellular contents of NAD constant in mammals (17Rongvaux A. Andris F. Van Gool F. Leo O. BioEssays. 2003; 25: 683-690Crossref PubMed Scopus (233) Google Scholar), the supplementation of Nam does not seem so effective in elevating cellular NAD contents beyond the basal level (18Revollo J.R. Grimm A.A. Imai S. J. Biol. Chem. 2004; 279: 50754-50763Abstract Full Text Full Text PDF PubMed Scopus (775) Google Scholar). nicotinamide nicotinic acid NA phosphoribosyltransferase NA mononucleotide NA adenine dinucleotide Nam phosphoribosyltransferase Nam mononucleotide 5-phosphoribosyl 1-pyrophosphate electrospray ionization mass spectrometry reverse transcription short interfering RNA NA, the other substrate of the salvage pathway, is converted by NA phosphoribosyltransferase (NAPRT) to NA mononucleotide (NaMN), which is then converted into NA adenine dinucleotide (NaAD), and lastly into NAD (Fig. 1). In mammals, which lack nicotinamidase (17Rongvaux A. Andris F. Van Gool F. Leo O. BioEssays. 2003; 25: 683-690Crossref PubMed Scopus (233) Google Scholar), NA seems to be derived primarily from the extracellular sources. Contrary to Nam, exogenously added NA has been clearly shown to be a better precursor in NAD biosynthesis than Nam and markedly increases NAD levels in mammalian tissues including liver, kidney, and heart in classical studies (19Collins P.B. Chaykin S. J. Biol. Chem. 1972; 247: 778-783Abstract Full Text PDF PubMed Google Scholar, 20Lin L.-F.H. Henderson L.M. J. Biol. Chem. 1972; 247: 8023-8030Abstract Full Text PDF PubMed Google Scholar, 21Williams G.T. Lau K.M. Coote J.M. Johnstone A.P. Exp. Cell Res. 1985; 160: 419-426Crossref PubMed Scopus (32) Google Scholar, 22Jackson T.M. Rawling J.M. Roebuck B.D. Kirkland J.B. J. Nutr. 1995; 125: 1455-1461PubMed Google Scholar). The tissue-specific increase in NAD levels by the addition of NA seems to correlate well with relatively high NAPRT activities in these tissues (23Shibata K. Hayakawa T. Iwai K. Agric. Biol. Chem. 1986; 50: 3037-3041Google Scholar), suggesting an important role of the enzyme in the NA-induced increase in cellular NAD levels. However, whether the enzyme indeed mediates the increases in cellular NAD levels and the role of the NA pathway in regulating biological processes through altering cellular NAD contents remain largely unknown, since the enzyme NAPRT has not been identified molecularly in mammals. In the present study we identified and characterized human NAPRT molecularly and showed using short interfering RNA (siRNA) knockdown of NAPRT that the enzyme is essential for the effects of NA to elevate NAD contents in human cells and protect the cells against oxidative stress. Given the critical roles of NAD in regulating cell functions, the strong capability to increase cellular NAD contents of NA, which has long been used for the treatment of hyperlipidemia (24Carlson L.A. J. Intern. Med. 2005; 258: 94-114Crossref PubMed Scopus (504) Google Scholar), may have clinical relevance. Materials—α-32PdCTP (3000 Ci/mmol) was purchased from Amersham Biosciences. carboxyl-14CNA (50 mCi/mmol) and carbonyl-14CNam (50 mCi/mmol) were from American Radiolabeled Chemical Inc. (St. Louis, MO). NA and Nam were from Nacalai Tesque (Kyoto, Japan) and Wako Pure Chemical Industries (Osaka, Japan), respectively. NAD and 5-phosphoribosyl 1-pyrophosphate (PRPP) were from Oriental Yeast (Tokyo, Japan) and Sigma, respectively. Cell Culture—Human hepatoma HepG2, kidney epithelia HEK293, and cervical carcinoma HeLa cells were cultured in Eagle's minimum essential medium (Sigma) and human promyelocytic HL60 cells in RPMI1640 medium (Sigma). These media were supplemented with 10% fetal bovine serum and antibiotics. Cloning and Expression of Human NAPRT and NamPRT cDNAs—Full-length human NAPRT cDNA was determined from a candidate sequence in a public data base (GenBank™ accession number AAH06284) and 5′- as well as 3′-flanking parts of the sequence obtained using rapid amplification of cDNA ends (see supplemental methods). Human NAPRT cDNA and the coding region of human NamPRT were ligated into pET22b (Novagen, Madison, WI) and pET15b (Novagen) to produce C- and N-terminal His6-tagged proteins, respectively. The recombinant proteins expressed in Escherichia coli BL21 (DE3) cells were purified with His-Bind resin (Novagen) (25Hara N. Yamada K. Terashima M. Osago H. Shimoyama M. Tsuchiya M. J. Biol. Chem. 2003; 278: 10914-10921Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). To express human NAPRT in eucaryotic