In conclusion, our work shows that NR is a powerful supplement to boost NAD+ levels, activate sirtuin signalling and improve mitochondrial function, suggesting that this vitamin could be used to prevent and treat the mitochondrial decline that is a hallmark of many diseases associated with aging. Very recent work, showing that intraperitoneal administration of NMN could improve the metabolic damage induced by high fat feeding (Yoshino et al., 2011) further supports this concept. To date, however, only NR has been identified as a naturally occurring component of the human diet (Bieganowski and Brenner, 2004). Furthermore, NR protects against metabolic dysfunction at lower concentrations than those reported for NMN and we proved that it is effective after oral administration when mixed with food, which is in contrast to NMN which is injected intraperitoneally (Yoshino et al., 2011). So, how might NR and NMN biology intertwine and impact on NAD+ synthesis in mammalian physiology? Recent observations suggest that, at least for some cell types, NMN needs to be converted into NR through dephosphorylation performed by extracellular 5′-nucleotidases in order to penetrate into the cell (Nikiforov et al., 2011). Given that the presence of NMN in plasma is debated (Hara et al., 2011), the most likely scenario is that NR, rather than NMN, is the entity directly taken up by the cell, to then become metabolized in the cytosolic compartment to NMN. The lack of NRK1 and NRK2 in the mitochondrial compartment (Nikiforov et al., 2011) supports that NMN might act as the precursor entering the mitochondria or other compartments for further metabolism into NAD+ by the various NMNAT enzymes (see Fig.7 for a scheme).
Our data, combined with the evidence that other NAD+ precursors can improve age-related insulin resistance (Yoshino et al., 2011) and that NR increases yeast replicative lifespan (Belenky et al., 2007), therefore warrant future investigations to see if boosting NAD+ levels by NR supplementation might also improve the health- and lifespan of humans.

Experimental Procedures

Materials

All chemicals and reagents were purchased from Sigma-Aldrich unless stated otherwise. Nicotinamide Riboside was custom synthesized as previously described (Yang et al., 2007b) and was kindly provided by Amazentis and Merck Research Laboratories.

Animal experiments

8 weeks-old male C57Bl/6J mice were purchased from Charles River and powder chow (D12450B) and high fat (D12492) diets were from Research Diets Inc (New Brunswick, NJ, USA). 80 ml of water per kg of powder CD were used to make food pellets. 40 ml of water per kg of powder HFD were used to make food pellets. For NR, NMN and NA supplemented diets, the appropriate amount of these compounds was added to the water used to create the pellets, taking into account the differences in the daily intake of each diet. Mice were housed separately, had ad libitum access to water and food and were kept under a 12h dark-light cycle. Mice were fed with homemade pellets from 10 weeks of age. To make the pellets, the powder food was mixed with water (vehicle) or with NR. All animal experiments were carried according to national Swiss and EU ethical guidelines and approved by the local animal experimentation committee of the Canton de Vaud under license #2279.

Animal phenotyping

Mice were weighed and the food consumption was measured each week on the same day. Most clinical tests were carried out according to standard operational procedures (SOPs) established and validated within the Eumorphia program (Champy et al., 2008). Body composition was determined by Echo-MRI (Echo Medical Systems, Houston, TX, USA) and oxygen consumption (VO2), respiratory exchange ratios (RER), food intake and activity levels were monitored by indirect calorimetry using the comprehensive laboratory animal monitoring system (Columbus Instruments, Columbus, OH, USA). Treadmill, cold tests and hyperinsulinemic-euglycemic clamps were performed as described (Lagouge et al., 2006). Glucose and insulin tolerance was analyzed by measuring blood glucose and insulin following intraperitoneal injection of 2 g/kg glucose or 0.25 U insulin/kg, respectively, after an overnight fast. Plasma insulin was determined in heparinized plasma samples using specific ELISA kits (Mercodia). All animals were sacrificed at 13:00, after a 6 hr fast, using CO2 inhalation. Blood samples were collected in heparinized tubes and plasma was isolated after centrifugation. All plasma parameters were measured using a Cobas c111 (Roche Diagnostics). Tissues were collected upon sacrifice and flash-frozen in liquid nitrogen.

