Impact of nicotinamide riboside supplementation on skeletal muscle mitochondria and whole-body glucose homeostasis: challenging the current hypothesis.

In recent decades, data from several studies have supported the notion that declines in mitochondrial structure and function are strongly linked to obesity, insulin resistance, and type 2 diabetes in both rodent and human tissues. Further, impaired NAD+ metabolism and NAD+-mediated sirtuin signalling are also implicated in ageing and the above metabolic diseases (Dollerup et al. 2020). Currently, lifestyle manipulations such as energy restriction and exercise remain the most effective interventions known to improve metabolic health and promote mitochondrial biogenesis and function in human skeletal muscle (Hesselink et al. 2016). Hence, therapeutically targeting NAD+ to combat ageing and metabolic disease has garnered interest in recent years. In this regard, the nutritional supplement nicotinamide riboside (NR), a naturally occurring vitamin B3 and a precursor of NAD+, is attracting attention. NAD+ and its reduced form, NADH, are major regulators of energy metabolism in various tissues, including skeletal muscle, which require NAD+ for the catalytic activities for beta-oxidation, glycolysis, and the Kreb's cycle. In addition to its role in cellular energy metabolism, NAD+ has also been extensively investigated for its contribution to DNA repair, sirtuin activity, and mitochondrial biogenesis and function (Fang et al. 2017). Interestingly, preclinical studies demonstrated that supplementing NAD+ precursors, such as NR, enhanced oxidative metabolism and protected against obesity and insulin resistance in mice fed a high-fat diet (Fang et al. 2017). While recent data have shown NR supplementation increases NAD+ bioavailability and is well tolerated in humans (Martens et al. 2018), it remains unclear whether NR improves insulin resistance or attenuates mitochondrial dysfunction in humans. In a recently published series of papers, Dollerup and colleagues (Dollerup et al. 2018, 2019, 2020) investigated the effects of 12 weeks of NR supplementation on safety, insulin sensitivity, endocrine response, and mitochondrial function in 40 males with obesity randomized to receive NR or placebo. The most recent report from this randomized control trial was published in the February 2020 issue of The Journal of Physiology and focused on evaluating the effects of the NR supplementation on skeletal muscle mitochondrial content, respiration, and morphology in men with obesity and insulin resistance (Dollerup et al. 2020). Dollerup et al. utilized both quantitative and qualitative measures of mitochondrial function prior to and following NR supplementation at 1000 mg twice/day for 12 weeks. Skeletal muscle biopsies were collected before and during a hyperinsulinaemic-euglycaemic clamp to quantitate both basal and insulin-stimulated effects on the mitochondria. A portion of the basal biopsy was used to measure mitochondrial respiration by Oroboros and the insulin-stimulated biopsy was prepared for confocal light microscopy analysis. Western blot analysis and qRT-PCR were completed on the frozen muscle tissue, as were NAD(H) and NADP(H) measurements, and lipid content was assessed by 1H-magnetic resonance spectroscopy. Mitochondrial abundance was quantified via citrate synthase activity, morphological analysis (completed in 40% of the samples) was visualized by confocal imaging, and mitochondrial networks were scored based on fragmentation, distribution homogeneity throughout the fibre, and periodicity of the organization. Increased concentrations of NAD+-derived metabolites were detected in the urine of the men receiving NR, which suggests the supplements were absorbed, metabolized and excreted. While NAD+-metabolite concentrations were elevated in the urine, no changes in NAD+, NADH, NADP+ or NADPH were detected in the skeletal muscle of the NR group. In fact, NAMPT (a NAD+ biosynthetic enzyme) protein abundance was decreased by 14% in the supplemented group; however, the mRNA levels remained unchanged. It was concluded that this reduction may signify adaptation to the supraphysiological dose of NR, thereby reducing the reliance on the NAD+ salvage pathway in which NAMPT facilitates the rate-limiting step. No indication of NR supplementation affecting sirtuin activity was observed, and specifically, no changes in global protein acetylation were detected as quantified by levels of total acetylated lysine residues. This was also true in