cells as a fusion protein with a His6 tag, human NAPRT cDNA cloned into pcDNA3His6 (25Hara N. Yamada K. Terashima M. Osago H. Shimoyama M. Tsuchiya M. J. Biol. Chem. 2003; 278: 10914-10921Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) (pcDNA3-NAPRT) was transfected into the cells as described (25Hara N. Yamada K. Terashima M. Osago H. Shimoyama M. Tsuchiya M. J. Biol. Chem. 2003; 278: 10914-10921Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Sequences of expression plasmids and PCR fragments were confirmed by entire sequencing in both directions. Knockdown of NAPRT—siRNAs specific for human NAPRT were synthesized against two regions of human NAPRT cDNA (nucleotides 829–1634 and 445–935 for NAPRT siRNA1 and −2, respectively) using X-tremeGENE siRNA Dicer kit (Roche Applied Science). Hypoxanthine-guanine phosphoribosyltransferase specific siRNA, generated using the hypoxanthine-guanine phosphoribosyltransferase template included in the kit, was used as control siRNA. NAPRT and hypoxanthine-guanine phosphoribosyltransferase siRNAs (at final concentrations of 13–26 nm) were transfected into HEK293 cells using X-tremeGENE siRNA transfection reagent (Roche Applied Science). Two days after transfection cells were subjected to assays as indicated. The control siRNA reduced hypoxanthine-guanine phosphoribosyltransferase, but not NAPRT, transcript levels in HEK293 cells (data not shown). Preparation of Lysates from Culture Cells and Animal Tissues—Cultured human cells were collected and sonicated in buffer containing 0.5 m NaCl, 20 mm Tris-Cl– (pH 7.5), and 10% glycerol. Tissues were removed from female Wistar rats, washed with 0.9% NaCl, 10 mm Tris-Cl– (pH 7.5), 0.5 mm dithiothreitol, 1 mm EDTA, 1 mm EGTA, and protease inhibitor mixture (Roche Applied Science), and homogenized in the buffer. After centrifugation of these homogenates, the supernatants (whole cell lysates or tissue extracts) were subjected to enzyme assay or Western blot analysis as described below. Enzyme Assays—Unless otherwise stated, NAPRT activity was determined by measuring the formation of NaMN from NA and PRPP using thin layer chromatography (TLC). Enzyme preparations were incubated with 14CNA (50 mCi/mmol) and PRPP as indicated in standard reaction mixtures (50 μl) containing 50 mm Tris-Cl– (pH 7.5), 10 mm MgCl2, 2.5 mm dithiothreitol, 1 mm ATP, and 25 μg of bovine serum albumin. After incubating at 37 °C for the indicated times, the reaction was terminated by heating in a boiling water bath for 60 s. Proteins were removed by centrifugation, and reaction products were separated on silica gel sheets (Merck) using an isobutyric acid-5% ammonium hydroxide-water mixture (66:10:19, v/v/v) as a solvent and visualized and quantified using a bio-imaging analyzer BAS 2000 (Fujifilm, Tokyo, Japan). In some cases recombinant human NAPRT was incubated with 1 mm NA, quinolinic acid, or Nam in the presence of 0.6 mm PRPP in standard reaction mixture at 37 °C for 2 h. The reaction product (NaMN or NMN) was quantified by electrospray ionization mass spectrometry (ESI-MS) as described below. For kinetic analyses, purified recombinant NAPRT was incubated with the specified concentrations of NA and PRPP in standard reaction mixture with or without NAD as indicated at 37 °C, and the amount of NaMN formed was determined by TLC assay. Kinetic parameters were determined by analysis of a Lineweaver-Burk plot of the initial rate of NaMN synthesis. For determination of NamPRT activity, purified recombinant NamPRT was incubated with 14CNam (50 mCi/mmol) and PRPP in the presence or absence of NAD in the same standard reaction mixture as for NAPRT assay, and NMN formation was determined by TLC assay. Determination of Molecular Mass of Catalytically Active Human NAPRT—Purified recombinant human NAPRT was electrophoresed on non-denaturing polyacrylamide gel as described previously (25Hara N. Yamada K. Terashima M. Osago H. Shimoyama M. Tsuchiya M. J. Biol. Chem. 2003; 278: 10914-10921Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar). Gel sliced into 2-mm pieces was incubated with 50 μm 14CNA and 0.3 mm PRPP in standard reaction mixture (100 μl) at 37 °C for 3 h. The amount of NaMN formed in the reaction mixture was determined by TLC assay. Determination of NAD Synthesis by Human Cells—Cellular contents of NAD and related compounds were simultaneously determined by ESI-MS analysis, as described previously (26Yamada K. Hara N. Shibata T. Osago H. Tsuchiya M. Anal. Biochem. 