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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. [ABSTRACT FROM AUTHOR]

Background and aims: Physical 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. Methods: Healthy 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. Results: The 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. Conclusions: 12 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. [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 CO2 from palmitic acid (PA), whereas release of β-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 β-oxidation of palmitic acid, whereas EPA increased only β-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 β-oxidation; 2) EPA promoted accumulation of TAG, enhanced β-oxidation, and increased glucose oxidation; and 3) TTA improved complete palmitic acid oxidation in diabetic myotubes, opposed increased lipid accumulation, and increased glucose oxidation. Diabetes 58:527-535, 2009 [ABSTRACT FROM AUTHOR]

We recently described a primarily reduced palmitate oxidation in myotubes established from type 2 diabetic subjects, whereas triacylglycerol (TAG) accumulation seemed to be adaptive. However, it is still uncertain whether these changes are similar for saturated and unsaturated fatty acids and whether high concentrations of glucose and/or insulin may change this picture. Studies of palmitic acid and oleic acid metabolism in human myotubes established from control and type 2 diabetic subjects under conditions of acute high concentrations of insulin and/or glucose may solve these questions. Total oleic acid and palmitic acid uptake in myotubes was increased during acute insulin stimulation (P < 0.01) but not under acute, high-glucose concentrations, and no differences were found between the groups. Type 2 diabetic myotubes expressed a reduced palmitic acid oxidation to carbon dioxide (P ≤ 0.04), whereas oleic acid oxidation showed no differences between myotubes from both groups. High glucose concentrations decreased oleic acid oxidation (P ≤ 0.03). Lipid distribution was not different in diabetic and control myotubes when palmitic acid and oleic acid incorporation into cellular lipids was compared. Myotubes that were exposed to palmitic acid showed an increased palmitic acid incorporation into diacylglycerol (DAG) and TAG compared with myotubes that were exposed to oleic acid (P < 0.05) expressing an increased intracellular free fatty acid (FFA) level (P < 0.05). Lipid distribution was not affected by high glucose, whereas insulin increased FFAs, DAG, and TAG (P < 0.05). De novo lipid synthesis from glucose in both diabetic and control myotubes was of the same magnitude independent of glucose and insulin concentrations. These results indicate that palmitic acid and oleic acid are utilized in the same pattern in diabetic and control myotubes even though palmitic acid oxidation is primarily reduced in diabetic cells. Palmitic acid and oleic acid are handled differently by myotubes: Palmitic acid seems to accumulate as DAG and TAG, whereas oleic acid accumulates as intracellular FFAs. These observations indicate that oleic acid is preferable as fatty acid as it accumulates to a lesser extent as DAG and TAG than palmitic acid. Neither acute hyperglycemia nor de novo lipid synthesis from glucose seems central to the TAG accumulation in obesity or type 2 diabetes. [ABSTRACT FROM AUTHOR]

Insulin resistance in skeletal muscle in vivo is associated with reduced lipid oxidation and lipid accumulation. It is still uncertain whether changes in lipid metabolism represent an adaptive compensation at the cellular level or a direct expression of a genetic trait. Studies of palmitate metabolism in human myotubes established from control and type 2 diabetic subjects may solve this problem, as genetic defects are preserved and expressed in vitro. In this study, total uptake of palmitic acid was similar in myotubes established from both control and type 2 diabetic subjects under basal conditions and acute insulin stimulation. Myotubes established from diabetic subjects expressed a primary reduced palmitic acid oxidation to carbon dioxide with a concomitantly increased esterification of palmitic acid into phospholipids compared with control myotubes under basal conditions. Triacylglycerol (TAG) content and the incorporation of palmitic acid into diacylglycerol (DAG) and TAG at basal conditions did not vary between the groups. Acute insulin treatment significantly increased palmitate uptake and incorporation of palmitic acid into DAG and TAG in myotubes established from both study groups, but no difference was found in myotubes established from control and diabetic subjects. These results indicate that the reduced lipid oxidation in diabetic skeletal muscle in vivo may be of genetic origin; it also appears that TAG metabolism is not primarily affected in diabetic muscles under basal physiological conditions. [ABSTRACT FROM AUTHOR]

Background: Skeletal muscle adapts in reaction to contractile activity to efficiently utilize energy substrates, primarily glucose and free fatty acids (FA). Inactivity leads to atrophy and a change in energy utilization in individuals with spinal cord injury (SCI). The present study aimed to characterize possible inactivity‐related differences in the energy metabolism between skeletal muscle cells cultured from satellite cells isolated 1‐ and 12‐months post‐SCI. Methods: To characterize inactivity‐related disturbances in spinal cord injury, we studied skeletal muscle cells isolated from SCI subjects. Cell cultures were established from biopsy samples from musculus vastus lateralis from subjects with SCI 1 and 12 months after the injury. The myoblasts were proliferated and differentiated into myotubes before fatty acid and glucose metabolism were assessed and gene and protein expressions were measured. Results: The results showed that glucose uptake was increased, while oleic acid oxidation was reduced at 12 months compared to 1 month. mRNA expressions of PPARGC1α, the master regulator of mitochondrial biogenesis, and MYH2, a determinant of muscle fiber type, were significantly reduced at 12 months. Proteomic analysis showed reduced expression of several mitochondrial proteins. Conclusion: In conclusion, skeletal muscle cells isolated from immobilized subjects 12 months compared to 1 month after SCI showed reduced fatty acid metabolism and reduced expression of mitochondrial proteins, indicating an increased loss of oxidative capacity with time after injury. [ABSTRACT FROM AUTHOR]

