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IntroductionKrill oil is a dietary supplement derived from Antarctic krill; a small crustacean found in the ocean. Krill oil is a rich source of omega-3 fatty acids, specifically eicosapentaenoic acid and docosahexaenoic acid, as well as the antioxidant astaxanthin. The aim of this study was to investigate the effects of krill oil supplementation, compared to placebo oil (high oleic sunflower oil added astaxanthin), in vivo on energy metabolism and substrate turnover in human skeletal muscle cells.MethodsSkeletal muscle cells (myotubes) were obtained before and after a 7-week krill oil or placebo oil intervention, and glucose and oleic acid metabolism and leucine accumulation, as well as effects of different stimuli in vitro, were studied in the myotubes. The functional data were combined with proteomic and transcriptomic analyses.ResultsIn vivo intervention with krill oil increased oleic acid oxidation and leucine accumulation in skeletal muscle cells, however no effects were observed on glucose metabolism. The krill oil-intervention-induced increase in oleic acid oxidation correlated negatively with changes in serum low-density lipoprotein (LDL) concentration. In addition, myotubes were also exposed to krill oil in vitro. The in vitro study revealed that 24 h of krill oil treatment increased both glucose and oleic acid metabolism in myotubes, enhancing energy substrate utilization. Transcriptomic analysis comparing myotubes obtained before and after krill oil supplementation identified differentially expressed genes associated with e.g., glycolysis/gluconeogenesis, metabolic pathways and calcium signaling pathway, while proteomic analysis demonstrated upregulation of e.g., LDL-receptor in myotubes obtained after the krill oil intervention.ConclusionThese findings suggest that krill oil intervention promotes increased fuel metabolism and protein synthesis in human skeletal muscle cells, with potential implications for metabolic health.

Introduction: Skeletal muscle is a major contributor to whole-body energy homeostasis and the utilization of fatty acids and glucose. At present, 2D cell models have been the most used cellular models to study skeletal muscle energy metabolism. However, the transferability of the results to in vivo might be limited. This project aimed to develop and characterize a skeletal muscle 3D cell model (myospheres) as an easy and low-cost tool to study molecular mechanisms of energy metabolism.Methods and results: We demonstrated that human primary myoblasts form myospheres without external matrix support and carry structural and molecular characteristics of mature skeletal muscle after 10 days of differentiation. We found significant metabolic differences between the 2D myotubes model and myospheres. In particular, myospheres showed increased lipid oxidative metabolism than the 2D myotubes model, which oxidized relatively more glucose and accumulated more oleic acid.Discussion and conclusion: These analyses demonstrate model differences that can have an impact and should be taken into consideration for studying energy metabolism and metabolic disorders in skeletal muscle.

Obesity and physical inactivity have a profound impact on skeletal muscle metabolism. In the present work, we have investigated differences in protein expression and energy metabolism in primary human skeletal muscle cells established from lean donors (BMI30 kg/m2). Furthermore, we have studied the effect of fatty acid pretreatment on energy metabolism in myotubes from these donor groups. Alterations in protein expression were investigated using proteomic analysis, and energy metabolism was studied using radiolabeled substrates. Gene Ontology enrichment analysis showed that glycolytic, apoptotic, and hypoxia pathways were upregulated, whereas the pentose phosphate pathway was downregulated in myotubes from donors with obesity compared to myotubes from lean donors. Moreover, fatty acid, glucose, and amino acid uptake were increased in myotubes from individuals with obesity. However, fatty acid oxidation was reduced, glucose oxidation was increased in myotubes from subjects with obesity compared to cells from lean. Pretreatment of myotubes with palmitic acid (PA) or eicosapentaenoic acid (EPA) for 24 h increased glucose oxidation and oleic acid uptake. EPA pretreatment increased the glucose and fatty acid uptake and reduced leucine fractional oxidation in myotubes from donors with obesity. In conclusion, these results suggest that myotubes from individuals with obesity showed increased fatty acid, glucose, and amino acid uptake compared to cells from lean donors. Furthermore, myotubes from individuals with obesity had reduced fatty acid oxidative capacity, increased glucose oxidation, and a higher glycolytic reserve capacity compared to cells from lean donors. Fatty acid pretreatment enhances glucose metabolism, and EPA reduces oleic acid and leucine fractional oxidation in myotubes from donor with obesity, suggesting increased metabolic flexibility after EPA treatment.

Electrical pulse stimulation (EPS) has proven to be a useful tool to interrogate cell-specific responses to muscle contraction. In the present study, we aimed to uncover networks of signaling pathways and regulatory molecules responsible for the metabolic effects of exercise in human skeletal muscle cells exposed to chronic EPS. Differentiated myotubes from young male subjects were exposed to EPS protocol 1 (i.e. 2 ms, 10 V, and 0.1 Hz for 24 h), whereas myotubes from middle-aged women and men were exposed to protocol 2 (i.e. 2 ms, 30 V, and 1 Hz for 48 h). Fuel handling as well as the transcriptome, cellular proteome, and secreted proteins of EPS-treated myotubes from young male subjects were analyzed using a combination of high-throughput RNA sequencing, high-resolution liquid chromatography-tandem mass spectrometry, oxidation assay, and immunoblotting. The data showed that oxidative metabolism was enhanced in EPS-exposed myotubes from young male subjects. Moreover, a total of 81 differentially regulated proteins and 952 differentially expressed genes (DEGs) were observed in these cells after EPS protocol 1. We also found 61 overlapping genes while comparing the DEGs to mRNA expression in myotubes from the middle-aged group exposed to protocol 2, assessed by microarray. Gene ontology (GO) analysis indicated that significantly regulated proteins and genes were enriched in biological processes related to glycolytic pathways, positive regulation of fatty acid oxidation, and oxidative phosphorylation, as well as muscle contraction, autophagy/mitophagy, and oxidative stress. Additionally, proteomic identification of secreted proteins revealed extracellular levels of 137 proteins were changed in myotubes from young male subjects exposed to EPS protocol 1. Selected putative myokines were measured using ELISA or multiplex assay to validate the results. Collectively, our data provides new insight into the transcriptome, proteome and secreted proteins alterations following in vitro exercise and is a valuable resource for understanding the molecular mechanisms and regulatory molecules mediating the beneficial metabolic effects of exercise.