Co-Ingestion of Branched-Chain Amino Acids and Carbohydrate Stimulates Myofibrillar Protein Synthesis Following Resistance Exercise in Trained Young Men.

Branched-chain amino acids (BCAA) and carbohydrate (CHO) are commonly recommended postexercise supplements. However, no study has examined the interaction of CHO and BCAA ingestion on myofibrillar protein synthesis (MyoPS) rates following exercise. We aimed to determine the response of MyoPS to the co-ingestion of BCAA and CHO following an acute bout of resistance exercise. Ten resistance-trained young men completed two trials in counterbalanced order, ingesting isocaloric drinks containing either 30.6-g CHO plus 5.6-g BCAA (B + C) or 34.7-g CHO alone following a bout of unilateral, leg resistance exercise. MyoPS was measured postexercise with a primed, constant infusion of L-[ring13C6] phenylalanine and collection of muscle biopsies pre- and 4 hr postdrink ingestion. Blood samples were collected at time points before and after drink ingestion. Serum insulin concentrations increased to a similar extent in both trials (p > .05), peaking at 30 min postdrink ingestion. Plasma leucine (514 ± 34 nmol/L), isoleucine (282 ± 23 nmol/L), and valine (687 ± 33 nmol/L) concentrations peaked at 0.5 hr postdrink in B + C and remained elevated for 3 hr during exercise recovery. MyoPS was ∼15% greater (95% confidence interval [-0.002, 0.028], p = .039, Cohen's d = 0.63) in B + C (0.128%/hr ± 0.011%/hr) than CHO alone (0.115%/hr ± 0.011%/hr) over the 4 hr postexercise period. Co-ingestion of BCAA and CHO augments the acute response of MyoPS to resistance exercise in trained young males.

Association between vitamin D deficiency and exercise capacity in patients with CKD, a cross-sectional analysis.

BACKGROUND: Evidence is growing for a role of vitamin D in regulating skeletal muscle mass, strength and functional capacity. Given the role the kidneys play in activating total vitamin D, and the high prevalence of vitamin D deficiency in Chronic Kidney Disease (CKD), it is possible that deficiency contributes to the low levels of physical function and muscle mass in these patients. METHODS: This is a secondary cross-sectional analysis of previously published interventional study, with in vitro follow up work. 34 CKD patients at stages G3b-5 (eGFR 25.5 ± 8.3 mL/min/1.73m2; age 61 ± 12 years) were recruited, with a sub-group (n = 20) also donating a muscle biopsy. Vitamin D and associated metabolites were analysed in plasma by liquid chromatography tandem-mass spectroscopy and correlated to a range of physiological tests of muscle size, function, exercise capacity and body composition. The effects of 1α,25(OH)2D3 supplementation on myogenesis and myotube size was investigated in primary skeletal muscle cells from vitamin D deficient donors. RESULTS: In vivo, there was no association between total or active vitamin D and muscle size or strength, but a significant correlation with V̇O2Peak was seen with total vitamin D (25OHD). in vitro, 1α,25(OH)2D3 supplementation reduced IL-6 mRNA expression, but had no effect upon proliferation, differentiation or myotube diameter. CONCLUSIONS: Vitamin D deficiency is not a prominent factor driving the loss of muscle mass in CKD, but may play a role in reduced exercise capacity.

Nicotinamide riboside supplementation does not alter whole-body or skeletal muscle metabolic responses to a single bout of endurance exercise.