Primary human myotubes represent an alternative system to intact skeletal muscle for the study of human diseases related to changes in muscle energy metabolism. This work aimed to study if fatty acid and glucose metabolism in human myotubes in vitro were related to muscle of origin, donor gender, age, or body mass index (BMI). Myotubes from a total of 82 donors were established from three different skeletal muscles, i.e., musculus vastus lateralis, musculus obliquus internus abdominis, and musculi interspinales, and cellular energy metabolism was evaluated. Multiple linear regression analyses showed that donor age had a significant effect on glucose and oleic acid oxidation after correcting for gender, BMI, and muscle of origin. Donor BMI was the only significant contributor to cellular oleic acid uptake, whereas cellular glucose uptake did not rely on any of the variables examined. Despite the effect of age on substrate oxidation, cellular mRNA expression of pyruvate dehydrogenase kinase 4 (PDK4) and peroxisome proliferator–activated receptor gamma coactivator 1 alpha (PPARGC1A) did not correlate with donor age. In conclusion, donor age significantly impacts substrate oxidation in cultured human myotubes, whereas donor BMI affects cellular oleic acid uptake. [ABSTRACT FROM AUTHOR]

Background: Parenteral (intravenous) nutrition is lifesaving for patients with intestinal failure, but long-term use of parenteral nutrition often leads to liver disease. SEFA-6179 is a synthetic medium-chain fatty acid analogue designed to target multiple fatty acid receptors regulating metabolic and inflammatory pathways. We hypothesized that SEFA-6179 would prevent hepatosteatosis and lipotoxicity in a murine model of parenteral nutrition-induced hepatosteatosis. Methods: Two in vivo experiments were conducted. In the first experiment, six-week-old male mice were provided an ad lib fat-free high carbohydrate diet (HCD) for 19 days with orogastric gavage of either fish oil, medium-chain triglycerides, or SEFA-6179 at a low (0.3mmol/kg) or high dose (0.6mmol/kg). In the second experiment, six-week-old mice were provided an ad lib fat-free high carbohydrate diet for 19 days with every other day tail vein injection of saline, soybean oil lipid emulsion, or fish oil lipid emulsion. Mice then received every other day orogastric gavage of medium-chain triglyceride vehicle or SEFA-6179 (0.6mmol/kg). Hepatosteatosis was assessed by a blinded pathologist using an established rodent steatosis score. Hepatic lipid metabolites were assessed using ultra-high-performance liquid chromatography-mass spectrometry. Effects of SEFA-6179 on fatty acid oxidation, lipogenesis, and fatty acid uptake in human liver cells were assessed in vitro. Results: In the first experiment, mice receiving the HCD with either saline or medium-chain triglyceride treatment developed macrovesicular steatosis, while mice receiving fish oil or SEFA-6179 retained normal liver histology. In the second experiment, mice receiving a high carbohydrate diet with intravenous saline or soybean oil lipid emulsion, along with medium chain triglyceride vehicle treatment, developed macrovescular steatosis. Treatment with SEFA-6179 prevented steatosis. In each experiment, SEFA-6179 treatment decreased arachidonic acid metabolites as well as key molecules (diacylglycerol, ceramides) involved in lipotoxicity. SEFA-6179 increased both β- and complete fatty oxidation in human liver cells, while having no impact on lipogenesis or fatty acid uptake. Conclusions: SEFA-6179 treatment prevented hepatosteatosis and decreased toxic lipid metabolites in a murine model of parenteral nutrition-induced hepatosteatosis. An increase in both β- and complete hepatic fatty acid oxidation may underlie the reduction in steatosis. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Although long-chain omega-3 fatty acids (LCn-3FAs) regulate inflammatory pathways of relevance to non-alcoholic steatohepatitis (NASH), their susceptibility to peroxidation may limit their therapeutic potential. We compared the metabolism of eicosapentaenoic acid (EPA) with an engineered EPA derivative (icosabutate) in human hepatocytes in vitro and their effects on hepatic glutathione metabolism, oxidised lipids, inflammation, and fibrosis in a dietary mouse model of NASH, and in patients prone to fatty liver disease. Oxidation rates and cellular partitioning of EPA and icosabutate were compared in primary human hepatocytes. Comparative effects of delayed treatment with either low- (56 mg/kg) or high-dose (112 mg/kg) icosabutate were compared with EPA (91 mg/kg) or a glucagon-like peptide 1 receptor agonist in a choline-deficient (CD), L-amino acid-defined NASH mouse model. To assess the translational potential of these findings, effects on elevated liver enzymes and fibrosis-4 (FIB-4) score were assessed in overweight, hyperlipidaemic patients at an increased risk of NASH. In contrast to EPA, icosabutate resisted oxidation and incorporation into hepatocytes. Icosabutate also reduced inflammation and fibrosis in conjunction with a reversal of CD diet-induced changes in the hepatic lipidome. EPA had minimal effect on any parameter and even worsened fibrosis in association with depletion of hepatic glutathione. In dyslipidaemic patients at risk of NASH, icosabutate rapidly normalised elevated plasma ALT, GGT and AST and reduced FIB-4 in patients with elevated ALT and/or AST. Icosabutate does not accumulate in hepatocytes and confers beneficial effects on hepatic oxidative stress, inflammation and fibrosis in mice. In conjunction with reductions in markers of liver injury in hyperlipidaemic patients, these findings suggest that structural engineering of LCn-3FAs offers a novel approach for the treatment of NASH. Long-chain omega-3 fatty acids are involved in multiple pathways regulating hepatic inflammation and fibrosis, but their susceptibility to peroxidation and use as an energy source may limit their clinical efficacy. Herein, we show that a structurally modified omega-3 fatty acid, icosabutate, overcame these challenges and had markedly improved antifibrotic efficacy in a mouse model of non-alcoholic steatohepatitis. A hepatoprotective effect of icosabutate was also observed in patients with elevated circulating lipids, in whom it led to rapid reductions in markers of liver injury. [Display omitted] • The susceptibility of PUFAs to peroxidation may limit their efficacy in NASH. • Icosabutate, a modified PUFA derivative, does not accumulate in liver cells. • Icosabutate, but not eicosapentaenoic acid, reduces inflammation and fibrosis in mice. • Markers of liver injury are reduced after icosabutate treatment in humans. • Activation of FFAR4 (GPR120) may underlie the beneficial effects of icosabutate. [ABSTRACT FROM AUTHOR]

