Analysis of skeletal muscle metabolome: Evaluation of extraction methods for targeted metabolite quantification using liquid chromatography tandem mass spectrometry

Functional metabolomics of skeletal muscle involves the simultaneous identification and quantification of a large number of metabolites. For this purpose, the extraction of metabolites from animal tissues is a crucial technical step that needs to be optimized. In this work, five extraction methods for skeletal muscle metabolome analysis using liquid chromatography tandem mass spectrometry (LC-MS/MS) were tested. Bird skeletal muscles sampled postmortem and quenched in liquid nitrogen were used. Three replicates of the same sample were extracted using the following solvent systems of varying polarity: boiling water (BW, +100 °C), cold pure methanol (CPM, −80 °C), methanol/chloroform/water (MCW, −20 °C), boiling ethanol (BE, +80 °C), and perchloric acid (PCA, −20 °C). Three injections by extraction were performed. The BW extraction showed the highest recovery of metabolites with the lowest variability (<10%) except for creatine-phosphate (creatine-P). Considering yield (area of the peaks), reproducibility, and ease, the current experiment drew a scale for the muscle metabolome extraction starting from the best to the least convenient: BW > MCW > CPM > PCA ? BE. In addition, the semiquantification of metabolites in two muscles showing different metabolic and contractile properties was carried out after BW extraction and showed expected differences in metabolite contents, thereby validating the technique for biological investigations. In conclusion, the BW extraction is recommended for analysis of skeletal muscle metabolome except for creatine-P, which was poorly recovered with this technique.     

Expression and functional activity of ryanodine receptors (RyRs) during skeletal muscle development

Two isoforms of ryanodine receptors are expressed in skeletal muscles, RyR1 and RyR3. We investigated the relative level of expression of RyRs in developing murine skeletal muscles using [3H]ryanodine binding and immunoprecipitation experiments. In the diaphragm RyR3 accounted for 11% of total RyRs in 5-day-old mice and for 3% of total RyRs in 60-day-old mice. In hindlimb muscles, RyR3 accounted for 3% and 1% of total RyRs in 5-day-old and adult mice, respectively. The activity of RyR1 channels in native microsomal vesicles from murine muscles was found to be as low as 35% of that measured after CHAPS exposure, while no inhibition was observed for RyR3. CHAPS sensitivity of recombinant RyR1 and RyR3 expressed in HEK293 cells was also investigated. The activity of recombinant RyR1 but not RyR3 channels was found to be inhibited in native conditions, suggesting that this property may not be dependent on a muscle environment.     

Bayesian flux balance analysis applied to a skeletal muscle metabolic model

In this article, the steady state condition for the multi-compartment models for cellular metabolism is considered. The problem is to estimate the reaction and transport fluxes, as well as the concentrations in venous blood when the stoichiometry and bound constraints for the fluxes and the concentrations are given. The problem has been addressed previously by a number of authors, and optimization-based approaches as well as extreme pathway analysis have been proposed. These approaches are briefly discussed here. The main emphasis of this work is a Bayesian statistical approach to the flux balance analysis (FBA). We show how the bound constraints and optimality conditions such as maximizing the oxidative phosphorylation flux can be incorporated into the model in the Bayesian framework by proper construction of the prior densities. We propose an effective Markov chain Monte Carlo (MCMC) scheme to explore the posterior densities, and compare the results with those obtained via the previously studied linear programming (LP) approach. The proposed methodology, which is applied here to a two-compartment model for skeletal muscle metabolism, can be extended to more complex models.     

Insulin signaling and glucose transport in insulin resistant human skeletal muscle

Insulin increases glucose uptake and metabolism in skeletal muscle by signal transduction via protein phosphorylation cascades. Insulin action on signal transduction is impaired in skeletal muscle from Type 2 diabetic subjects, underscoring the contribution of molecular defects to the insulin resistant phenotype. This review summarizes recent work to identify downstream intermediates in the insulin signaling pathways governing glucose homeostasis, in an attempt to characterize the molecular mechanism accounting for skeletal muscle insulin resistance in Type 2 diabetes. Furthermore, the effects of pharmaceutical treatment of Type 2 diabetic patients on insulin signaling and glucose uptake are discussed. The identification and characterization of pathways governing insulin action on glucose metabolism will facilitate the development of strategies to improve insulin sensitivity in an effort to prevent and treat Type 2 diabetes mellitus.     

Localisation of lymphatic vessels and vascular endothelial growth factors-C and -D in human and mouse skeletal muscle with immunohistochemistry

The present study was aimed to localise lymphatic vessels and their growth factors in human and mouse skeletal muscle with immunohistochemistry and specific antibodies (VEGFR-3, LYVE-1, VEGF-C and VEGF-D). The largest lymphatic vessels were found in perimysial connective tissue next to the arteries and veins, as has been shown earlier with electron microscopy. As a new finding, we also found small LYVE-1 positive vessels in the capillary bed between muscle fibres. These vessels were located next to CD31 positive blood capillaries and were of the same size, but fewer in number. In addition, we described the localisation of the two main lymphangiogenic growth factor proteins, vascular endothelial growth factor-C and -D. Both proteins were expressed in skeletal muscle at mRNA and protein levels. VEGF-D was located under the sarcolemma in some of the muscle fibres, in the endothelia of larger blood vessels and in fibroblasts. VEGF-C protein was localised to the nerves and muscle spindles, to fibroblasts and surrounding connective tissue, but was not found in muscle fibres or endothelial cells. Our results are the first to suggest the presence of lymphatic capillaries throughout the skeletal muscle, and to present the localisation of VEGF-C and -D in the muscles. Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Increasing NAD Synthesis in Muscle via Nicotinamide Phosphoribosyltransferase Is Not Sufficient to Promote Oxidative Metabolism

