Higher lipid turnover and oxidation in cultured human myotubes from athletic versus sedentary young male subjects

Abstract

In this study we compared fatty acid (FA) metabolism in myotubes established from athletic and sedentary young subjects. Six healthy sedentary (maximal oxygen uptake (VO_2max) ≤ 46 ml/kg/min) and six healthy athletic (VO_2max > 60 ml/kg/min) young men were included. Myoblasts were cultured and differentiated to myotubes from satellite cells isolated from biopsy of musculus vastus lateralis. FA metabolism was studied in myotubes using [¹⁴C]oleic acid. Lipid distribution was assessed by thin layer chromatography, and FA accumulation, lipolysis and re-esterification were measured by scintillation proximity assay. Gene and protein expressions were studied. Myotubes from athletic subjects showed lower FA accumulation, lower incorporation of FA into total lipids, triacylglycerol (TAG), diacylglycerol and cholesteryl ester, higher TAG-related lipolysis and re-esterification, and higher complete oxidation and incomplete β-oxidation of FA compared to myotubes from sedentary subjects. mRNA expression of the mitochondrial electron transport chain complex III gene UQCRB was higher in cells from athletic compared to sedentary. Myotubes established from athletic subjects have higher lipid turnover and oxidation compared to myotubes from sedentary subjects. Our findings suggest that cultured myotubes retain some of the phenotypic traits of their donors.

Figure 1

Lower oleic acid accumulation and incorporation into lipids in myotubes from athletic subjects. Satellite cells isolated from biopsies from musculus vastus lateralis from athletic and sedentary subjects were cultured and differentiated into myotubes. (a) Fatty acid accumulation of [¹⁴C]oleic acid (100 µM) during the last 24 h of the differentiation period was measured with SPA. (b) Myotubes were treated with [¹⁴C]oleic acid (100 µM) for 24 h, and lipids were thereafter extracted from the cells, separated by TLC and quantified by liquid scintillation. CE, cholesteryl ester; DAG, diacylglycerol; FFA, free fatty acid; PL, phospholipid; TAG, triacylglycerol. Data are presented as means ± SEM (n = 6 in each group) in nmol/mg cell protein. *Statistically significant versus sedentary (p < 0.05, a: Linear mixed-model analysis, SPSS, b: Mann-Whitney test).

Figure 2

Higher TAG-related lipolysis and re-esterification in myotubes from athletic subjects. (a) Lipolysis was measured over 6 h after 24 h incubation with [¹⁴C]oleic acid. Lipolysis was related to the labelled triacylglycerol (TAG) pool in the cells as measured by TLC. (b) Re-esterification was measured over 6 h after 24 h incubation with [¹⁴C]oleic acid. Re-esterification of oleic acid was calculated from lipolysis measurements as [triacsin C present (total lipolysis) – triacsin C absent (basal lipolysis)]. Fatty acid re-esterification was related to the labelled TAG pool in the cells as measured by TLC. Data are presented as means ± SEM (n = 6 in each group). *Statistically significant versus sedentary (p < 0.05, linear mixed-model analysis, SPSS. (c) RNA was isolated and mRNA reversely transcribed before expressions of diacylglycerol acyltransferase (DGAT) 1, DGAT2, fatty acid binding protein (FABP) 4, and G0/G1 switch gene 2 (G0S2) were assessed by qPCR. Values are presented as means ± SEM (n = 6 in each group), and corrected for the average of the housekeeping gene acidic ribosomal phosphoprotein P0 (RPLP0). The results were normalized to the results of the mRNA expression for myotubes from sedentary subjects. (d,e) Protein expressions of adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL) and phosphorylated HSL at serine 660 (HSL^Ser660) were analysed by immunoblotting of protein isolated from cell lysates. d, representative immunoblots. e, quantified expressions of the proteins. All values were corrected for the housekeeping control β-actin, and are presented as means ± SEM (n = 6 in each group). All samples were derived at the same time, from the same experiment, and processed in parallel. Full-length blots are presented in Supplementary Fig. S2.

Figure 3

Higher oleic acid metabolism in myotubes from athletic subjects. Satellite cells isolated from biopsies from musculus vastus lateralis from athletic and sedentary subjects were cultured and differentiated into myotubes. CO₂ (complete fatty acid oxidation) and acid-soluble metabolites (ASMs, representing incomplete fatty acid β-oxidation) were measured after extracellular treatment (100 µM [¹⁴C]oleic acid for 4 h) and intracellular treatment (prelabelling with a combination of 100 µM and 400 µM [¹⁴C]oleic acid for 24 h). (a) CO₂ from extracellular (acute) and intracellular (prelabelled) [¹⁴C]oleic acid. (b) ASM from extracellular and intracellular [¹⁴C]oleic acid. Values are presented as means ± SEM (n = 6 in each group) in nmol/mg cell protein. *Statistically significant versus sedentary (p < 0.05, a: Mann-Whitney test, b: Linear mixed-model analysis, SPSS).

Figure 4

Minor differences in mRNA expression in myotubes from the two groups. Satellite cells isolated from biopsies from musculus vastus lateralis from athletic and sedentary subjects were cultured and differentiated into myotubes. RNA was isolated and mRNA reversely transcribed before expressions of selected genes were assessed by qPCR. Values are presented as means ± SEM (n = 6 in each group), and corrected for the average of the housekeeping gene acidic ribosomal phosphoprotein P0 (RPLP0). The results were normalized to the results of the mRNA expression for myotubes from sedentary subjects. (a) mRNA expression of FA utilization. (b) mRNA expression of genes encoding for enzymes of the electron transport chain which drives the oxidative phosphorylation. *Statistically significant versus sedentary (p < 0.05, Mann-Whitney test). ATP5F1A, ATP-synthase F1 subunit α; ATP5MC2, ATP-synthase membrane subunit c locus 2; CD36, fatty acid translocase; COX4I1, cytochrome c oxidase subunit 4I1; COX5B, cytochrome c oxidase subunit 5B; CPT1A, carnitine palmitoyltransferase 1A; CPT1B, carnitine palmitoyltransferase 1B; NDUFA8, NADH-ubiquinone oxidoreductase core subunit A8; NDUFS1, NADH-ubiquinone oxidoreductase core subunit S1; PLIN2, perilipin 2; PLIN3, perilipin 3; PPARD, peroxisome proliferator-activated receptor δ; SDHB, succinate dehydrogenase complex iron sulphur subunit B; UQCR11, ubiquinol-cytochrome c reductase complex III subunit XI; UQCRB, ubiquinol-cytochrome c reductase binding protein.