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]

Abstract: The substitution of fish oil with plant-derived oil in diets for carnivorous fish, such as Atlantic salmon, has previously revealed the potentially deleterious supranuclear accumulation of lipid droplets in intestinal cells (enterocytes) which may compromise gut integrity, and consequently, fish health. This suggests that unfamiliar dietary lipid sources may have a significant impact on intestinal lipid metabolism, however, the mode of lipid resynthesis is largely unknown in teleost fish intestine. The present study aimed at characterising three key lipogenic enzymes involved in the biosynthesis of triacylglycerol (TAG) and phosphatidylcholine (PC) in Atlantic salmon enterocytes: monoacylglycerol acyltransferase (MGAT), diacylglycerol acyltransferase (DGAT), and diacylglycerol cholinephosphotransferase (CPT). Furthermore, to investigate the dietary effect of plant oils on these enzymes, two experimental groups of fish were fed a diet with either capelin (fish oil) or vegetable oil (rapeseed oil:palm oil:linseed oil, 55:30:15 w/w) as the lipid source. The monoacylglycerol (MAG) pathway was highly active in the intestinal mucosa of Atlantic salmon as demonstrated by MGAT activity (7 nmol [1-14C]palmitoyl-CoA incorporated min−1 mg protein−1) and DGAT activity (4 nmol [1-14C]palmitoyl-CoA incorporated min−1 mg protein−1), with MGAT appearing to also provide adequate production of sn-1,2-diacylglycerol for potential utilisation in PC synthesis via CPT activity (0.4 nmol CDP-[14C]choline incorporated min−1 mg protein−1). Both DGAT and CPT specific activity values were comparable to reported mammalian equivalents, although MGAT activity was lower. Nevertheless, MGAT appeared not to be the rate-limiting step in salmon intestinal TAG synthesis. The homology between piscine and mammalian enzymes was established by similar stimulation and inhibition profiles by a variety of tested cofactors and isomeric substrates. The low dietary n-3/n-6 PUFA ratio presented in the vegetable oil diet did not significantly affect the activities of MGAT, DGAT, or CPT under optimised assay conditions, or in vivo intestinal mucosa lipid class composition, when compared to a standard fish oil diet. [Copyright &y& Elsevier]

Skeletal muscle plays an important role in glycaemic control and metabolic homeostasis, making it a tissue of interest with respect to type 2 diabetes mellitus. The aim of the present study was to determine if ligands of Toll-like receptors (TLRs) could have an impact on energy metabolism and myokine expression and secretion in cultured human skeletal muscle cells. The myotubes expressed mRNA for TLRs 1–6. TLR3, TLR4, TLR5 and TLR6 ligands (TLRLs) increased glucose metabolism. Furthermore, TLR4L and TLR5L increased oleic acid metabolism. The metabolic effects of TLRLs were not evident until after at least 24 h pre-incubation of the cells and here the metabolic effects were more evident for the metabolism of glucose than oleic acid, with a shift towards effects on oleic acid metabolism after chronic exposure (168 h). However, the stimulatory effect of TLRLs on myokine expression and secretion was detected after only 6 h, where TLR3-6L stimulated secretion of interleukin-6 (IL-6). TLR5L also increased secretion of interleukin-8 (IL-8), while TLR6L also increased secretion of granulocyte–macrophage colony stimulating factor (GM-CSF). Pre-incubation of the myotubes with IL-6 for 24 h increased oleic acid oxidation but had no effect on glucose metabolism. Thus IL-6 did not mimic all the metabolic effects of the TLRLs, implying metabolic effects beyond the actions of this myokine. [ABSTRACT FROM AUTHOR]

To investigate the importance of factors influencing substrate availability for triacylglycerol biosynthesis on lipoprotein metabolism, the effects of two opposite-acting sulphur-substituted fatty acid analogues, tetradecylthioacetic acid and tetradecylthiopropionic acid were studied. Administration of tetradecylthioacetic acid to rats resulted in a reduction of plasma levels of triacylglycerols (44%) and cholesterol (26%). This was accompanied by a reduction in very-low-density lipoprotein (VLDL) triacylglycerols (48 %), VLDL cholesterol (36 %), low-density lipoprotein (LDL) cholesterol (36 %) and high-density lipoprotein (HDL) triacylglycerols (50%), whereas HDL cholesterol levels did not change. Subsequently, the HDL/LDL-cholesterol ratio increased by 40%. The cholesterol-lowering effect was accompanied by a reduction in hydroxymethylglutaryl CoA (HMG-CoA) reductase activity (37%). Both mitochondrial and peroxisomal fatty acid oxidation increased (1.7-fold and 5.3-fold, respectively). Furthermore, there was a significant negative correlation between plasma triacylglycerols and mitochondrial fatty acid oxidation. Hepatic triacylglycerol synthesis was retarded, as indicated by a decrease in VLDL triacylglycerol secretion (40%.), and by a reduced liver triacylglycerol content (29%). The activities of lipoprotein lipase and hepatic lipase m post-heparin plasma were not affected. Microsomal and cytosolic phosphatidate phosphohydrolase activities were inhibited (28% and 70%, respectively). Hepatic malonyl-CoA levels decreased by 29% and the total activity of acetyl-CoA carboxylase was reduced (23%). In hepatocytes treated with tetradecylthioacetic acid, mitochondrial fatty acid oxidation increased markedly (100%) and triacylglycerol secretion was reduced (40%). In tetradecylthiopropionic-acid-treated rats, a significant increase in both plasma and VLDL triacylglycerols was found (46% and 72 %, respectively) but VLDL triacylglycerol secretion was unaffected. However, no effect on either plasma or lipoprotein cholesterol levels was seen. Mitochondrial fatty acid oxidation was decreased by 50% and hepatic triacylglycerol levels increased by 33 %. In hepatocytes exposed to tetradecylthiopropionic acid, triacylglycerol synthesis increased (100%) while triacylglycerol secretion and fatty acid oxidation remained unaltered. The results illustrate that lipoprotein triacylglycerol levels can be modulated by changes in the availability of fatty acid substrate for triacylglycerol biosynthesis, mainly by affecting mitochondrial fatty acid oxidation. In addition, we demonstrate that suppression, of rat hepatic HMG-CoA reductase activity during treatment with tetradecylthioacetic acid may contribute to a cholesterol-lowering effect. [ABSTRACT FROM AUTHOR]