KEY POINTS: Acute nicotinamide riboside (NR) supplementation does not alter substrate metabolism at rest, during or in recovery from endurance exercise. NR does not alter NAD+ -sensitive signalling pathways in human skeletal muscle. NR supplementation and acute exercise influence the NAD+ metabolome. ABSTRACT: Oral supplementation of the NAD+ precursor nicotinamide riboside (NR) has been reported to alter metabolism alongside increasing sirtuin (SIRT) signalling and mitochondrial biogenesis in rodent skeletal muscle. However, whether NR supplementation can elicit a similar response in human skeletal muscle is unclear. This study assessed the effect of 7-day NR supplementation on whole-body metabolism and exercise-induced mitochondrial biogenic signalling in skeletal muscle. Eight male participants (age: 23 ± 4 years, V̇O2peak 46.5 ± 4.4 ml kg-1  min-1 ) received 1 week of NR or cellulose placebo (PLA) supplementation (1000 mg day-1 ). Muscle biopsies were collected from the medial vastus lateralis prior to supplementation and pre-, immediately post- and 3 h post-exercise (1 h of 60% Wmax cycling) performed following the supplementation period. There was no effect of NR supplementation on substrate utilisation at rest or during exercise or on skeletal muscle mitochondrial respiration. Global acetylation, auto-PARylation of poly ADP-ribose polymerase 1 (PARP1), acetylation of Tumour protein 53 (p53)Lys382 and Manganese superoxide dismutase (MnSOD)Lys122 were also unaffected by NR supplementation or exercise. NR supplementation did not increase skeletal muscle NAD+ concentration, but it did increase the concentration of deaminated NAD+ precursors nicotinic acid riboside (NAR) and nicotinic acid mononucleotide (NAM) and methylated nicotinamide breakdown products (Me2PY and Me4PY), demonstrating the skeletal muscle bioavailability of NR supplementation. In summary, 1 week of NR supplementation does not alter whole-body metabolism or skeletal muscle signal transduction pathways implicated in the mitochondrial adaptation to endurance exercise.

High-dose leucine supplementation does not prevent muscle atrophy or strength loss over 7 days of immobilization in healthy young males.

BACKGROUND: Unavoidable periods of disuse lead to muscle atrophy and functional decline. Preventing such declines can reduce the risk of re-injury and improve recovery of normal physiological functioning. OBJECTIVES: We aimed to determine the effectiveness of high-dose leucine supplementation on muscle morphology and strength during 7 d of unilateral lower-limb immobilization, and the role of myofibrillar (MyoPS) and mitochondrial (MitoPS) protein synthesis in disuse atrophy. METHODS: Sixteen healthy males (mean ± SEM age: 23 ± 1 y) underwent 7 d of unilateral lower-limb immobilization, with thrice-daily leucine (LEU; n = 8) or placebo (PLA; n = 8) supplementation (15 g/d). Before and after immobilization, muscle strength and compartmental tissue composition were assessed. A primed continuous infusion of l-[ring-13C6]-phenylalanine with serial muscle biopsies was used to determine postabsorptive and postprandial (20 g milk protein) MyoPS and MitoPS, fiber morphology, markers of protein turnover, and mitochondrial function between the control leg (CTL) and the immobilized leg (IMB). RESULTS: Leg fat-free mass was reduced in IMB (mean ± SEM: -3.6% ± 0.5%; P = 0.030) but not CTL with no difference between supplementation groups. Isometric knee extensor strength declined to a greater extent in IMB (-27.9% ± 4.4%) than in CTL (-14.3% ± 4.4%; P = 0.043) with no difference between groups. In response to 20 g milk protein, postprandial MyoPS rates were significantly lower in IMB than in CTL (-22% ± 4%; P < 0.01) in both LEU and PLA. Postabsorptive MyoPS rates did not differ between legs or groups. Postabsorptive MitoPS rates were significantly lower in IMB than in CTL (-14% ± 5%; P < 0.01) and postprandial MitoPS rates significantly declined in response to 20 g milk protein ingestion (CTL: -10% ± 8%; IMB: -15% ± 10%; P = 0.039), with no differences between legs or groups. There were no significant differences in measures of mitochondrial respiration between legs, but peroxisome proliferator-activated receptor γ coactivator 1-α and oxidative phosphorylation complex II and III were significantly lower in IMB than in CTL (P < 0.05), with no differences between groups. CONCLUSIONS: High-dose leucine supplementation (15 g/d) does not appear to attenuate any functional declines associated with 7 d of limb immobilization in young, healthy males.This trial was registered at clinicaltrials.gov as NCT03762278.

Nicotinamide Riboside Augments the Aged Human Skeletal Muscle NAD+ Metabolome and Induces Transcriptomic and Anti-inflammatory Signatures.

