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We investigated the effects of PGC-1α (peroxisome proliferator-activated receptor γ coactivator-1α) overexpression on the oxidative capacity of human skeletal muscle cells ex vivo. PGC-1α overexpression increased the oxidation rate of palmitic acid and mRNA expression of genes regulating lipid metabolism, mitochondrial biogenesis, and function in human myotubes. Basal and insulin-stimulated deoxyglucose uptake were decreased, possibly due to upregulation of PDK4 mRNA. Expression of fast fibertype gene marker (MHCIIa) was decreased. Compared to skeletal muscle in vivo, PGC-1α overexpression increased expression of several genes, which were down regulated during the process of cell isolation and culturing. In conclusion, PGC-1α overexpression increased oxidative capacity of cultured myotubes by improving lipid metabolism, increasing expression of genes involved in regulation of mitochondrial function and biogenesis, and decreasing expression of MHCIIa. These results suggest that therapies aimed at increasing PGC-1α expression may have utility in treatment of obesity and obesity-related diseases. [ABSTRACT FROM AUTHOR]

Objective: Increased availability of fatty acids is important for accumulation of intracellular lipids and development of insulin resistance in human myotubes. It is unknown whether different types of fatty acids like eicosapentaenoic acid (EPA) or tetradecylthioacetic acid (TTA) influence these processes.Research Design and Methods: We examined fatty acid and glucose metabolism and gene expression in cultured human skeletal muscle cells from control and type 2 diabetic individuals after 4 days of preincubation with EPA or TTA.Results: Type 2 diabetes myotubes exhibited reduced formation of CO(2) from palmitic acid (PA), whereas release of beta-oxidation products was unchanged at baseline but significantly increased with respect to control myotubes after preincubation with TTA and EPA. Preincubation with TTA enhanced both complete (CO2) and beta-oxidation of palmitic acid, whereas EPA increased only beta-oxidation significantly. EPA markedly enhanced triacylglycerol (TAG) accumulation in myotubes, more pronounced in type 2 diabetes cells. TAG accumulation and fatty acid oxidation were inversely correlated only after EPA preincubation, and total level of acyl-CoA was reduced. Glucose oxidation (CO2 formation) was enhanced and lactate production decreased after chronic exposure to EPA and TTA, whereas glucose uptake and storage were unchanged. EPA and especially TTA increased the expression of genes involved in fatty acid uptake, activation, accumulation, and oxidation.Conclusions: Our results suggest that 1) mitochondrial dysfunction in diabetic myotubes is caused by disturbances downstream of fatty acid beta-oxidation; 2) EPA promoted accumulation of TAG, enhanced beta-oxidation, and increased glucose oxidation; and 3) TTA improved complete palmitic acid oxidation in diabetic myotubes, opposed increased lipid accumulation, and increased glucose oxidation. [ABSTRACT FROM AUTHOR]

Work in cold environments may have a significant impact on occupational health. In these and similar situations, cold exposure localized to the extremities may reduce the temperature of underlying tissues. To investigate the molecular effects of cold exposure in muscle, and since adequate methods were missing, we established two experimental cold exposure models: 1) In vitro exposure to cold (18°C) or control temperature (37°C) of cultured human skeletal muscle cells (myotubes); and 2) unilateral cold exposure of hind limb skeletal muscle in anesthetized rats (intramuscular temperature 18°C), with contralateral control (37°C). This methodology enables studies of muscle responses to local cold exposures at the level of gene expression, but also other molecular outcomes. Graphical abstract., Competing Interests: Competing interestsThe authors have no financial and non-financial competing interests., (Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.)

We hypothesised that skeletal muscles of healthy young people have a large variation in oxidative capacity and fibre-type composition, and aimed therefore to investigate glucose metabolism in biopsies and myotubes isolated from musculus vastus lateralis from healthy males with varying degrees of maximal oxygen uptake. Trained and intermediary trained subjects showed higher carbohydrate oxidation in vivo. Fibre-type distribution in biopsies and myotubes did not differ between groups. There was no correlation between fibre-type I expression in biopsies and myotubes. Myotubes from trained had higher deoxyglucose accumulation and fractional glucose oxidation (glucose oxidation relative to glucose uptake), and were also more sensitive to the suppressive action of acutely added oleic acid to the cells. Despite lack of correlation of fibre types between skeletal muscle biopsies and cultured cells, myotubes from trained subjects retained some of their phenotypes in vitro with respect to enhanced glucose metabolism and metabolic flexibility.

Exercise improves insulin sensitivity and oxidative capacity in skeletal muscles. However, the effect of exercise on substrate oxidation is less clear in obese and type 2 diabetic subjects than in lean subjects. We investigated glucose and lipid metabolism and gene expression after 48 h with low-frequency electrical pulse stimulation (EPS), as an in vitro model of exercise, in cultured myotubes established from lean nondiabetic subjects and severely obese subjects (BMI ≥ 40 kg/m(2)) with and without type 2 diabetes. EPS induced an increase in insulin sensitivity but did not improve lipid oxidation in myotubes from severely obese subjects. Thus, EPS-induced increases in insulin sensitivity and lipid oxidation were positively and negatively correlated to BMI of the subjects, respectively. EPS enhanced oxidative capacity of glucose in myotubes from all subjects. Furthermore, EPS reduced mRNA expression of slow fiber-type marker (MYH7) in myotubes from diabetic subjects; however, the protein expression of this marker was not significantly affected by EPS in either of the donor groups. On the contrary, mRNA levels of interleukin-6 (IL-6) and IL-8 were unaffected by EPS in myotubes from diabetic subjects, while IL-6 mRNA expression was increased in myotubes from nondiabetic subjects. EPS-stimulated mRNA expression levels of MYH7, IL-6, and IL-8 correlated negatively with subjects' HbA1c and/or fasting plasma glucose, suggesting an effect linked to the diabetic phenotype. Taken together, these data show that myotubes from different donor groups respond differently to EPS, suggesting that this effect may reflect the in vivo characteristics of the donor groups., (Copyright © 2015 the American Physiological Society.)