Contraction-induced adaptations in skeletal muscles are well characterized in vivo, but the underlying cellular mechanisms are still not completely understood. Cultured human myotubes represent an essential model system for human skeletal muscle that can be modulated ex vivo, but they are quiescent and do not contract unless being stimulated. Stimulation can be achieved by innervation of human myotubes in vitro by co-culturing with embryonic rat spinal cord, or by replacing motor neuron activation by electrical pulse stimulation (EPS). Effects of these two in vitro approaches, innervation and EPS, were characterized with respects to the expression of myosin heavy chains (MyHCs) and metabolism of glucose and oleic acid in cultured human myotubes. Adherent human myotubes were either innervated with rat spinal cord segments or exposed to EPS. The expression pattern of MyHCs was assessed by quantitative polymerase chain reaction, immunoblotting, and immunofluorescence, while the metabolism of glucose and oleic acid were studied using radiolabelled substrates. Innervation and EPS promoted differentiation towards different fiber types in human myotubes. Expression of the slow MyHC-1 isoform was reduced in innervated myotubes, whereas it remained unaltered in EPS-treated cells. Expression of both fast isoforms (MyHC-2A and MyHC-2X) tended to decrease in EPS-treated cells. Both approaches induced a more oxidative phenotype, reflected in increased CO2 production from both glucose and oleic acid. Novelty: Innervation and EPS favour differentiation into different fiber types in human myotubes. Both innervation and EPS promote a metabolically more oxidative phenotype in human myotubes. [ABSTRACT FROM AUTHOR]

Denervation reduces the abundance of Na+,K+-ATPase (NKA) in skeletal muscle, while reinnervation increases it. Primary human skeletal muscle cells, the most widely used model to study human skeletal muscle in vitro, are usually cultured as myoblasts or myotubes without neurons and typically do not contract spontaneously, which might affect their ability to express and regulate NKA. We determined how differentiation, de novo innervation, and electrical pulse stimulation affect expression of NKA (α and β) subunits and NKA regulators FXYD1 (phospholemman) and FXYD5 (dysadherin). Differentiation of myoblasts into myotubes under low serum conditions increased expression of myogenic markers CD56 (NCAM1), desmin, myosin heavy chains, dihydropyridine receptor subunit α1S, and SERCA2 as well as NKAα2 and FXYD1, while it decreased expression of FXYD5 mRNA. Myotubes, which were innervated de novo by motor neurons in co-culture with the embryonic rat spinal cord explants, started to contract spontaneously within 7–10 days. A short-term co-culture (10–11 days) promoted mRNA expression of myokines, such as IL-6, IL-7, IL-8, and IL-15, but did not affect mRNA expression of NKA, FXYDs, or myokines, such as musclin, cathepsin B, meteorin-like protein, or SPARC. A long-term co-culture (21 days) increased the protein abundance of NKAα1, NKAα2, FXYD1, and phospho-FXYD1Ser68 without attendant changes in mRNA levels. Suppression of neuromuscular transmission with α-bungarotoxin or tubocurarine for 24 h did not alter NKA or FXYD mRNA expression. Electrical pulse stimulation (48 h) of non-innervated myotubes promoted mRNA expression of NKAβ2, NKAβ3, FXYD1, and FXYD5. In conclusion, low serum concentration promotes NKAα2 and FXYD1 expression, while de novo innervation is not essential for upregulation of NKAα2 and FXYD1 mRNA in cultured myotubes. Finally, although innervation and EPS both stimulate contractions of myotubes, they exert distinct effects on the expression of NKA and FXYDs. [ABSTRACT FROM AUTHOR]

A fatty acid analogue, 2-(tridec-12-yn-1-ylthio)acetic acid (1-triple TTA), was previously shown to have hypolipidemic effects in rats by targeting mitochondrial activity predominantly in liver. This study aimed to determine if 1-triple TTA could influence carbohydrate metabolism. Male Wistar rats were treated for three weeks with oral supplementation of 100 mg/kg body weight 1-triple TTA. Blood glucose and insulin levels, and liver carbohydrate metabolism gene expression and enzyme activities were determined. In addition, human myotubes and Huh7 liver cells were treated with 1-triple TTA, and glucose and fatty acid oxidation were determined. The level of plasma insulin was significantly reduced in 1-triple TTA-treated rats, resulting in a 32% reduction in the insulin/glucose ratio. The hepatic glucose and glycogen levels were lowered by 22% and 49%, respectively, compared to control. This was accompanied by lower hepatic gene expression of phosphenolpyruvate carboxykinase, the rate-limiting enzyme in gluconeogenesis, and Hnf4A, a regulator of gluconeogenesis. Gene expression of pyruvate kinase, catalysing the final step of glycolysis, was also reduced by 1-triple TTA. In addition, pyruvate dehydrogenase activity was reduced, accompanied by 10-15-fold increased gene expression of its regulator pyruvate dehydrogenase kinase 4 compared to control, suggesting reduced entry of pyruvate into the TCA cycle. Indeed, the NADPH-generating enzyme malic enzyme 1 (ME1) catalysing production of pyruvate from malate, was increased 13-fold at the gene expression level. Despite the decreased glycogen level, genes involved in glycogen synthesis were not affected in livers of 1-triple TTA treated rats. In contrast, the pentose phosphate pathway seemed to be increased as the hepatic gene expression of glucose-6-phosphate dehydrogenase (G6PD) was higher in 1-triple TTA treated rats compared to controls. In human Huh7 liver cells, but not in myotubes, 1-triple-TTA reduced glucose oxidation and induced fatty acid oxidation, in line with previous observations of increased hepatic mitochondrial palmitoyl-CoA oxidation in rats. Importantly, this work recognizes the liver as an important organ in glucose homeostasis. The mitochondrially targeted fatty acid analogue 1-triple TTA seemed to lower hepatic glucose and glycogen levels by inhibition of gluconeogenesis. This was also linked to a reduction in glucose oxidation accompanied by reduced PHD activity and stimulation of ME1 and G6PD, favouring a shift from glucose- to fatty acid oxidation. The reduced plasma insulin/glucose ratio indicate that 1-triple TTA may improve glucose tolerance in rats. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

