Background and aimsPhysical activity has preventive as well as therapeutic benefits for overweight subjects. In this study we aimed to examine effects of in vivo exercise on in vitro metabolic adaptations by studying energy metabolism in cultured myotubes isolated from biopsies taken before and after 12 weeks of extensive endurance and strength training, from healthy sedentary normal weight and overweight men.MethodsHealthy sedentary men, aged 40–62 years, with normal weight (body mass index (BMI) < 25 kg/m2) or overweight (BMI ≥ 25 kg/m2) were included. Fatty acid and glucose metabolism were studied in myotubes using [14C]oleic acid and [14C]glucose, respectively. Gene and protein expressions, as well as DNA methylation were measured for selected genes.ResultsThe 12-week training intervention improved endurance, strength and insulin sensitivity in vivo, and reduced the participants’ body weight. Biopsy-derived cultured human myotubes after exercise showed increased total cellular oleic acid uptake (30%), oxidation (46%) and lipid accumulation (34%), as well as increased fractional glucose oxidation (14%) compared to cultures established prior to exercise. Most of these exercise-induced increases were significant in the overweight group, whereas the normal weight group showed no change in oleic acid or glucose metabolism.Conclusions12 weeks of combined endurance and strength training promoted increased lipid and glucose metabolism in biopsy-derived cultured human myotubes, showing that training in vivo are able to induce changes in human myotubes that are discernible in vitro.
Diacylglycerol acyltransferases (DGAT) 1 and 2 catalyse the final step in triacylglycerol (TAG) synthesis, the esterification of fatty acyl-CoA to diacylglycerol. Despite catalysing the same reaction and being present in the same cell types, they exhibit different functions on lipid metabolism in various tissues. Yet, their roles in skeletal muscle remain poorly defined. In this study, we investigated how selective inhibitors of DGAT1 and DGAT2 affected lipid metabolism in human primary skeletal muscle cells. The results showed that DGAT1 was dominant in human skeletal muscle cells utilizing fatty acids (FAs) derived from various sources, both exogenously supplied FA, de novo synthesised fA, or fA derived from lipolysis, to generate TAG, as well as being involved in de novo synthesis of tAG. on the other hand, DGAT2 seemed to be specialised for de novo synthesis of TAG from glycerol-3-posphate only. Interestingly, DGAT activities were also important for regulating FA oxidation, indicating a key role in balancing FAs between storage in TAG and efficient utilization through oxidation. Finally, we observed that inhibition of DGAT enzymes could potentially alter glucose-FA interactions in skeletal muscle. In summary, treatment with DGAT1 or DGAT2 specific inhibitors resulted in different responses on lipid metabolism in human myotubes, indicating that the two enzymes play distinct roles in TAG metabolism in skeletal muscle.Skeletal muscle utilizes both carbohydrates and fat as energy sources. Approximately 50-60% of the free fatty acids (FFAs) taken up by skeletal muscle are stored as triacylglycerol (TAG) in lipid droplets (LDs) 1 . TAG, which is a neutral lipid, consists of a glycerol backbone and three FAs attached by ester bonds. The terminal and only committed step of TAG synthesis, the esterification of fatty acyl-CoA to diacylglycerol (DAG), is catalysed by the enzymes diacylglycerol acyltransferase (DGAT) 1 and 2 2-4 . Both DGAT enzymes reside at the endoplasmic reticulum 5,6 , though DGAT2 is also found to co-localize with LDs and mitochondria in cultured fibroblasts and adipocytes, in contrast to DGAT1 5,6 . Although the two isozymes catalyse the same reaction, there are several differences between them. They share no sequence homology with each other, belong to unrelated families of proteins 4 and overexpression of the two isozymes in rat hepatoma cells give rise to LDs with markedly different morphology (size) and intracellular distribution 7 . In addition, they are non-redundant in some functions, which are reflected by the phenotype of mice lacking DGAT1 or DGAT2. Whereas Dgat1 −/− mice are viable with a favourable metabolic phenotype showing an increased insulin and leptin sensitivity and resistance to diet-induced obesity, Dgat2 −/− mice die shortly after birth; they are lipopenic, have a defect in the skin barrier leading to rapid dehydration 8-10 , and are possibly unable to utilize glucose in brown adipocytes for thermoregulation 11 . TAG formation can occur in two ways, namely from re-esterificati...
In this study we compared fatty acid (FA) metabolism in myotubes established from athletic and sedentary young subjects. Six healthy sedentary (maximal oxygen uptake (VO2max) ≤ 46 ml/kg/min) and six healthy athletic (VO2max > 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.
It has previously been shown that pretreatment of differentiated human skeletal muscle cells (myotubes) with eicosapentaenoic acid (EPA) promoted increased uptake of fatty acids and increased triacylglycerol accumulation, compared to pretreatment with oleic acid (OA) and palmitic acid (PA). The aim of the present study was to examine whether EPA could affect substrate cycling in human skeletal muscle cells by altering lipolysis rate of intracellular TAG and re-esterification of fatty acids. Fatty acid metabolism was studied in human myotubes using a mixture of fatty acids, consisting of radiolabelled oleic acid as tracer (14C-OA) together with EPA or PA. Co-incubation of myotubes with EPA increased cell-accumulation and incomplete fatty acid oxidation of 14C-OA compared to co-incubation with PA. Lipid distribution showed higher incorporation of 14C-OA into all cellular lipids after co-incubation with EPA relative to PA, with most markedly increases (3 to 4-fold) for diacylglycerol and triacylglycerol. Further, the increases in cellular lipids after co-incubation with EPA were accompanied by higher lipolysis and fatty acid re-esterification rate. Correspondingly, basal respiration, proton leak and maximal respiration were significantly increased in cells exposed to EPA compared to PA. Microarray and Gene Ontology (GO) enrichment analysis showed that EPA, related to PA, significantly changed i.e. the GO terms “Neutral lipid metabolic process” and “Regulation of lipid storage”. Finally, an inhibitor of diacylglycerol acyltransferase 1 decreased the effect of EPA to promote fatty acid accumulation. In conclusion, incubation of human myotubes with EPA, compared to PA, increased processes of fatty acid turnover and oxidation suggesting that EPA may activate futile substrate cycling of fatty acids in human myotubes. Increased TAG—FA cycling may be involved in the potentially favourable effects of long-chain polyunsaturated n-3 fatty acids on skeletal muscle and whole-body energy metabolism.
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