This study explores the pharmacokinetics of 22-S-hydroxycholesterol (22SHC) in vivo in rats. We also carried out a metabolic study to explore whether the beneficial effects observed of 22SHC on glucose and lipid metabolism in vitro could be seen in vivo in rats. In the pharmacokinetic study, rats were given 50 mg/kg of [³H]22-S-hydroxycholesterol before absorption, distribution and excretion were monitored. In the metabolic study, the effect of 22SHC (30 mg/kg/day for 3 weeks) in rats on body weight gain [chow and high-fat diet (HFD)], serum lipids triacylglycerol (TAG) content and gene expression in liver and skeletal muscle were examined. Results showed that 22SHC was well absorbed after oral administration and distributed to most organs and mainly excreted in feces. Rats receiving 22SHC gained less body weight than their controls regardless whether the animals received chow diet or HFD. Moreover, we observed that animals receiving HFD had elevated levels of serum TAG while this was not observed for animals on HFD supplemented with 22SHC. The amount of TAG in liver was reduced after 22SHC treatment in animals receiving either chow diet or HFD. Gene expression analysis revealed that two genes (carnitine palmitoyltransferase 2 and uncoupling protein 3) involved in fatty acid oxidation and energy dissipation were increased in liver. Ucp3 expression (both protein and mRNA level) was increased in skeletal muscle, but insulin-stimulated glucose uptake and TAG content were unchanged. In conclusion, 22SHC seems to be an interesting model substance in the search of treatments for disorders involving aberrations in lipid metabolism.

Regular physical activity protects against several types of diseases. This may involve altered secretion of signaling proteins from skeletal muscle. Our aim was to identify the most abundantly secreted proteins in cultures of human skeletal muscle cells and to monitor their expression in muscles of strength-training individuals. A total of 236 proteins were detected by proteome analysis in medium conditioned by cultured human myotubes, which was narrowed down to identification of 18 classically secreted proteins expressed in skeletal muscle, using the SignalP 3.0 and Human Genome Expression Profile databases together with a published mRNA-based reconstruction of the human skeletal muscle secretome. For 17 of the secreted proteins, expression was confirmed at the mRNA level in cultured human myotubes as well as in biopsies of human skeletal muscles. RT-PCR analyses showed that 15 of the secreted muscle proteins had significantly enhanced mRNA expression in m. vastus lateralis and/or m. trapezius after 11 wk of strength training among healthy volunteers. For example, secreted protein acidic and rich in cysteine, a secretory protein in the membrane fraction of skeletal muscle fibers, was increased 3- and 10-fold in m. vastus lateralis and m. trapezius, respectively. Identification of proteins secreted by skeletal muscle cells in vitro facilitated the discovery of novel responses in skeletal muscles of strength-training individuals.

We evaluated the effects of partly substituting lard with marine n-3 fatty acids (FA) on body composition and weight, adipose tissue distribution and gene expression in five adipose depots of male Wistar rats fed a high-fat diet. Rats were fed diets including lard (19.5 % lard) or n-3 FA (9.1 % lard and 10.4 % Triomar) for 7 weeks. Feed consumption and weight gain were similar, whereas plasma lipid concentrations were lower in the n-3 FA group. Magnetic resonance imaging revealed smaller visceral (mesenteric, perirenal and epididymal) adipose depots in the n-3 FA-fed animals (35, 44 and 32 % reductions, respectively). n-3 FA feeding increased mRNA expression of cytokines as well as chemokines in several adipose depots. Expression of Adipoq and Pparg was enhanced in the mesenteric adipose depots of the n-3 FA-fed rats, and fasting plasma insulin levels were lowered. Expression of the lipogenic enzymes Acaca and Fasn was increased in the visceral adipose depots, whereas Dgat1 was reduced in the perirenal and epididymal depots. Cpt2 mRNA expression was almost doubled in the mesenteric depot and liver. Carcass analyses showed similar body fat (%) in the two feeding groups, indicating that n-3 FA feeding led to redistribution of fat away from the visceral compartment.

The nuclear liver X receptors (LXRalpha and beta) are regulators of lipid and cholesterol metabolism. Oxysterols are known LXR ligands, but the functional role of hydroxycholesterols is at present unknown. In human myotubes, chronic exposure to the LXR ligand T0901317 promoted formation of diacylglycerol (DAG) and triacylglycerol (TAG), 22-R-hydroxycholesterol (22-R-HC) had no effect, and 22-S-hydroxycholesterol (22-S-HC) reduced the formation. In accordance with this, 22-HC and T0901317 regulated the expression of fatty acid transporter CD36, stearoyl-CoA desaturase-1, acyl-CoA synthetase long chain family member 1 and fatty acid synthase (FAS) differently; all genes were increased by T0901317, 22-R-HC did not change their expression level, while 22-S-HC reduced it. Transfection studies confirmed that the FAS promoter was activated by T0901317 and repressed by 22-S-HC through an LXR response element in the promoter. Both 22-R-HC and T0901317 increased gene expression of LXRalpha, sterol regulatory element-binding protein 1c and ATP-binding cassette transporter A1, while 22-S-HC had little effect. In summary, 22-R-HC regulated lipid metabolism and mRNA expression of some LXR target genes in human myotubes differently than T0901317. Moreover, 22-S-HC did not behave like an inactive ligand; it reduced synthesis of complex lipids and repressed certain genes involved in lipogenesis and lipid handling.