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. • TRPA1, TRPM8 and TRPV1 are expressed in rat muscle and human primary myotubes. • Activation of TRPA1 by ligustilide increased Ca2+ levels and glucose metabolism. • Activation of TRPM8 by icilin increased Ca2+ levels but decreased glucose metabolism. • Expression of thermogenic related genes were downregulated after TRPA1 activation. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Thio-ether fatty acids (THEFAs), including the parent 2-(tetradecylthio)acetic acid (TTA), are modified fatty acids (FAs) that have profound effects on lipid metabolism given that they are blocked for β-oxidation, and able to act as peroxisome proliferator-activated receptor (PPAR) agonists. Therefore, TTA in particular has been tested clinically for its therapeutic potential against metabolic syndrome related disorders. Here, we describe the preparation of THEFAs based on the TTA scaffold with either a double or a triple bond. These are tested in cultured human skeletal muscle cells (myotubes), either as free acid or following esterification as phospholipids, lysophospholipids or monoacylglycerols. Metabolic effects are assessed in terms of cellular bioavailabilities in myotubes, by FA substrate uptake and oxidation studies, and gene regulation studies with selected PPAR-regulated genes. We note that the inclusion of a triple bond promotes THEFA-mediated FA oxidation. Furthermore, esterification of THEFAs as lysophospholipids also promotes FA oxidation effects. Given that the apparent clinical benefits of TTA administration were offset by dose limitation and poor bioavailability, we discuss the possibility that a selection of our latest THEFAs and THEFA-containing lipids might be able to fulfill the therapeutic potential of the parent TTA while minimizing required doses for efficacy, side-effects and adverse reactions. [ABSTRACT FROM AUTHOR]

Glycosylated lysosomal membrane protein (GLMP) has been reported to enhance the expression from a peroxisome proliferator-activated receptor alpha (PPARα) responsive promoter, but also to be an integral lysosomal membrane protein. Using myotubes established from wild-type andGlmpgt/gtmice, the importance of GLMP in skeletal muscle was examined.Glmpgt/gtmyotubes expressed a more glycolytic phenotype than wild-type myotubes. Myotubes fromGlmpgt/gtmice metabolized glucose faster and had a larger pool of intracellular glycogen, while oleic acid uptake, storage and oxidation were significantly reduced. Gene expression analyses indicated lower expression of three PPAR-isoforms, a co-regulator of PPAR (PGC1α) and several genes important for lipid metabolism inGlmpgt/gtmyotubes. However, ablation of GLMP did not seem to substantially impair the response to PPAR agonists. In conclusion, myotubes established fromGlmpgt/gtmice were more glycolytic than myotubes from wild-type animals, in spite of no differences in muscle fiber typesin vivo. [ABSTRACT FROM PUBLISHER]

Circulating natriuretic peptide (NP) levels are reduced in obesity and predict the risk of type 2 diabetes (T2D). Since skeletal muscle was recently shown as a key target tissue of NP, we aimed to investigate muscle NP receptor (NPR) expression in the context of obesity and T2D. Muscle NPRA correlated positively with whole-body insulin sensitivity in humans and was strikingly downregulated in obese subjects and recovered in response to diet-induced weight loss. In addition, muscle NP clearance receptor (NPRC) increased in individuals with impaired glucose tolerance and T2D. Similar results were found in obese diabetic mice. Although no acute effect of brain NP (BNP) on insulin sensitivity was observed in lean mice, chronic BNP infusion improved blood glucose control and insulin sensitivity in skeletal muscle of obese and diabetic mice. This occurred in parallel with a reduced lipotoxic pressure in skeletal muscle due to an upregulation of lipid oxidative capacity. In addition, chronic NP treatment in human primary myotubes increased lipid oxidation in a PGC1α-dependent manner and reduced palmitate-induced lipotoxicity. Collectively, our data show that activation of NPRA signaling in skeletal muscle is important for the maintenance of long-term insulin sensitivity and has the potential to treat obesity-related metabolic disorders. [ABSTRACT FROM AUTHOR]

A decrease in skeletal muscle lipolysis and hormone sensitive-lipase (HSL) expression has been linked to insulin resistance in obesity. The purpose of this study was to identify potential intrinsic defects in lipid turnover and lipolysis in myotubes established from obese and type 2 diabetic subjects. Lipid trafficking and lipolysis were measured by pulse-chase assay with radiolabeled substrates in myotubes from non-obese/non-diabetic (lean), obese/non-diabetic (obese) and obese/diabetic (T2D) subjects. Lipolytic protein content and level of Akt phosphorylation were measured by Western blot. HSL was overexpressed by adenovirus-mediated gene delivery. Myotubes established from obese and T2D subjects had lower lipolysis (−30–40%) when compared to lean, using oleic acid as precursor. Similar observations were also seen for labelled glycerol. Incorporation of oleic acid into diacylglycerol (DAG) and free fatty acid (FFA) level was lower in T2D myotubes, and acetate incorporation into FFA and complex lipids was also lower in obese and/or T2D subjects. Both protein expression of HSL (but not ATGL) and changes in DAG during lipolysis were markedly lower in cells from obese and T2D when compared to lean subjects. Insulin-stimulated glycogen synthesis (−60%) and Akt phosphorylation (−90%) were lower in myotubes from T2D, however, overexpression of HSL in T2D myotubes did not rescue the diabetic phenotype. In conclusion, intrinsic defects in lipolysis and HSL expression co-exist with reduced insulin action in myotubes from obese T2D subjects. Despite reductions in intramyocellular lipolysis and HSL expression, overexpression of HSL did not rescue defects in insulin action in skeletal myotubes from obese T2D subjects. [ABSTRACT FROM AUTHOR]

