Uptake Of NAD Research Articles

SLC25A51 is a mammalian mitochondrial NAD+ transporter.

SummaryMitochondria require nicotinamide adenine dinucleotide (NAD+) in order to carry out the fundamental processes that fuel respiration and mediate cellular energy transduction. Mitochondrial NAD+ transporters have been identified in yeast and plants 1,2 but their very existence is controversial in mammals 3–5. Here we demonstrate that mammalian mitochondria are capable of taking up intact NAD+ and identify SLC25A51 (an essential 6,7 mitochondrial protein of previously unknown function, also known as MCART1) as a mammalian mitochondrial NAD+ transporter. Loss of SLC25A51 decreases mitochondrial but not whole-cell NAD+ content, impairs mitochondrial respiration, and blocks the uptake of NAD+ into isolated mitochondria. Conversely, overexpression of SLC25A51 or a nearly identical paralog, SLC25A52, increases mitochondrial NAD+ levels and restores NAD+ uptake into yeast mitochondria lacking endogenous NAD+ transporters. Together, these findings identify SLC25A51 as the first transporter capable of importing NAD+ into mammalian mitochondria.

Exogenous nicotinamide adenine dinucleotide regulates energy metabolism via hypothalamic connexin 43

BackgroundNicotinamide adenine dinucleotide (NAD)-dependent deacetylase SIRT1 is an important regulator of hypothalamic neuronal function. Thus, an adequate hypothalamic NAD content is critical for maintaining normal energy homeostasis. MethodsWe investigated whether NAD supplementation increases hypothalamic NAD levels and affects energy metabolism in mice. Furthermore, we investigated the mechanisms underlying the effects of exogenous NAD on central metabolism upon entering the hypothalamus. ResultsCentral and peripheral NAD administration suppressed fasting-induced hyperphagia and weight gain in mice. Extracellular NAD was imported into N1 hypothalamic neuronal cells in a connexin 43-dependent and CD73-independent manner. Consistent with the in vitro data, inhibition of hypothalamic connexin 43 blocked hypothalamic NAD uptake and NAD-induced anorexia. Exogenous NAD suppressed NPY and AgRP transcriptional activity, which was mediated by SIRT1 and FOXO1. ConclusionsExogenous NAD is effectively transported to the hypothalamus via a connexin 43-dependent mechanism and increases hypothalamic NAD content. Therefore, NAD supplementation is a potential therapeutic method for metabolic disorders characterized by hypothalamic NAD depletion.

High-Affinity Transporters for NAD+ Precursors in Candida glabrata Are Regulated by Hst1 and Induced in Response to Niacin Limitation

The yeast Candida glabrata is an opportunistic pathogen of humans. C. glabrata is a NAD(+) auxotroph, and its growth depends on the availability of niacin (environmental vitamin precursors of NAD(+)). We have previously shown that a virulence-associated adhesin, encoded by EPA6, is transcriptionally induced in response to niacin limitation. Here we used transcript profiling to characterize the transcriptional response to niacin limitation and the roles of the sirtuins Hst1, Hst2, and Sir2 in mediating this response. The majority of genes transcriptionally induced by niacin limitation are regulated by Hst1, suggesting that it is the primary sensor of niacin limitation in C. glabrata. We show that three highly induced genes, TNA1, TNR1, and TNR2, encode transporters which are necessary and sufficient for high-affinity uptake of NAD(+) precursors. Strikingly, if a tna1 tnr1 tnr2 mutant is starved for niacin, it exhibits an extended lag phase, suggesting a central role for the transporters in restoring NAD(+) homeostasis after niacin limitation. Lastly, we had previously shown that the adhesin encoded by EPA6 is induced during experimental urinary tract infection (UTI); we show here that EPA6 transcriptional induction during UTI is strongly enhanced in the tna1 tnr1 tnr2 mutant strain, implicating the transporters in the growth of C. glabrata during infection.

