Original Publication: Cantó C, Houtkooper RH, Pirinen E, et al. The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet induced obesity. Cell metabolism. 2012;15(6):838-847. doi:10.1016/j.cmet.2012.04.022.
Summary
As NAD+ is a rate-limiting co-substrate for the sirtuin enzymes, its modulation is emerging as a valuable tool to regulate sirtuin function and, consequently, oxidative metabolism. In line with this premise, decreased activity of PARP-1 or CD38 —both NAD+ consumers— increases NAD+ bioavailability, resulting in SIRT1 activation and protection against metabolic disease. Here we evaluated whether similar effects could be achieved by increasing the supply of nicotinamide riboside (NR), a recently described natural NAD+ precursor with the ability to increase NAD+ levels, Sir2-dependent gene silencing and replicative lifespan in yeast. We show that NR supplementation in mammalian cells and mouse tissues increases NAD+ levels and activates SIRT1 and SIRT3, culminating in enhanced oxidative metabolism and protection against high fat diet-induced metabolic abnormalities. Consequently, our results indicate that the natural vitamin, NR, could be used as a nutritional supplement to ameliorate metabolic and age-related disorders characterized by defective mitochondrial function.
Introduction
The administration of NAD+ precursors, mostly in the form of nicotinic acid (NA), has long been known to promote beneficial effects on blood lipid and cholesterol profiles and even to induce short-term improvement of type 2 diabetes (Karpe and Frayn, 2004). Unfortunately, NA treatment often leads to severe flushing, resulting in poor patient compliance (Bogan and Brenner, 2008). These side effects are mediated by the binding of NA to the GPR109A receptor (Benyo et al., 2005). We hence became interested in the possible therapeutic use of alternative NAD+ precursors that do not activate GPR109A.
NR was recently identified as a NAD+ precursor, with conserved metabolism from yeast to mammals (Bieganowski and Brenner, 2004). Importantly, NR is found in milk (Bieganowski and Brenner, 2004) constituting a dietary source for NAD+ production. Once it enters the cell, NR is metabolized into nicotinamide mononucleotide (NMN) by a phosphorylation step catalyzed by the nicotinamide riboside kinases (NRKs) (Bieganowski and Brenner, 2004). In contrast to NR, NMN has not yet been found in dietary constituents and its presence in serum is a matter of debate (Hara et al., 2011; Revollo et al., 2007). This highlights how NR might be an important vehicular form of an NAD+ precursor, whose levels could be modulated through nutrition.
The sirtuins are a family of enzymes that use NAD+ as a cosubstrate to catalyze the deacetylation and/or mono-ADP-ribosylation of target proteins. One of their major particularities is that their Km for NAD+ is relatively high, making NAD+ a rate-limiting substrate for their reaction (Canto and Auwerx, 2012). Initial work by yeast biologists indicated that the activity of Sir2 (the yeast SIRT1 homolog) as an NAD+-coupled enzyme could provide a link between metabolism and gene silencing (Imai et al., 2000a; Imai et al., 2000b). In this way, Sir2 was proposed to mediate metabolic transcriptional adaptations linked to situations of nutrient scarcity, which are generally coupled to increased NAD+ levels (for review, see Houtkooper et al., 2010). During the last decade, an overwhelming body of evidence indicates that the activity of mammalian sirtuins, most notably SIRT1 and SIRT3, have the ability to enhance fat oxidation and prevent against metabolic disease (Hirschey et al., 2011; Lagouge et al., 2006; Pfluger et al., 2008). Therefore, strategies aimed to increase intracellular NAD+ levels have gained interest in order to activate sirtuins and battle metabolic damage. Validation of this concept was achieved recently, by demonstrating that pharmacological and genetic approaches aimed to reduce the activity of major NAD+ consuming activities in the cell, such as PARP-1 (Bai et al., 2011b) and CD38 (Barbosa et al., 2007), prompted an increase in NAD+ bioavailability and enhanced SIRT1 activity, ultimately leading to effective protection against metabolic disease. In this work we aimed to test whether similar effects could be achieved through dietary supplementation with a natural NAD+ precursor, such as NR.
Results
NR increases intracellular and mitochondrial NAD+ content in mammalian cells and tissues
NR treatment dose-dependently increased intracellular NAD+ levels in murine and human cell lines (Fig.1A), with maximal effects at concentrations between 0.5 and 1 mM. In C2C12 myotubes, the Km for NR uptake was 172.3±17.6 μM, with a Vmax of 204.2±20.5 pmol/mg of protein/min. Unlike NA, both NR and another well-described NAD+ precursor, NMN (Revollo et al., 2007), did not activate GPR109A (Fig.1B), hence constituting valuable candidates to increase NAD+ levels without activating GPR109A. Strikingly, the ability of NR to increase intracellular NAD+ in mammalian cells was, at least, similar to that of these other precursors (Fig.1C). We next evaluated the efficacy of NR, NMN and NA to increase NAD+ in vivo by supplementing mouse chow with NR, NMN or NA at 400 mg/kg/day for one week. All compounds increased NAD+ levels in liver, but only NR and NA significantly enhanced muscle NAD+ content. (Fig.1D). These results illustrate how NR administration is a valid tool to boost NAD+ levels in mammalian cells and tissues without activating GPR109A.