Ablation of glycosylated lysosomal membrane protein (GLMP, formerly known as NCU-G1) has been shown to cause chronic liver injury which progresses into liver fibrosis in mice. Both lysosomal dysfunction and chronic liver injury can cause metabolic dysregulation. Glmpgt/gt mice (formerly known as Ncu-g1gt/gtmice) were studied between 3 weeks and 9 months of age. Body weight gain and feed efficiency of Glmpgt/gt mice were comparable to wild type siblings, only at the age of 9 months the Glmpgt/gt siblings had significantly reduced body weight. Reduced size of epididymal fat pads was accompanied by hepatosplenomegaly in Glmpgt/gt mice. Blood analysis revealed reduced levels of blood glucose, circulating triacylglycerol and non-esterified fatty acids in Glmpgt/gt mice. Increased flux of glucose, increased de novo lipogenesis and lipid accumulation were detected in Glmpgt/gt primary hepatocytes, as well as elevated triacylglycerol levels in Glmpgt/gt liver homogenates, compared to hepatocytes and liver from wild type mice. Gene expression analysis showed an increased expression of genes involved in fatty acid uptake and lipogenesis in Glmpgt/gt liver compared to wild type. Our findings are in agreement with the metabolic alterations observed in other mouse models lacking lysosomal proteins, and with alterations characteristic for advanced chronic liver injury. [ABSTRACT FROM AUTHOR]

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/m²) 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. [ABSTRACT FROM AUTHOR]

About 80% of patients with type 2 diabetes are classified as overweight. However, only about 1/3 of severely obese subjects have type 2 diabetes. This indicates that several severely obese individuals may possess certain characteristics that protect them against type 2 diabetes. We therefore hypothesized that this apparent paradox could be related to fundamental differences in skeletal muscle lipid handling. Energy metabolism and metabolic flexibility were examined in human myotubes derived from severely obese subjects without (BMI 44±7 kg/m2) and with type 2 diabetes (BMI 43±6 kg/m2). Lower insulin sensitivity was observed in myotubes from severely obese subjects with type 2 diabetes. Lipolysis rate was higher, and oleic acid accumulation, triacylglycerol content, and fatty acid adaptability were lower in myotubes from severely obese subjects with type 2 diabetes compared to severely obese non-diabetic subjects. There were no differences in lipid distribution and mRNA and protein expression of the lipases HSL and ATGL, the lipase cofactor CGI-58, or the lipid droplet proteins PLIN2 and PLIN3. Glucose and oleic acid oxidation were also similar in cells from the two groups. In conclusion, myotubes established from severely obese donors with established type 2 diabetes had lower ability for lipid accumulation and higher lipolysis rate than myotubes from severely obese donors without diabetes. This indicates that a difference in intramyocellular lipid turnover might be fundamental in evolving type 2 diabetes. [ABSTRACT FROM AUTHOR]

Lipid droplet-associated proteins such as perilipin 3 (PLIN3) and coatomer GTPase proteins (GBF1, ARF1, Sec23a, and ARFRP1) are expressed in skeletal muscle but little is known so far as to their regulation of lipolysis. We aimed here to explore the effects of lipolytic stimulation in vitro in primary human myotubes as well as in vivo following an acute exercise bout. In vitro lipolytic stimulation by epinephrine (100 μM) or by a lipolytic cocktail (30 μM palmitate, 4 μM forskolin, and 0.5 μM ionomycin, PFI) resulted in increases in PLIN3 protein content. Coatomer GTPases such as GBF1, ARF1, Sec23a, and ARFRP1 also increased in response to lipolytic stimuli. Furthermore, a long duration endurance exercise bout (20 males; age 24.0±4.5 y; BMI 23.6±1.8 kg/m2) increased PLIN3 protein in human skeletal muscle (p = 0.03) in proportion to ex vivo palmitate oxidation (r = 0.45, p = 0.04) and whole body in vivo fat oxidation (r = 0.52, p = 0.03). Protein content of ARF1 was increased (p = 0.04) while mRNA expression was increased for several other coatomers (GBF1, ARF1, and Sec23a, all p<0.05). These data provide novel observational insight into the possible relationships between lipolysis and PLIN3 along with these coatomoer GTPase proteins in human skeletal muscle. [ABSTRACT FROM AUTHOR]

The role of peroxisome proliferator-activated receptor δ (PPARδ) activation on global gene expression and mitochondrial fuel utilization were investigated in human myotubes. Only 21 genes were up-regulated and 3 genes were down-regulated after activation by the PPARδ agonist GW501516. Pathway analysis showed up-regulated mitochondrial fatty acid oxidation, TCA cycle and cholesterol biosynthesis. GW501516 increased oleic acid oxidation and mitochondrial oxidative capacity by 2-fold. Glucose uptake and oxidation were reduced, but total substrate oxidation was not affected, indicating a fuel switch from glucose to fatty acid. Cholesterol biosynthesis was increased, but lipid biosynthesis and mitochondrial content were not affected. This study confirmed that the principal effect of PPARδ activation was to increase mitochondrial fatty acid oxidative capacity. Our results further suggest that PPARδ activation reduced glucose utilization through a switch in mitochondrial substrate preference by up-regulating pyruvate dehydrogenase kinase isozyme 4 and genes involved in lipid metabolism and fatty acid oxidation. [ABSTRACT FROM AUTHOR]