Ca2+ Signaling Occurs via Second Messenger Release from Intraorganelle Synthesis Sites

SummaryCyclic ADP-ribose is an important Ca2+-mobilizing cytosolic messenger synthesized from β-NAD+ by ADP-ribosyl cyclases (ARCs). However, the focus upon ectocellular mammalian ARCs (CD38 and CD157) has led to confusion as to how extracellular enzymes generate intracellular messengers in response to stimuli. We have cloned and characterized three ARCs in the sea urchin egg and found that endogenous ARCβ and ARCγ are intracellular and located within the lumen of acidic, exocytotic vesicles, where they are optimally active. Intraorganelle ARCs are shielded from cytosolic substrate and targets by the organelle membrane, but this barrier is circumvented by nucleotide transport. We show that a β-NAD+ transporter provides ARC substrate that is converted luminally to cADPR, which, in turn, is shuttled out to the cytosol via a separate cADPR transporter. Moreover, nucleotide transport is integral to ARC activity physiologically because three transport inhibitors all inhibited the fertilization-induced Ca2+ wave that is dependent upon cADPR. This represents a novel signaling mechanism whereby an extracellular stimulus increases the concentration of a second messenger by promoting messenger transport from intraorganelle synthesis sites to the cytosol.

Characterization of NAD Uptake in Mammalian Cells

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.

Cooperative and non-cooperative conformational effects of the coenzyme on yeast glyceraldehyde-3-phosphate dehydrogenase.

Changes induced on addition of the cofactor, NAD, to glyceraldehyde-3-phosphate dehydrogenase from yeast have been investigated by hydrodynamic and spectroscopic techniques. As shown by light-scattering and sedimentation and diffusion studies, the tetrameric structure of the holoenzyme persists under all but strong dissociating conditions: there is thus no change in the quaternary structure on binding. On the other hand, binding is accompanied by an increase of some 4% in the sedimentation coefficient, which is too large to be explicable in terms of the increase in particle weight by bound ligand. A conformational change is also observed by optical rotatory dispersion and circular dichroism measurements. The magnitude of the Cotton effect associated with the aromatic chromophores of the protein, near 280 nm, increases, whereas the rotation in the neighbourhood of 233 nm undergoes a small diminution as the cofactor is added. It is shown that these changes are conformational in origin, rather than a consequence of NAD attachment per se. Increasing the temperature in the range 20–40° leads to a transition from non-cooperative to cooperative binding of NAD to the apoenzyme. At 40°, where NAD binding is weakly sigmoidal, the change in optical rotation is likewise sigmoidal and parallels the increase in the sedimentation coefficient. At 20° both the uptake of NAD and the structural changes are hyperbolic. In the cooperative situation the data are shown to be consistent with the two-state “concerted” model for ligand-binding and incompatible with a sequential mechanism.

PnuC and the Utilization of the Nicotinamide Riboside Analog 3-Aminopyridine inHaemophilus influenzae

The utilization pathway for the uptake of NAD and nicotinamide riboside was previously characterized for Haemophilus influenzae. We now report on the cellular location, topology, and substrate specificity of PnuC. pnuC of H. influenzae is only distantly related to pnuC of Escherichia coli and Salmonella enterica serovar Typhimurium. When E. coli PnuC was expressed in an H. influenzae pnuC mutant, it was able to take up only nicotinamide riboside and not nicotinamide mononucleotide. Therefore, we postulated that PnuC transporters in general possess specificity for nicotinamide riboside. Earlier studies showed that 3-aminopyridine derivatives (e.g., 3-aminopyridine adenine dinucleotide) are inhibitory for H. influenzae growth. By testing characterized strains with mutations in the NAD utilization pathway, we show that 3-aminopyridine riboside is inhibitory to H. influenzae and is taken up by the NAD-processing and nicotinamide riboside route. 3-Aminopyridine riboside is utilized effectively in a pnuC+ background. In addition, we demonstrate that 3-aminopyridine adenine dinucleotide resynthesis is produced by NadR. 3-Aminopyridine riboside-resistant H. influenzae isolates were characterized, and mutations in nadR could be detected. We also tested other species of the family Pasteurellaceae, Pasteurella multocida and Actinobacillus actinomycetemcomitans, and found that 3-aminopyridine riboside does not act as a growth inhibitor; hence, 3-aminopyridine riboside represents an anti-infective agent with a very narrow host range.