Histology and microscopy

Succinate dehydrogenase staining and transmission electron microscopy (TEM) was performed as described (Bai et al., 2011b). Mitochondrial size and cristae content was analyzed as previously described (St-Pierre et al., 2003).

Cell culture and transfection

Murine C2C12 myoblasts were grown and differentiated into myotubes as described (Canto et al., 2009). Murine Hepa1.6 and human HEK293T cells were cultured in DMEM (4,5 g/l glucose, 10% FCS). Human FOXO1 and SIRT1 siRNAs were obtained from Dharmacon (Thermo Scientific) and used following the instructions from the manufacturer. SIRT3 MEFs were established according to standard techniques (Picard et al., 2002) from conditional SIRT3−/− mice, whose generation will be described separately (Fernandez-Marcos et al., submitted). Deletion of the SIRT3 gene was induced via infection with adenovirus encoding for the Cre recombinase.

Gene expression and mitochondrial DNA abundance

RNA was extracted and quantified by qRT-PCR as described (Lagouge et al., 2006). The qPCR primers used have been previously described. Mitochondrial DNA abundance (mtDNA) was quantified as described (Lagouge et al., 2006) after isolating total DNA using a standard phenol extraction.

Immunoprecipitation, SDS-PAGE, Western blotting

Cells were lysed in lysis buffer (50 mM Tris, 150 mM KCl, EDTA 1 mM, NP40 1%, nicotinamide 5mM, Na-butyrate 1 mM, protease inhibitors pH7,4). Immunoprecipitation from cultured cells or tissue samples was performed exactly as described (Canto et al., 2009). For western blotting, proteins were separated by SDS-PAGE and transferred onto nitrocellulose membranes. Anti-Acetyl-Lysine antibodies were from Cell Signalling; FOXO1 and SOD2 antibodies were from Santa Cruz Biotechnology; SIRT1 and PGC-1α antibodies were from Millipore; Tubulin and Acetyl-tubulin antibodies were from Sigma Inc. Antibodies for mitochondrial markers were purchased from Mitosciences. Antibody detection reactions were developed by enhanced chemiluminescence (Amersham, Little Chalfont, UK).

NAD+, NADH and NAM determination

NAD+ levels were determined using a commercial kit (Enzychrom, BioAssays Systems, CA) and tissue samples were also measured by HPLC (Ramsey et al., 2009). Other NAD metabolites were determined as previously described (Yang and Sauve, 2006).

GPR109A – calcium mobilization assay

Ready-to-Assay™ GRP109A Nicotinic Acid Receptor Cells were used to measure calcium mobilization as specified by the manufacturer (Millipore). Calcium flux was determined using excitation at 340 and 380 nm in a fluorescence spectrophotometer (VictorX4, Perkin Elmer) in a 180 seconds time course, adding the ligand at 60 seconds. Internal validation was made using 0,1% Triton X-100 for total fluorophore release and 10 mM EGTA to chelate free calcium. Similarly, GPR109A specificity was internally validated using control cells devoid of GRP109A overexpression.

Liver triglyceride measurement

Liver triglycerides were measured from 20 mg of liver tissue using a variation of the Folch method, as described (Bai et al., 2011a).

Statistics

For the statistical analysis of the animal studies, all data was verified for normal distribution. To assess significance we performed Student’s t-test for independent samples. Values are expressed as mean ± SD unless otherwise specified.

Highlights

Acknowledgements

This work was supported by grants of the École Polytechnique Fédérale de Lausanne, Swiss National Science Foundation, the European Research Council Ideas programme (Sirtuins; ERC-2008-AdG231-118) and the Velux foundation. RHH has been supported by a Rubicon fellowship of the Netherlands Organization for Scientific Research and by an AMC postdoc fellowship. EP is funded by the Academy of Finland. JA is the Nestle chair in energy metabolism. AAS received grants to support this research from the Ellison Medical Foundation New Scholar Award 2007 and contract C023832 from NY State Spinal Cord Injury Board. The authors thank Robert Myers, Peter Meinke and Thomas Vogt at Merck Research Laboratories, Rahway and Charles Thomas at Amazentis, Lausanne, for the kind gift of NR, all the members of the Auwerx lab for inspiring discussions and Graham Knott of the BioEM facility at EPFL for EM imaging.

References