Satellite cells can be isolated from skeletal muscle biopsies, activated to proliferating myoblasts and differentiated into multinuclear myotubes in culture. These cell cultures represent a model system for intact human skeletal muscle and can be modulated ex vivo. The advantages of this system are that the most relevant genetic background is available for the investigation of human disease (as opposed to rodent cell cultures), the extracellular environment can be precisely controlled and the cells are not immortalized, thereby offering the possibility of studying innate characteristics of the donor. Limitations in differentiation status (fiber type) of the cells and energy metabolism can be improved by proper treatment, such as electrical pulse stimulation to mimic exercise. This review focuses on the way that human myotubes can be employed as a tool for studying metabolism in skeletal muscles, with special attention to changes in muscle energy metabolism in obesity and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Development of insulin resistance is positively associated with dietary saturated fatty acids and negatively associated with monounsaturated fatty acids. To clarify aspects of this difference we have compared the metabolism of oleic (OA, monounsaturated) and palmitic acids (PA, saturated) in human myotubes. Human myotubes were treated with 100μM OA or PA and the metabolism of [14C]-labeled fatty acid was studied. We observed that PA had a lower lipolysis rate than OA, despite a more than two-fold higher protein level of adipose triglyceride lipase after 24h incubation with PA. PA was less incorporated into triacylglycerol and more incorporated into phospholipids after 24h. Supporting this, incubation with compounds modifying lipolysis and reesterification pathways suggested a less influenced PA than OA metabolism. In addition, PA showed a lower accumulation than OA, though PA was oxidized to a relatively higher extent than OA. Gene set enrichment analysis revealed that 24h of PA treatment upregulated lipogenesis and fatty acid β-oxidation and downregulated oxidative phosphorylation compared to OA. The differences in lipid accumulation and lipolysis between OA and PA were eliminated in combination with eicosapentaenoic acid (polyunsaturated fatty acid). In conclusion, this study reveals that the two most abundant fatty acids in our diet are partitioned toward different metabolic pathways in muscle cells, and this may be relevant to understand the link between dietary fat and skeletal muscle insulin resistance. [Copyright &y& Elsevier]

Background and Aims: Physical exercise leads to substantial adaptive responses in skeletal muscles and plays a central role in a healthy life style. Since exercise induces major systemic responses, underlying cellular mechanisms are difficult to study in vivo. It was therefore desirable to develop an in vitro model that would resemble training in cultured human myotubes. Methods: Electrical pulse stimulation (EPS) was applied to adherent human myotubes. Cellular contents of ATP, phosphocreatine (PCr) and lactate were determined. Glucose and oleic acid metabolism were studied using radio-labeled substrates, and gene expression was analyzed using real-time RT-PCR. Mitochondrial content and function were measured by live imaging and determination of citrate synthase activity, respectively. Protein expression was assessed by electrophoresis and immunoblotting. Results: High-frequency, acute EPS increased deoxyglucose uptake and lactate production, while cell contents of both ATP and PCr decreased. Chronic, low-frequency EPS increased oxidative capacity of cultured myotubes by increasing glucose metabolism (uptake and oxidation) and complete fatty acid oxidation. mRNA expression level of pyruvate dehydrogenase complex 4 (PDK4) was significantly increased in EPS-treated cells, while mRNA expressions of interleukin 6 (IL-6), cytochrome C and carnitin palmitoyl transferase b (CPT1b) also tended to increase. Intensity of MitoTrackerHRed FM was doubled after 48 h of chronic, low-frequency EPS. Protein expression of a slow fiber type marker (MHCI) was increased in EPS-treated cells. Conclusions: Our results imply that in vitro EPS (acute, high-frequent as well as chronic, low-frequent) of human myotubes may be used to study effects of exercise. [ABSTRACT FROM AUTHOR]

Abstract: (±)-2-Fluoro-2-(2-methyl-4-(((4-methyl-2-(4-(trifluoromethyl)phenyl)thiazol-5-yl)methyl)thio)phenoxy)acetic acid (2a) has been prepared and subjected to biological testing against all three subtypes of the PPARs. This compound exhibited agonist effects with EC50 values of 560 and 55nM against PPARα and PPARδ, respectively, in a luciferase assay. Moreover, compound (±)-2a also exhibited potent ability to induce oleic acid oxidation in a human myotube cell assay with EC50 =3.7nM. Compound (±)-2a can be classified as a dual PPARα/δ agonist with a 10-fold higher potency against the PPARδ receptor than against the PPARα receptor. Molecular modeling studies revealed that both enantiomers of 2a bind to the PPARδ receptor with similar binding energies. [Copyright &y& Elsevier]

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. [ABSTRACT FROM AUTHOR]

Objective: Disturbances in lipid metabolism are strongly associated with insulin resistance and type 2 diabetes (T2D). We hypothesized that activation of cAMP/PKA and calcium signaling pathways in cultured human myotubes would provide further insight into regulation of lipid storage, lipolysis, lipid oxidation and insulin responsiveness. Methods: Human myoblasts were isolated from vastus lateralis, purified, cultured and differentiated into myotubes. All cells were incubated with palmitate during differentiation. Treatment cells were pulsed 1 hour each day with forskolin and ionomycin (PFI) during the final 3 days of differentiation to activate the cAMP/PKA and calcium signaling pathways. Control cells were not pulsed (control). Mitochondrial content, 14C lipid oxidation and storage were measured, as well as lipolysis and insulin-stimulated glycogen storage. Myotubes were stained for lipids and gene expression measured. Results: PFI increased oxidation of oleate and palmitate to CO2 (p,0.001), isoproterenol-stimulated lipolysis (p = 0.01), triacylglycerol (TAG) storage (p,0.05) and mitochondrial DNA copy number (p = 0.01) and related enzyme activities. Candidate gene and microarray analysis revealed increased expression of genes involved in lipolysis, TAG synthesis and mitochondrial biogenesis. PFI increased the organization of lipid droplets along the myofibrillar apparatus. These changes in lipid metabolism were associated with an increase in insulin-mediated glycogen storage (p,0.001). Conclusions: Activation of cAMP/PKA and calcium signaling pathways in myotubes induces a remodeling of lipid droplets and functional changes in lipid metabolism. These results provide a novel pharmacological approach to promote lipid metabolism and improve insulin responsiveness in myotubes, which may be of therapeutic importance for obesity and type 2 diabetes. [ABSTRACT FROM AUTHOR]