NADP and NAD utilization in Haemophilus influenzae

Exogenous NAD utilization or pyridine nucleotide cycle metabolism is used by many bacteria to maintain NAD turnover and to limit energy-dependent de novo NAD synthesis. The genus Haemophilus includes several important pathogenic bacterial species that require NAD as an essential growth factor. The molecular mechanisms of NAD uptake and processing are understood only in part for Haemophilus. In this report, we present data showing that the outer membrane lipoprotein e(P4), encoded by the hel gene, and an exported 5'-nucleotidase (HI0206), assigned as nadN, are necessary for NAD and NADP utilization. Lipoprotein e(P4) is characterized as an acid phosphatase that uses NADP as substrate. Its phosphatase activity is inhibited by compounds such as adenosine or NMN. The nadN gene product was characterized as an NAD-nucleotidase, responsible for the hydrolysis of NAD. H. influenzae hel and nadN mutants had defined growth deficiencies. For growth, the uptake and processing of the essential cofactors NADP and NAD required e(P4) and 5'-nucleotidase. In addition, adenosine was identified as a potent growth inhibitor of wild-type H. influenzae strains, when NADP was used as the sole source of nicotinamide-ribosyl.

Metabolic fate of extracellular NAD in human skin fibroblasts.

Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.

Electrotransfer of [ 32P]NAD allows labeling of ADP-ribosylated proteins in intact Chinese hamster ovary cells

CHO-K1D cells electroporated in buffers containing [ 32P]NAD incorporated the label in a voltage-dependent manner. Electroporation with 650 V/cm at 1460 μF in Ham's F12 medium supplemented with 10 m m Hepes, pH 7.1, resulted in a >20-fold increase in [ 32P]NAD uptake, while decreasing relative cellular survival by only 6%. Exposure of cells to gamma irradiation (20 Gy) prior to electroporation increased the steady-state level of poly(ADP-ribosylated) nuclear proteins two- to fourfold over that of unirradiated control cells. These data indicate that electrotransfer of [ 32P]NAD is a simple and rapid means of labeling the cellular NAD pool and should prove useful in the analysis of the relationship between poly(ADP-ribosylation) of nuclear proteins and DNA repair.

Reactions of the intracellular NADpool in the yeast S. cerevisiae after UV-C- or X-ray irradiation.

The reaction of the intracellular NADpool after irradiation of cells either with UV-C light or with X-rays was studied in four different strains of the yeast S. cerevisiae. We found neither in wildtype strains nor in radiation sensitive mutants remarkable changes in the NADpool within 2 h after irradiation. Preculture of cells in medium enriched with nicotinic acid, a precursor of NAD, influenced the intracellular NAD concentration only to a small extend in all strains, but enhanced the radiation resistance against UV-C significantly in one rad6 mutant strain. The uptake of NAD and NAC by all strains before and after irradiation with UV-C and X-ray was tested also. NAD generally is taken up by the cells to a very low extent before and after irradiation without irradiation-dose dependency. NAC is taken up by all strains before and after irradiation. Only the rad6 mutant exhibited an irradiation-dose dependent NAC-uptake after UV-C irradiation.

An electrophysiological analysis of the uptake of noradrenaline at sympathetic nerve terminals.

1. An electrophysiological analysis has been made of the uptake of NAd in the sympathetic nerve terminals of the isolated vas deferens of the mouse. The amplitude of the excitatory junction potentials (e.j.p.s) recorded intracellularly in smooth muscle cells was taken as a measure of the NAd output per impulse from the terminals of sympathetic axons.2. Neuronal uptake blockers (desipramine and cocaine) greatly depressed the amplitude of all e.j.p.s after the first in a short train (< 100) at high frequencies (>/= 1 Hz).3. Blocking neuronal uptake did not affect the time course of decline in amplitude of the e.j.p. during long trains of stimulation over several minutes, apart from the immediate depression in the e.j.p. following the first few impulses.4. The time course of decline of the e.j.p. amplitude during continual stimulation, when both neuronal uptake and synthesis was blocked, was similar to that when only synthesis was blocked, apart from the immediate depression following the first few impulses.5. Phenoxybenzamine reversed the normal depression in e.j.p. amplitude observed at high frequencies (>/= 1 Hz) to facilitation. This facilitation lasted for several minutes of high frequency stimulation.6. A model has been proposed of the sympathetic nerve terminal, in which NAd is released by each nerve impulse in a train from a small pool in the nerve terminal, which is principally replenished by uptake of the NAd released by the immediately preceding impulses in the train. The pool is replenished to a less extent by transmitter located in two stores in the terminal which are in turn replenished by transmitter synthesis.