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.
(2010). Insulin-stimulated glycogen synthesis and glycogen synthase activation after electrical stimulation of epitrochlearis muscles with different initial glycogen contents. Archives of Physiology and Biochemistry: Vol. 116, No. 3, pp. 116-127.
Glycogen content and contraction strongly regulate glycogen synthase (GS) activity, and the aim of the present study was to explore their effects and interaction on GS phosphorylation and kinetic properties. Glycogen content in rat epitrochlearis muscles was manipulated in vivo. After manipulation, incubated muscles with normal glycogen [NG; 210.9 +/- 7.1 mmol/kg dry weight (dw)], low glycogen (LG; 108.1 +/- 4.5 mmol/ kg dw), and high glycogen (HG; 482.7 +/- 42.1 mmol/kg dw) were contracted or rested before the studies of GS kinetic properties and GS phosphorylation (using phospho-specific antibodies). LG decreased and HG increased GS K(m) for UDP-glucose (LG: 0.27 +/- 0.02 < NG: 0.71 +/- 0.06 < HG: 1.11 +/- 0.12 mM; P < 0.001). In addition, GS fractional activity inversely correlated with glycogen content (R = -0.70; P < 0.001; n = 44). Contraction decreased K(m) for UDP-glucose (LG: 0.14 +/- 0.01 = NG: 0.16 +/- 0.01 < HG: 0.33 +/- 0.03 mM; P < 0.001) and increased GS fractional activity, and these effects were observed independently of glycogen content. In rested muscles, GS Ser(641) and Ser(7) phosphorylation was decreased in LG and increased in HG compared with NG. GSK-3beta Ser(9) and AMPKalpha Thr(172) phosphorylation was not modulated by glycogen content in rested muscles. Contraction decreased phosphorylation of GS Ser(641) at all glycogen contents. However, contraction increased GS Ser(7) phosphorylation even though GS was strongly activated. In conclusion, glycogen content regulates GS affinity for UDP-glucose and low affinity for UDP-glucose in muscles with high glycogen content may reduce glycogen accumulation. Contraction increases GS affinity for UDP-glucose independently of glycogen content and creates a unique phosphorylation pattern.
Atrogin-1 and Muscle-specific RING finger protein 1 (MuRF1) are highly expressed in multiple conditions of skeletal muscle atrophy. The phosphoinositide 3-kinase (PI3K)/Akt/forkhead box (FoxO) signaling pathway is well known to regulate Atrogin-1 and MuRF1 gene expressions. However, Akt activation also activates the mechanistic target of rapamycin complex 1 (mTORC1), which induces skeletal muscle hypertrophy. Whether mTORC1-dependent signaling has a role in regulating Atrogin-1 and/or MuRF1 gene and protein expression is currently unclear. In this study, we showed that activation of insulin-mediated Akt signaling suppresses both Atrogin-1 and MuRF1 protein contents and that inhibition of Akt increases both Atrogin-1 and MuRF1 protein contents in C2C12 myotubes. Interestingly, inhibition of mTORC1 with a specific mTORC1 inhibitor, rapamycin, increased Atrogin-1, but not MuRF1, protein content. Furthermore, activation of AMP-activated protein kinase (AMPK), a negative regulator of the mTORC1 signaling pathway, also showed distinct time-dependent changes between Atrogin-1 and MuRF1 protein contents, suggesting differential regulatory mechanisms between Atrogin-1 and MuRF1 protein content. To further explore the downstream of mTORC1 signaling, we employed a specific S6K1 inhibitor, PF-4708671. We found that Atrogin-1 protein content was dose-dependently increased with PF-4708671 treatment, whereas MuRF1 protein content was decreased at 50 μM of PF-4708671 treatment. However, MuRF1 protein content was unexpectedly increased by PF-4708671 treatment for a longer period. Overall, our results indicate that Atrogin-1 and MuRF1 protein contents are regulated by different mechanisms, the downstream of Akt, and that Atrogin-1 protein content can be regulated by the rapamycin-sensitive mTOR-S6K1-dependent signaling pathway.
PIKfyve (FYVE domain-containing phosphatidylinositol 3-phosphate 5-kinase), the lipid kinase that phosphorylates PtdIns3P to PtdIns(3,5)P2, has been implicated in insulin-stimulated glucose uptake. We investigated whether PIKfyve could also be involved in contraction/AMPK (AMP-activated protein kinase)-stimulated glucose uptake in skeletal muscle. Incubation of rat epitrochlearis muscles with YM201636, a selective PIKfyve inhibitor, reduced contraction- and AICAriboside (5-amino-4-imidazolecarboxamide riboside)-stimulated glucose uptake. Consistently, PIKfyve knockdown in C2C12 myotubes reduced AICAriboside-stimulated glucose transport. Furthermore, muscle contraction increased PtdIns(3,5)P2 levels and PIKfyve phosphorylation. AMPK phosphorylated PIKfyve at Ser307 both in vitro and in intact cells. Following subcellular fractionation, PIKfyve recovery in a crude intracellular membrane fraction was increased in contracting versus resting muscles. Also in opossum kidney cells, wild-type, but not S307A mutant, PIKfyve was recruited to endosomal vesicles in response to AMPK activation. We propose that PIKfyve activity is required for the stimulation of skeletal muscle glucose uptake by contraction/AMPK activation. PIKfyve is a new AMPK substrate whose phosphorylation at Ser307 could promote PIKfyve translocation to endosomes for PtdIns(3,5)P2 synthesis to facilitate GLUT4 (glucose transporter 4) translocation.
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