2006; 352: 282-285Crossref PubMed Scopus (131) Google Scholar), using a triple quadrupole mass spectrometer (API3000, Applied Biosystems, Foster City, CA). Based on NAD contents and packed volumes of human cells, basal cellular concentrations of NAD were calculated as 503 ± 104 μm for HEK293 cells, 546 ± 46 μm for HeLa cells, and 597 ± 90 μm for HL60 cells (mean ± S.D. of three separate experiments). HepG2 cells transfected as indicated were incubated with 1 μCi/ml 14CNA for 6 h. After incubation, pyridine nucleotides were extracted from each cell pellet as described (7Hasmann M. Schemainda I. Cancer Res. 2003; 63: 7436-7442PubMed Google Scholar), separated on silica gel sheets using the solvent as described above, and quantified by BAS 2000. mRNA Analysis—NAPRT gene expression was determined in various Balb/c mouse tissues by Northern blot analysis (25Hara N. Yamada K. Terashima M. Osago H. Shimoyama M. Tsuchiya M. J. Biol. Chem. 2003; 278: 10914-10921Abstract Full Text Full Text PDF PubMed Scopus (57) Google Scholar) using NAPRT cDNA probe (corresponding to amino acids 200–521 of mouse NAPRT in supplemental Fig. 1) labeled with α-32PdCTP. Mouse NAPRT cDNA fragment was amplified from Balb/c mouse tissue total RNA by reverse transcription (RT)-PCR using primers 5′-GTG AGG TGA ATG TCA TTG GC-3′ (sense) and 5′-ACA GTG CGA CCG GAT ACA CT-3′ (antisense). Western Blot Analysis—Polyclonal anti-human NAPRT antibodies were generated by immunizing a mouse with purified recombinant human NAPRT and purified on the recombinant enzyme blotted to a polyvinylidene fluoride membrane (Millipore, Bedford, MA). NAPRT was immunodetected with anti-human NAPRT and peroxidase-conjugated anti-mouse IgG (MBL, Nagoya, Japan) antibodies, as described previously (27Hara N. Tsuchiya M. Shimoyama M. J. Biol. Chem. 1996; 271: 29552-29555Abstract Full Text Full Text PDF PubMed Scopus (34) Google Scholar). Protein loading was assessed using rabbit anti-actin (Sigma) and anti-rabbit IgG (MBL) antibodies. Determination of Cytotoxicity—HEK293 cells untransfected, mock-transfected, or transfected with hypoxanthine-guanine phosphoribosyltransferase or human NAPRT siRNAs were seeded at 2 × 104 cells/well in 96-well and to the cells were in Eagle's minimum essential medium with or without exogenously added NA or Nam as indicated and then further incubated in the presence or absence of with or without added NA or After incubation, reagent was added to the to the Japan) to cellular activity M. H. M. K. Y. K. Watanabe M. In 1995; Scholar). After 2 of incubation, at was a was calculated as described T. Yamada K. Hara N. K. Osago H. Terashima M. T. T. Tsuchiya M. J. 2003; PubMed Scopus Google Scholar). assay was in Cloning of Human NAPRT on a candidate sequence of human NAPRT in a public data base (GenBank™ accession number we determined 5′- as well as 3′-flanking parts of the sequence using rapid amplification of cDNA ends and obtained a human NAPRT cDNA (GenBank™ accession number a protein of amino acids (see supplemental Fig. 1). The of NAPRT not significant to the Expression and of Human protein by the expressed in E. coli as His6-tagged recombinant protein and purified on has a molecular mass of than the calculated from the (Fig. In human cell and antibodies against purified recombinant human NAPRT endogenous proteins with a molecular mass of as well as the expressed enzyme and shown in Fig. recombinant human protein the formation of in the presence of 50 μm 14CNA and 50 μm and the of NaMN formed were with those of NA The of either PRPP or from the reaction mixture in a of NaMN synthesis (data not shown). These results indicate that the cDNA NAPRT in human NAPRT not the formation of NMN or NaMN from Nam or quinolinic acid (data not shown). purified recombinant NAPRT was by non-denaturing and NAPRT activity was determined in gel the activity of human NAPRT with a protein of (Fig. suggesting that the human enzyme may as a as described for the enzyme J. Biol. Chem. Full Text PDF PubMed Google Scholar, J. L.S. J. Biol. Chem. Full Text PDF PubMed Google Scholar, T. Shibata K. Iwai K. Agric. Biol. Chem. of expression of NAPRT on cellular NAD biosynthesis in the presence of NA in human cells. HepG2 cells were transfected with either pcDNA3His6 or as indicated. cell lysates from these cells were subjected to Western blot analysis with or anti-actin antibodies as indicated. The lysates from × cells were incubated with 50 μm 14CNA and 0.3 mm PRPP for 2 h. The amount of NaMN formed was determined by TLC assay and HepG2 cells