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...

Understanding the metabolic processes in energy metabolism, particularly during fasted exercise, is a growing area of research. Previous work has focused on measuring metabolites pre and post exercise. This can provide information about the final state of energy metabolism in the participants, but it does not show how these processes vary during the exercise and any subsequent post-exercise period. To address this, the work described here took fasted participants and subjected them to an exercise and rest protocol under laboratory settings, which allowed for breath and blood sampling both pre, during and post exercise. Analysis of the data produced from both the physiological measurements and the untargeted metabolomics measurements showed clear switching between glycolytic and ketolytic metabolism, with the liquid chromatography-mass spectrometry (LC-MS) data showing the separate stages of ketolytic metabolism, notably the transport, release and breakdown of long chain fatty acids. Several signals, putatively identified as short peptides, were observed to change in a pattern similar to that of the ketolytic metabolites. This work highlights the power of untargeted metabolomic methods as an investigative tool for exercise science, both to follow known processes in a more complete way and discover possible novel biomarkers.

Background Mitochondrial dysfunction is a hallmark of both critical illness and propofol infusion syndrome and its severity seems to be proportional to the doses of noradrenaline, which patients are receiving. We comprehensively studied the effects of noradrenaline on cellular bioenergetics and mitochondrial biology in human skeletal muscle cells with and without propofol-induced mitochondrial dysfunction. Methods Human skeletal muscle cells were isolated from vastus lateralis biopsies from patients undergoing elective hip replacement surgery (n = 14) or healthy volunteers (n = 4). After long-term (96 h) exposure to propofol (10 µg/mL), noradrenaline (100 µM), or both, energy metabolism was assessed by extracellular flux analysis and substrate oxidation assays using [14C] palmitic and [14C(U)] lactic acid. Mitochondrial membrane potential, morphology and reactive oxygen species production were analysed by confocal laser scanning microscopy. Mitochondrial mass was assessed both spectrophotometrically and by confocal laser scanning microscopy. Results Propofol moderately reduced mitochondrial mass and induced bioenergetic dysfunction, such as a reduction of maximum electron transfer chain capacity, ATP synthesis and profound inhibition of exogenous fatty acid oxidation. Noradrenaline exposure increased mitochondrial network size and turnover in both propofol treated and untreated cells as apparent from increased co-localization with lysosomes. After adjustment to mitochondrial mass, noradrenaline did not affect mitochondrial functional parameters in naïve cells, but it significantly reduced the degree of mitochondrial dysfunction induced by propofol co-exposure. The fatty acid oxidation capacity was restored almost completely by noradrenaline co-exposure, most likely due to restoration of the capacity to transfer long-chain fatty acid to mitochondria. Both propofol and noradrenaline reduced mitochondrial membrane potential and increased reactive oxygen species production, but their effects were not additive. Conclusions Noradrenaline prevents rather than aggravates propofol-induced impairment of mitochondrial functions in human skeletal muscle cells. Its effects on bioenergetic dysfunctions of other origins, such as sepsis, remain to be demonstrated.

Objective In vivo studies have reported several beneficial metabolic effects of β-adrenergic receptor agonist administration in skeletal muscle, including increased glucose uptake, fatty acid metabolism, lipolysis and mitochondrial biogenesis. Although these effects have been widely studied in vivo , the in vitro data are limited to mouse and rat cell lines. Therefore, we sought to discover the effects of the β 2 -adrenergic receptor agonist terbutaline on metabolism and protein synthesis in human primary skeletal muscle cells. Methods Human cultured myotubes were exposed to terbutaline in various concentrations (0.01–30 ​μM) for 4 or 96 ​h. Thereafter uptake of [ 14 C]deoxy-D-glucose, oxydation of [ 14 C]glucose and [ 14 C]oleic acid were measured. Incorporation of [ 14 C]leucine, gene expression by qPCR and proteomics analyses by mass spectrometry by the STAGE-TIP method were performed after 96 ​h exposure to 1 and 10 ​μM of terbutaline. Results The results showed that 4 ​h treatment with terbutaline in concentrations up to 1 ​μM increased glucose uptake in human myotubes, but also decreased both glucose and oleic acid oxidation along with oleic acid uptake in concentrations of 10–30 ​μM. Moreover, administration of terbutaline for 96 ​h increased glucose uptake (in terbutaline concentrations up to 1 ​μM) and oxidation (1 ​μM), as well as oleic acid oxidation (0.1–30 ​μM), leucine incorporation into cellular protein (1–10 ​μM) and upregulated several pathways related to mitochondrial metabolism (1 ​μM). Data are available via ProteomeXchange with identifier PXD024063. Conclusion These results suggest that β 2 -adrenergic receptor have direct effects in human skeletal muscle affecting fuel metabolism and net protein synthesis, effects that might be favourable for both type 2 diabetes and muscle wasting disorders.

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