insulin-stimulated skeletal muscle biopsies obtained during a hyperinsulinaemic-euglycaemic clamp (Dollerup et al. 2018). Next, Dollerup et al. sought to measure mitochondrial respiration but found no changes in the skeletal muscle oxidative capacity in the supplemented men nor was the abundance of mitochondria affected. Lipid deposition was unaltered in the muscle of supplemented subjects. Lastly, to further understand the reduction in NAMPT protein levels, linear regression analyses were completed, and significant positive relationships were reported between the protein levels of complex I, succinate dehydrogenase, SIRT3, global protein levels and insulin sensitivity (M-value). On the other hand, fasting insulin and C-peptide from an oral glucose tolerance test (Dollerup et al. 2019) correlated negatively with skeletal muscle NAMPT protein levels. Taken together, these findings collected by Dollerup and colleagues challenge the current thinking that NAD+ precursor supplementation improves markers of mitochondrial health in human skeletal muscle (Dollerup et al. 2020). Further, these results align with a recently published randomized, placebo-controlled crossover study, demonstrating no effect of NR supplementation (1 g/day for 21 days) in a small cohort of elderly men 70–80 years old (Elhassan et al. 2019). Specifically, no changes were detected in NAD+ levels or skeletal muscle mitochondrial bioenergetics, and RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondrial pathways. These data do not corroborate with the existing preclinical data from rodent models (Fang et al. 2017). As such, NR supplementation of 1000 mg twice/day may not be efficacious in enhancing human skeletal muscle mitochondrial health and glucose homeostasis in middle-aged males with obesity and insulin resistance. The studies from Dollerup et al (Dollerup et al. 2018, 2019, 2020) indicate that further scientific evidence is required before recommending the use of NR supplementation or other NAD+ precursors. The data presented in this study do not support the hypothesis that NR enhances mitochondrial respiration and function in human skeletal muscle. However, the current study only investigated middle-aged males with insulin resistance and obesity, who were otherwise healthy. Future studies should examine whether NR may be effective in treating populations with differences in age, sex, and more severe metabolic disease, such as established type 2 diabetes. The pharmacokinetics of NR in humans and its bioavailability are poorly understood and may explain the lack of effects in skeletal muscle in humans. The authors highlight that limited amounts of NAD+ in skeletal muscle are generated directly from NR in mice, using isotope-labelled NR tracer studies (Liu et al. 2018); whether similar effects are seen in humans is unknown. Additionally, yet to be understood is whether tissue specificity of NR supplementation exists in humans, or whether NR exerts beneficial effects in other tissues such as the adipose, liver or immune cells. While in vitro studies have demonstrated positive effects of NR on mitochondria in various human cell types (leukocytes and fibroblasts), it is reasonable to state that limited tissue bioavailability of orally supplemented NR may account for the lack of effects observed in vivo. Hence, it is questionable whether NR exerts direct effects on skeletal muscle or whether there is poor bioavailability at the level of the tissue and further research is thus required. It would also be interesting to explore whether other supplementation strategies aimed at boosting NAD+ metabolism, such as nicotinamide mononucleotide, have different effects compared with NR supplementation. Taken together, the findings by Dollerup et al. certainly challenge the existing preclinical data in murine models and show little benefit of NR supplementation in enhancing mitochondrial respiration and whole-body glucose homeostasis in middle-aged men with obesity and insulin resistance. However, further studies are required to establish whether tissue specificity of NR supplementation exists in humans and if NR supplementation may be beneficial in other populations with differences in age, sex or disease state. None declared Both Mary Moore and Justine Mucinski have contributed equally to the writing of this Journal Club article. None Dr R. Scott Rector and Dr Elizabeth J. Parks, University of Missouri, Columbia, USA, are acknowledged for their helpful discussion and insightful feedback on this article.