Nicotinamide adenine dinucleotide (NAD+) is modulated by conditions of metabolic stress and has been reported to decline with aging in preclinical models, but human data are sparse. Nicotinamide riboside (NR) supplementation ameliorates metabolic dysfunction in rodents. We aimed to establish whether oral NR supplementation in aged participants can increase the skeletal muscle NAD+ metabolome and if it can alter muscle mitochondrial bioenergetics. We supplemented 12 aged men with 1 g NR per day for 21 days in a placebo-controlled, randomized, double-blind, crossover trial. Targeted metabolomics showed that NR elevated the muscle NAD+ metabolome, evident by increased nicotinic acid adenine dinucleotide and nicotinamide clearance products. Muscle RNA sequencing revealed NR-mediated downregulation of energy metabolism and mitochondria pathways, without altering mitochondrial bioenergetics. NR also depressed levels of circulating inflammatory cytokines. Our data establish that oral NR is available to aged human muscle and identify anti-inflammatory effects of NR.

Differential responses of myoblasts and myotubes to photobiomodulation are associated with mitochondrial number.

OBJECTIVE: Photobiomodulation (PBM) is the application of light to promote tissue healing. Current indications suggest PBM induces its beneficial effects in vivo through upregulation of mitochondrial activity. However, how mitochondrial content influences such PBM responses have yet to be evaluated. Hence, the current study assessed the biological response of cells to PBM with varying mitochondrial contents. METHODS: DNA was isolated from myoblasts and myotubes (differentiated myoblasts), and mitochondrial DNA (mtDNA) was amplified and quantified using a microplate assay. Cells were seeded in 96-wellplates, incubated overnight and subsequently irradiated using a light-emitting diode array (400, 450, 525, 660, 740, 810, 830 and white light, 24 mW/cm2 , 30-240 seconds, 0.72-5.76J/cm2 ). The effects of PBM on markers of mitochondrial activity including reactive-oxygen-species and real-time mitochondrial respiration (Seahorse XFe96) assays were assessed 8 hours post-irradiation. Datasets were analysed using general linear model followed by one-way analysis of variance (and post hoc-Tukey tests); P = 0.05). RESULTS: Myotubes exhibited mtDNA levels 86% greater than myoblasts (P < 0.001). Irradiation of myotubes at 400, 450 or 810 nm induced 53%, 29% and 47% increases (relative to non-irradiated control) in maximal respiratory rates, respectively (P < 0.001). Conversely, irradiation of myoblasts at 400 or 450 nm had no significant effect on maximal respiratory rates. CONCLUSION: This study suggests that mitochondrial content may influence cellular responses to PBM and as such explain the variability of PBM responses seen in the literature.

PPARα-independent effects of nitrate supplementation on skeletal muscle metabolism in hypoxia.

Hypoxia is a feature of many disease states where convective oxygen delivery is impaired, and is known to suppress oxidative metabolism. Acclimation to hypoxia thus requires metabolic remodelling, however hypoxia tolerance may be aided by dietary nitrate supplementation. Nitrate improves tissue oxygenation and has been shown to modulate skeletal muscle tissue metabolism via transcriptional changes, including through the activation of peroxisome proliferator-activated receptor alpha (PPARα), a master regulator of fat metabolism. Here we investigated whether nitrate supplementation protects skeletal muscle mitochondrial function in hypoxia and whether PPARα is required for this effect. Wild-type and PPARα knockout (PPARα-/-) mice were supplemented with sodium nitrate via the drinking water or sodium chloride as control, and exposed to environmental hypoxia (10% O2) or normoxia for 4 weeks. Hypoxia suppressed mitochondrial respiratory function in mouse soleus, an effect partially alleviated through nitrate supplementation, but occurring independently of PPARα. Specifically, hypoxia resulted in 26% lower mass specific fatty acid-supported LEAK respiration and 23% lower pyruvate-supported oxidative phosphorylation capacity. Hypoxia also resulted in 24% lower citrate synthase activity in mouse soleus, possibly indicating a loss of mitochondrial content. These changes were not seen, however, in hypoxic mice when supplemented with dietary nitrate, indicating a nitrate dependent preservation of mitochondrial function. Moreover, this was observed in both wild-type and PPARα-/- mice. Our results support the notion that nitrate supplementation can aid hypoxia tolerance and indicate that nitrate can exert effects independently of PPARα.

Lipid remodeling and an altered membrane-associated proteome may drive the differential effects of EPA and DHA treatment on skeletal muscle glucose uptake and protein accretion.