The NAD biosynthetic precursors nicotinamide mononucleotide and nicotinamide riboside are reported to confer resistance to metabolic defects induced by high fat feeding in part by promoting oxidative metabolism in skeletal muscle. Similar effects are obtained by germ line deletion of major NAD-consuming enzymes, suggesting that the bioavailability of NAD is limiting for maximal oxidative capacity. However, because of their systemic nature, the degree to which these interventions exert cell- or tissue-autonomous effects is unclear. Here, we report a tissue-specific approach to increase NAD biosynthesis only in muscle by overexpressing nicotinamide phosphoribosyltransferase, the rate-limiting enzyme in the salvage pathway that converts nicotinamide to NAD (mNAMPT mice). These mice display a ∼50% increase in skeletal muscle NAD levels, comparable with the effects of dietary NAD precursors, exercise regimens, or loss of poly(ADP-ribose) polymerases yet surprisingly do not exhibit changes in muscle mitochondrial biogenesis or mitochondrial function and are equally susceptible to the metabolic consequences of high fat feeding. We further report that chronic elevation of muscle NAD in vivo does not perturb the NAD/NADH redox ratio. These studies reveal for the first time the metabolic effects of tissue-specific increases in NAD synthesis and suggest that critical sites of action for supplemental NAD precursors reside outside of the heart and skeletal muscle.     

High CO2 Levels Cause Skeletal Muscle Atrophy via AMP-activated Kinase (AMPK), FoxO3a Protein,and Muscle-specific Ring Finger Protein 1 (MuRF1)

Patients with chronic obstructive pulmonary disease, acute lung injury, and critical care illness may develop hypercapnia. Many of these patients often have muscle dysfunction which increases morbidity and impairs their quality of life. Here, we investigated whether hypercapnia leads to skeletal muscle atrophy. Mice exposed to high CO₂ had decreased skeletal muscle wet weight, fiber diameter, and strength. Cultured myotubes exposed to high CO₂ had reduced fiber diameter, protein/DNA ratios, and anabolic capacity. High CO₂ induced the expression of MuRF1 in vivo and in vitro, whereas MuRF1^−/− mice exposed to high CO₂ did not develop muscle atrophy. AMP-activated kinase (AMPK), a metabolic sensor, was activated in myotubes exposed to high CO₂, and loss-of-function studies showed that the AMPKα2 isoform is necessary for muscle-specific ring finger protein 1 (MuRF1) up-regulation and myofiber size reduction. High CO₂ induced AMPKα2 activation, triggering the phosphorylation and nuclear translocation of FoxO3a, and leading to an increase in MuRF1 expression and myotube atrophy. Accordingly, we provide evidence that high CO₂ activates skeletal muscle atrophy via AMPKα2-FoxO3a-MuRF1, which is of biological and potentially clinical significance in patients with lung diseases and hypercapnia.     

Acetylation of TUG Protein Promotes the Accumulation of GLUT4 Glucose Transporters in an Insulin-responsive Intracellular Compartment

Insulin causes the exocytic translocation of GLUT4 glucose transporters to stimulate glucose uptake in fat and muscle. Previous results support a model in which TUG traps GLUT4 in intracellular, insulin-responsive vesicles termed GLUT4 storage vesicles (GSVs). Insulin triggers TUG cleavage to release the GSVs; GLUT4 then recycles through endosomes during ongoing insulin exposure. The TUG C terminus binds a GSV anchoring site comprising Golgin-160 and possibly other proteins. Here, we report that the TUG C terminus is acetylated. The TUG C-terminal peptide bound the Golgin-160-associated protein, ACBD3 (acyl-CoA-binding domain-containing 3), and acetylation reduced binding of TUG to ACBD3 but not to Golgin-160. Mutation of the acetylated residues impaired insulin-responsive GLUT4 trafficking in 3T3-L1 adipocytes. ACBD3 overexpression enhanced the translocation of GSV cargos, GLUT4 and insulin-regulated aminopeptidase (IRAP), and ACBD3 was required for intracellular retention of these cargos in unstimulated cells. Sirtuin 2 (SIRT2), a NAD⁺-dependent deacetylase, bound TUG and deacetylated the TUG peptide. SIRT2 overexpression reduced TUG acetylation and redistributed GLUT4 and IRAP to the plasma membrane in 3T3-L1 adipocytes. Mutation of the acetylated residues in TUG abrogated these effects. In mice, SIRT2 deletion increased TUG acetylation and proteolytic processing. During glucose tolerance tests, glucose disposal was enhanced in SIRT2 knock-out mice, compared with wild type controls, without any effect on insulin concentrations. Together, these data support a model in which TUG acetylation modulates its interaction with Golgi matrix proteins and is regulated by SIRT2. Moreover, acetylation of TUG enhances its function to trap GSVs within unstimulated cells and enhances insulin-stimulated glucose uptake.