transfected as indicated × cells on were incubated in the presence of 20 μm 14CNA for 6 h. After incubation, pyridine nucleotides were extracted from these cells, separated by TLC and quantified as described The of and of NAD and of formed in and the represent the ± S.D. of at three separate otherwise of NAPRT expression in mouse and 10 μg of total RNA from the indicated mouse tissues on gel was using Northern blot with a mouse NAPRT cDNA The blot was then and with a cDNA The of and RNA indicated by from mouse tissues were subjected to for NAPRT and tissue from were subjected to Western blot analysis with anti-human NAPRT antibodies The blot was then and with anti-actin antibodies Molecular indicated on the from were incubated with 50 μm 14CNA and 0.3 mm PRPP for 1 and the amount of NaMN formed was determined by TLC assay To whether the human enzyme mediates NAD biosynthesis from NA, we for human cell the expression of NAPRT is not We that is the with human hepatoma HepG2 cells (data not NAPRT protein levels and activity were in HepG2 cells transfected with (Fig. In HepG2 cells transfected with human NAPRT cDNA NAPRT activity as well as a protein with a molecular mass of (Fig. The expressed protein was cells (see supplemental Fig. the of human enzyme in We cultured these transfected HepG2 cells in the presence of 14CNA and determined the of compounds formed in these cells. of the compounds in cell by TLC revealed that the amount of 14CNAD was in NAPRT cells than in those transfected with the and was observed in cells expressing human NAPRT and these was not The small amount of 14CNAD observed in cells a of NAPRT activity in the cells. these indicate that the cDNA identified here indeed NAPRT protein in human cells and that the enzyme mediates the NAD biosynthesis from NA in the cells. of the tissue of NAPRT, Northern blot analysis was with total RNA from various mouse shown in Fig. a was in the small intestine, liver, kidney, and with these revealed a expression of the NAPRT gene in the three tissues and a expression in other tissues including the heart (Fig. In tissues Western blot analysis revealed the of NAPRT protein in the kidney and (Fig. with these activity of NAPRT was in these two and significant activity was also in other tissues including the heart (Fig. in these tissues demonstrated the presence of endogenous NAPRT protein and enzyme activity in the (data not shown). NAPRT transcript was observed in the small in mice and but we not NAPRT protein in the small in (Fig. due to of the enzyme the of tissue of NA in Culture NAD that human NAPRT mediates NAD biosynthesis from NA, we whether the of NAD biosynthesis from NA elevate NAD levels. HEK293 cells were cultured in the presence of exogenously added NA, and the total cellular contents of NAD as well as related compounds were determined by ESI-MS shown in Fig. the addition of NA in medium markedly the total cellular NAD contents in a as 1 NA the cellular NAD increase beyond the basal level was observed with μm The increase in NAD contents well with the of in the cells (Fig. In of added Nam not increase cellular NAD levels (Fig. and these the of were the of However, Nam concentration in medium was to cellular NAD contents were to ± that of the control (mean ± S.D. of three separate and a small but significant amount of cellular was ± cells, ± S.D. of three separate experiments). To whether the increase in cellular NAD contents in the presence of NA is mediated by NAPRT activity, we the effects of the knockdown of NAPRT expression on NAD contents in HEK293 cells. with cells transfected with control siRNA, cells transfected with siRNAs specific for NAPRT significant in NAPRT enzyme activity as well as NAPRT protein and these cells were cultured in the presence of NA, the of increase in NAD (Fig. and (Fig. contents was markedly reduced in NAPRT cells. Knockdown of NAPRT expression not basal NAD contents obtained in the absence of added NA (Fig. These indicate that cellular NAD contents be by the addition of NA, but not Nam, at the in medium and that NAPRT activity mediates increases in NAD contents in human cells. Human NAPRT by that NA was a better precursor to increase total NAD contents than Nam in HEK293 cells may be by the lack of feedback inhibition of NAPRT, but not by NAD L.S. O. M. J. 14: Google Scholar, J. M. H. J. Biochem. PubMed Scopus Google Scholar, M.C. Biochem. Res. PubMed Scopus Google Scholar). To we kinetic analyses in the presence or absence of NAD using the recombinant human