In striated muscle, eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have differential effects on the metabolism of glucose and differential effects on the metabolism of protein. We have shown that, despite similar incorporation, treatment of C2C12 myotubes (CM) with EPA but not DHA improves glucose uptake and protein accretion. We hypothesized that these differential effects of EPA and DHA may be due to divergent shifts in lipidomic profiles leading to altered proteomic profiles. We therefore carried out an assessment of the impact of treating CM with EPA and DHA on lipidomic and proteomic profiles. Fatty acid methyl esters (FAME) analysis revealed that both EPA and DHA led to similar but substantials changes in fatty acid profiles with the exception of arachidonic acid, which was decreased only by DHA, and docosapentanoic acid (DPA), which was increased only by EPA treatment. Global lipidomic analysis showed that EPA and DHA induced large alterations in the cellular lipid profiles and in particular, the phospholipid classes. Subsequent targeted analysis confirmed that the most differentially regulated species were phosphatidylcholines and phosphatidylethanolamines containing long-chain fatty acids with five (EPA treatment) or six (DHA treatment) double bonds. As these are typically membrane-associated lipid species we hypothesized that these treatments differentially altered the membrane-associated proteome. Stable isotope labeling by amino acids in cell culture (SILAC)-based proteomics of the membrane fraction revealed significant divergence in the effects of EPA and DHA on the membrane-associated proteome. We conclude that the EPA-specific increase in polyunsaturated long-chain fatty acids in the phospholipid fraction is associated with an altered membrane-associated proteome and these may be critical events in the metabolic remodeling induced by EPA treatment.

Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells.

OBJECTIVE: Augmenting 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. METHODS: We 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. RESULTS: Skeletal 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. CONCLUSIONS: These 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.

Supplementation of a suboptimal protein dose with leucine or essential amino acids: effects on myofibrillar protein synthesis at rest and following resistance exercise in men.

Leucine is a nutrient regulator of muscle protein synthesis by activating mTOR and possibly other proteins in this pathway. The purpose of this study was to examine the role of leucine in the regulation of human myofibrillar protein synthesis (MPS). Twenty-four males completed an acute bout of unilateral resistance exercise prior to consuming either: a dose (25 g) of whey protein (WHEY); 6.25 g whey protein with total leucine equivalent to WHEY (LEU); or 6.25 g whey protein with total essential amino acids (EAAs) equivalent to WHEY for all EAAs except leucine (EAA-LEU). Measures of MPS, signalling through mTOR, and amino acid transporter (AAT) mRNA abundance were made while fasted (FAST), and following feeding under rested (FED) and post-exercise (EX-FED) conditions. Leucinaemia was equivalent between WHEY and LEU and elevated compared to EAA-LEU (P=0.001). MPS was increased above FAST at 1­3 h post-exercise in both FED (P <0.001) and EX-FED (P <0.001) conditions with no treatment effect.At 3­5 h, only WHEY remained significantly elevated above FAST in EX-FED(WHEY 184% vs. LEU 55% and EAA-LEU 35%; P =0.036). AAT mRNA abundance was increased above FAST after feeding and exercise with no effect of leucinaemia. In summary, a low dose of whey protein supplemented with leucine or all other essential amino acids was as effective as a complete protein (WHEY) in stimulating postprandial MPS; however only WHEY was able to sustain increased rates of MPS post-exercise and may therefore be most suited to increase exercise-induced muscle protein accretion.

Resveratrol ameliorates aging-related metabolic phenotypes by inhibiting cAMP phosphodiesterases.

Resveratrol, a polyphenol in red wine, has been reported as a calorie restriction mimetic with potential antiaging and antidiabetogenic properties. It is widely consumed as a nutritional supplement, but its mechanism of action remains a mystery. Here, we report that the metabolic effects of resveratrol result from competitive inhibition of cAMP-degrading phosphodiesterases, leading to elevated cAMP levels. The resulting activation of Epac1, a cAMP effector protein, increases intracellular Ca(2+) levels and activates the CamKKß-AMPK pathway via phospholipase C and the ryanodine receptor Ca(2+)-release channel. As a consequence, resveratrol increases NAD(+) and the activity of Sirt1. Inhibiting PDE4 with rolipram reproduces all of the metabolic benefits of resveratrol, including prevention of diet-induced obesity and an increase in mitochondrial function, physical stamina, and glucose tolerance in mice. Therefore, administration of PDE4 inhibitors may also protect against and ameliorate the symptoms of metabolic diseases associated with aging.