Highlights d NR supplementation in aged subjects augments the skeletal muscle NAD + metabolome d NR supplementation does not affect skeletal muscle mitochondrial bioenergetics d NR supplementation reduces levels of circulating inflammatory cytokines
ObjectiveAugmenting nicotinamide adenine dinucleotide (NAD+) availability may protect skeletal muscle from age-related metabolic decline. Dietary supplementation of NAD+ precursors nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) appear efficacious in elevating muscle NAD+. Here we sought to identify the pathways skeletal muscle cells utilize to synthesize NAD+ from NMN and NR and provide insight into mechanisms of muscle metabolic homeostasis.MethodsWe exploited expression profiling of muscle NAD+ biosynthetic pathways, single and double nicotinamide riboside kinase 1/2 (NRK1/2) loss-of-function mice, and pharmacological inhibition of muscle NAD+ recycling to evaluate NMN and NR utilization.ResultsSkeletal muscle cells primarily rely on nicotinamide phosphoribosyltransferase (NAMPT), NRK1, and NRK2 for salvage biosynthesis of NAD+. NAMPT inhibition depletes muscle NAD+ availability and can be rescued by NR and NMN as the preferred precursors for elevating muscle cell NAD+ in a pathway that depends on NRK1 and NRK2. Nrk2 knockout mice develop normally and show subtle alterations to their NAD+ metabolome and expression of related genes. NRK1, NRK2, and double KO myotubes revealed redundancy in the NRK dependent metabolism of NR to NAD+. Significantly, these models revealed that NMN supplementation is also dependent upon NRK activity to enhance NAD+ availability.ConclusionsThese results identify skeletal muscle cells as requiring NAMPT to maintain NAD+ availability and reveal that NRK1 and 2 display overlapping function in salvage of exogenous NR and NMN to augment intracellular NAD+ availability.
Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD+ availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD+ as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD+ homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD+ depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD+ metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U-13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD+ excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells results in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD+ depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity is increased in response to low NAD+, which is rapidly reversed with repletion of the NAD+ pool using NR. NAD+ depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reverses these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD+ depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD+ precursor usage in models of skeletal muscle physiology.
Tel: + 44(0)121 414 3917, email: g.g.lavery@bham.ac.uk 26 27 2012), reduction of blood glucose, hepatic steatosis, and neuropathy on high fat diet 64 (Trammell et al. 2016), improvement of cardiac function in genetic cardiomyopathy (Diguet et 65 al., 2018), and prevention of cortical neuronal degeneration (Vaur et al., 2017). Depletion of 66 the enzyme nicotinamide phosphoribosyltransferase (NAMPT), rate-limiting for NAD + 67 biosynthesis, in mouse skeletal muscle severely diminishes NAD + levels and induces 68 sarcopenia. Oral repletion of NAD + with NR in this model rescued pathology in skeletal 69 muscle in a cell-autonomous manner (Frederick et al., 2016). However, recent data in mice 70 tracing NAD + fluxes questioned whether oral NR has the ability to access muscle (Liu et al., 2018). Thus, whether oral NR can augment the human skeletal muscle NAD + metabolome is 72 currently unknown. 73A decline in NAD + availability and signalling appears to occur as part of the aging process in 74 many species (Gomes et al., 2013; Mouchiroud et al., 2013), though there is a paucity of 75 data to confirm that this is the case in human aging. NR and nicotinamide mononucleotide 76 (NMN) are reported to extend life span (Zhang et al., 2016) and enhance metabolism in 77 aged mice (Mills et al., 2016). To date, NR supplementation studies in humans have been 78 reported, focussing on cardiovascular (Martens et al., 2018), systemic metabolic (Dollerup et 79 al., 2018), and safety (Conze et al, 2019) end-points, but have not addressed advanced 80 aging, tissue metabolomic changes, or effects on muscle metabolism and function. 81Herein, we set out to study if oral NR is available to aged human skeletal muscle and 82 whether potential effects on muscle metabolism can be detected. We conducted a 21-day 83 NR supplementation intervention in a cohort of 70 -80 year old men in a randomized, 84 double-blind, placebo-controlled crossover trial. We demonstrate that NR augments the 85 skeletal muscle NAD + metabolome inducing a gene expression signature suggestive of 86 downregulation of energy metabolism pathways, but without affecting muscle mitochondrial 87 bioenergetics or metabolism. Additionally, NR suppresses specific circulating inflammatory 88 cytokines levels. In an additional study, we used 31 P magnetic resonance spectroscopy 89 (MRS) and show that NAD + decline is not associated with chronological aging per se in 90 either human muscle or brain. 91 92 5 RESULTS 93Oral NR is safe and well tolerated in aged adults 94Twelve aged (median age 75 years) and marginally overweight (median BMI 26.6 kg/m 2 ; 95 range 21 -30), but otherwise healthy men were recruited and orally supplemented with NR 1 96 g per day for 21-days in a randomized, double-blind, placebo-controlled crossover design 97 with 21-days washout period between phases. Baseline characteristics of participants are 98 included in Suppl Table 1. NR chloride (Niagen ®) and placebo were provided as 250 mg 99 capsules (ChromaDex, Inc.) and subjects were instructed to take ...
Background: Skeletal muscle is central to whole body metabolic homeostasis, with age and disease impairing its ability to function appropriately to maintain health. Inadequate NAD + availability is proposed to contribute to pathophysiology by impairing metabolic energy pathway use. Despite the importance of NAD + as a vital redox cofactor in energy production pathways being well-established, the wider impact of disrupted NAD + homeostasis on these pathways is unknown. Methods: We utilised skeletal muscle myotube models to induce NAD + depletion, repletion and excess and conducted metabolic tracing to provide comprehensive and detailed analysis of the consequences of altered NAD + metabolism on central carbon metabolic pathways. We used stable isotope tracers, [1,2-13C] D-glucose and [U- 13C] glutamine, and conducted combined 2D-1H,13C-heteronuclear single quantum coherence (HSQC) NMR spectroscopy and GC-MS analysis. Results: NAD + excess driven by nicotinamide riboside (NR) supplementation within skeletal muscle cells resulted in enhanced nicotinamide clearance, but had no effect on energy homeostasis or central carbon metabolism. Nicotinamide phosphoribosyltransferase (NAMPT) inhibition induced NAD + depletion and resulted in equilibration of metabolites upstream of glyceraldehyde phosphate dehydrogenase (GAPDH). Aspartate production through glycolysis and TCA cycle activity was increased in response to low NAD +, which was rapidly reversed with repletion of the NAD + pool using NR. NAD + depletion reversibly inhibits cytosolic GAPDH activity, but retains mitochondrial oxidative metabolism, suggesting differential effects of this treatment on sub-cellular pyridine pools. When supplemented, NR efficiently reversed these metabolic consequences. However, the functional relevance of increased aspartate levels after NAD + depletion remains unclear, and requires further investigation. Conclusions: These data highlight the need to consider carbon metabolism and clearance pathways when investigating NAD + precursor usage in models of skeletal muscle physiology.
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