The roles of DGAT1 and DGAT2 in human myotubes are dependent on donor patho-physiological background.

The roles of DGAT1 and DGAT2 in lipid metabolism and insulin responsiveness of human skeletal muscle were studied using cryosections and myotubes prepared from muscle biopsies from control, athlete, and impaired glucose regulation (IGR) cohorts of men. The previously observed increases in intramuscular triacylglycerol (IMTG) in athletes and IGR were shown to be related to an increase in lipid droplet (LD) area in type I fibers in athletes but, conversely, in type II fibers in IGR subjects. Specific inhibition of both diacylglycerol acyltransferase (DGAT) 1 and 2 decreased fatty acid (FA) uptake by myotubes, whereas only DGAT2 inhibition also decreased fatty acid oxidation. Fatty acid uptake in myotubes was negatively correlated with the lactate thresholds of the respective donors. DGAT2 inhibition lowered acetate uptake and oxidation in myotubes from all cohorts whereas DGAT1 inhibition had no effect. A positive correlation between acetate oxidation in myotubes and resting metabolic rate (RMR) from fatty acid oxidation in vivo was observed. Myotubes from athletes and IGR had higher rates of de novo lipogenesis from acetate that were normalized by DGAT2 inhibition. Moreover, DGAT2 inhibition in myotubes also resulted in increased insulin-induced Akt phosphorylation. The differential effects of DGAT1 and DGAT2 inhibition suggest that the specialized role of DGAT2 in esterifying nascent diacylglycerols and de novo synthesized FA is associated with synthesis of a pool of triacylglycerol, which upon hydrolysis results in effectors that promote mitochondrial fatty acid oxidation but decrease insulin signaling in skeletal muscle cells.

Functional expression of the thermally activated transient receptor potential channels TRPA1 and TRPM8 in human myotubes.

Transient potential (TRP) ion channels expressed in primary sensory neurons act as the initial detectors of environmental cold and heat, information which controls muscle energy expenditure. We hypothesize that non-neuronal TRPs have direct cellular responses to thermal exposure, also affecting cellular metabolism. In the present study we show expression of TRPA1, TRPM8 and TRPV1 in rat skeletal muscle and human primary myotubes by qPCR. Effects of TRP activity on metabolism in human myotubes were studied using radiolabeled glucose. FURA-2 was used for Ca2+ imaging. TRPA1, TRPM8 and TRPV1 were expressed at low levels in primary human myotubes and in m. gastrocnemius, m. soleus, and m. trapezius from rat. Activation of TRPA1 by ligustilide resulted in an increased glucose uptake and oxidation in human myotubes, whereas activation of TRPM8 by menthol and icilin significantly decreased glucose uptake and oxidation. Activation of heat sensing TRPV1 by capsaicin had no effect on glucose metabolism. Agonist-induced increases in intracellular Ca2+ levels by ligustilide and icilin in human myotubes confirmed a direct activation of TRPA1 and TRPM8, respectively. The mRNA expression of some genes involved in thermogenesis, i.e. peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), uncoupling protein (UCP) 1 and UCP3, were downregulated in human myotubes following TRPA1 activation, while the mRNA expression of TRPM8 and TRPA1 were downregulated following TRPM8 activation by menthol and icilin, respectively. Cold exposure (18 °C) of cultured myotubes followed by a short recovery period had no effect on glucose uptake and oxidation in the basal situation, however when TRPA1 and TRPM8 channels were chemically inhibited a temperature-induced difference in glucose metabolism was found. In conclusion, mRNA of TRPA1, TRPM8 and TRPV1 are expressed in rat skeletal muscle and human skeletal muscle cells. Modulation of TRPA1 and TRPM8 by chemical agents induced changes in Ca2+ levels and glucose metabolism in human skeletal muscle cells, indicating functional receptors.

Primary defects in lipid handling and resistance to exercise in myotubes from obese donors with and without type 2 diabetes.

Several studies have shown that human primary myotubes retain the metabolic characteristic of their donors in vitro. We have demonstrated, along with other researchers, a reduced lipid turnover and fat oxidation rate in myotubes derived from obese donors with and without type 2 diabetes (T2D). Because exercise is known to increase fat oxidative capacity in skeletal muscle, we investigated if in vitro exercise could restore primary defects in lipid handling in myotubes of obese individuals with and without T2D compared with lean nondiabetic donors. Primary myotubes cultures were derived from biopsies of lean, obese, and T2D subjects. One single bout of long-duration exercise was mimicked in vitro by electrical pulse stimulation (EPS) for 24 h. Lipid handling was measured using radiolabeled palmitate, metabolic gene expression by real-time qPCR, and proteins by Western blot. We first showed that myotubes from obese and T2D donors had increased uptake and incomplete oxidation of palmitate. This was associated with reduced mitochondrial respiratory chain complex II, III, and IV protein expression in myotubes from obese and T2D subjects. EPS stimulated palmitate oxidation in lean donors, while myotubes from obese and T2D donors were refractory to this effect. Interestingly, EPS increased total palmitate uptake in myotubes from lean donors while myotubes from T2D donors had a reduced rate of palmitate uptake into complex lipids and triacylglycerols. Novelty Myotubes from obese and T2D donors are characterized by primary defects in palmitic acid handling. Both obese and T2D myotubes are partially refractory to the beneficial effect of exercise on lipid handling.

Upregulated PDK4 expression is a sensitive marker of increased fatty acid oxidation.

Fatty acid oxidation is a central fueling pathway for mitochondrial ATP production. Regulation occurs through multiple nutrient- and energy-sensitive molecular mechanisms. We explored if upregulated mRNA expression of the mitochondrial enzyme pyruvate dehydrogenase kinase 4 (PDK4) may be used as a surrogate marker of increased mitochondrial fatty acid oxidation, by indicating an overall shift from glucose to fatty acids as the preferred oxidation fuel. The association between fatty acid oxidation and PDK4 expression was studied in different contexts of metabolic adaption. In rats treated with the modified fatty acid tetradecylthioacetic acid (TTA), Pdk4 was upregulated simultaneously with fatty acid oxidation genes in liver and heart, whereas muscle and white adipose tissue remained unaffected. In MDA-MB-231 cells, fatty acid oxidation increased nearly three-fold upon peroxisome proliferator-activated receptor α (PPARα, PPARA) overexpression, and four-fold upon TTA-treatment. PDK4 expression was highly increased under these conditions. Further, overexpression of PDK4 caused increased fatty acid oxidation in these cells. Pharmacological activators of PPARα and AMPK had minor effects, while the mTOR inhibitor rapamycin potentiated the effect of TTA. There were minor changes in mitochondrial respiration, glycolytic function, and mitochondrial biogenesis under conditions of increased fatty acid oxidation. TTA was found to act as a mild uncoupler, which is likely to contribute to the metabolic effects. Repeated experiments with HeLa cells supported these findings. In summary, PDK4 upregulation implies an overarching metabolic shift towards increased utilization of fatty acids as energy fuel, and thus constitutes a sensitive marker of enhanced fatty acid oxidation.

Effects of Propofol on Cellular Bioenergetics in Human Skeletal Muscle Cells.

OBJECTIVES: Propofol may adversely affect the function of mitochondria and the clinical features of propofol infusion syndrome suggest that this may be linked to propofol-related bioenergetic failure. We aimed to assess the effect of therapeutic propofol concentrations on energy metabolism in human skeletal muscle cells. DESIGN: In vitro study on human skeletal muscle cells. SETTINGS: University research laboratories. SUBJECTS: Patients undergoing hip surgery and healthy volunteers. INTERVENTIONS: Vastus lateralis biopsies were processed to obtain cultured myotubes, which were exposed to a range of 1-10 µg/mL propofol for 96 hours. MEASUREMENTS AND MAIN RESULTS: Extracellular flux analysis was used to measure global mitochondrial functional indices, glycolysis, fatty acid oxidation, and the functional capacities of individual complexes of electron transfer chain. In addition, we used [1-C]palmitate to measure fatty acid oxidation and spectrophotometry to assess activities of individual electron transfer chain complexes II-IV. Although cell survival and basal oxygen consumption rate were only affected by 10 µg/mL of propofol, concentrations as low as 1 µg/mL reduced spare electron transfer chain capacity. Uncoupling effects of propofol were mild, and not dependent on concentration. There was no inhibition of any respiratory complexes with low dose propofol, but we found a profound inhibition of fatty acid oxidation. Addition of extra fatty acids into the media counteracted the propofol effects on electron transfer chain, suggesting inhibition of fatty acid oxidation as the causative mechanism of reduced spare electron transfer chain capacity. Whether these metabolic in vitro changes are observable in other organs and at the whole-body level remains to be investigated. CONCLUSIONS: Concentrations of propofol seen in plasma of sedated patients in ICU cause a significant inhibition of fatty acid oxidation in human skeletal muscle cells and reduce spare capacity of electron transfer chain in mitochondria.