OBJECTIVE--Insulin resistance is associated with elevated content of skeletal muscle lipids, including triacylglycerols (TAGs) and diacylglycerols (DAGs). DAGs are by-products of lipolysis consecutive to TAG hydrolysis by adipose triglyceride lipase (ATGL) and are subsequently hydrolyzed by hormone-sensitive lipase (HSL). We hypothesized that an imbalance of ATGL relative to HSL (expression or activity) may contribute to DAG accumulation and insulin resistance. RESEARCH DESIGN AND METHODS--We first measured lipase expression in vastus lateralis biopsies of young lean (n = 9), young obese (n = 9), and obese-matched type 2 diabetic (n = 8) subjects. We next investigated in vitro in human primary myotubes the impact of altered lipase expression/activity on lipid content and insulin signaling. RESULTS--Muscle ATGL protein was negatively associated with whole-body insulin sensitivity in our population (r = -0.55, P = 0.005), whereas muscle HSL protein was reduced in obese subjects. We next showed that adenovirus-mediated ATGL overexpression in human primary myotubes induced DAG and ceramide accumulation. ATGL overexpression reduced insulin-stimulated glycogen synthesis (-30%, P < 0.05) and disrupted insulin signaling at Ser1101 of the insulin receptor substrate-1 and downstream Akt activation at Ser473. These defects were fully rescued by nonselective protein kinase C inhibition or concomitant HSL overexpression to restore a proper lipolytic balance. We show that selective HSL inhibition induces DAG accumulation and insulin resistance. CONCLUSIONS--Altogether, the data indicate that altered ATGL and HSL expression in skeletal muscle could promote DAG accumulation and disrupt insulin signaling and action. Targeting skeletal muscle lipases may constitute an interesting strategy to improve insulin sensitivity in obesity and type 2 diabetes. [ABSTRACT FROM AUTHOR]

Abstract: Skeletal muscle of insulin resistant individuals is characterized by lower fasting lipid oxidation and reduced ability to switch between lipid and glucose oxidation. The purpose of the present study was to examine if chronic hyperglycemia would impair metabolic switching of myotubes. Human myotubes were treated with or without chronic hyperglycemia (20mmol/l glucose for 4days), and metabolism of [14C]oleic acid (OA) and [14C]glucose was studied. Myotubes exposed to chronic hyperglycemia showed a significantly reduced OA uptake and oxidation to CO2, whereas acid-soluble metabolites were increased compared to normoglycemic cells (5.5mmol/l glucose). Glucose suppressibility, the ability of acute glucose (5mmol/l) to suppress lipid oxidation, was 50% in normoglycemic cells and reduced to 21% by hyperglycemia. Adaptability, the capacity to increase lipid oxidation with increasing fatty acid availability, was not affected by hyperglycemia. Glucose uptake and oxidation were reduced by about 40% after hyperglycemia, and oxidation of glucose in presence of mitochondrial uncouplers showed that net and maximal oxidative capacities were significantly reduced. Hyperglycemia also abolished insulin-stimulated glucose uptake. Moreover, ATP concentration was reduced by 25% after hyperglycemia. However, none of the measured mitochondrial genes were downregulated nor was mitochondrial DNA content. Microarray and real-time RT-PCR showed that no genes were significantly regulated by chronic hyperglycemia. Addition of chronic lactate reduced both glucose and OA oxidation to the same extent as hyperglycemia. In conclusion, chronic hyperglycemia reduced substrate oxidation in skeletal muscle cells and impaired metabolic switching. The effect is most likely due to an induced mitochondrial dysfunction. [ABSTRACT FROM AUTHOR]

Obesity and insulin resistance are related to both enlarged intramyocellular triacylglycerol stores and accumulation of lipid intermediates. We investigated how lipid overflow can change the oxidation of intramyocellular lipids (ICLOX) and intramyocellular lipid storage (ICL). These experiments were extended by comparing these processes in primary cultured myotubes established from healthy lean and obese type 2 diabetic (T2D) individuals, two extremes in a range of metabolic phenotypes. ICLs were prelabeled for 2 days with 100 µM [14C]oleic acid (OA). ICLOX was studied using a 14CO2 trapping system and measured under various conditions of extracellular OA (5 or 100 µM) and glucose (0.1 or 5.0 mM) and the absence or presence of mitochondrial uncoupling [carbony1 cyanide p-trifluoromethoxyphenylhydrazone (FCCP)]. First, increased extracellular OA availability (5 vs. 100 µM) reduced ICLOX by 37%. No differences in total lipolysis were observed between low and high OA availability. Uncoupling with FCCP restored ICLOX to basal levels during high OA availability. Mitochondrial mass was positively related to ICLOX, but only in myotubes from lean individuals. In all, a lower mitochondrial mass and lower ICLOX were related to a higher cell-associated OA accumulation. Second, myotubes established from obese T2D individuals showed reduced ICLOX. ICLOX remained lower during uncoupling (P < 0.001), even with comparable mitochondrial mass, suggesting decreased mitochondrial function. Furthermore, the variation in ICLOX in vitro was significantly related to the in vivo fasting respiratory quotient of all subjects (P < 0.02). In conclusion, the rate of ICLOX is dependent on the availability of extracellular fatty acids and mitochondrial function rather than mitochondrial mass. [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]

Abstract: The nuclear liver X receptors (LXRα and β) 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 LXRα, 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. [Copyright &y& Elsevier]

Liver X receptors (LXRs) are important regulators of cholesterol and lipid metabolism and are also involved in glucose metabolism. However, the functional role of LXRs in human skeletal muscle is at present unknown. This study demonstrates that chronic ligand activation of LXRs by a synthetic LXR agonist increases the uptake, distribution into complex cellular lipids, and oxidation of palmitate as well as the uptake and oxidation of glucose in cultured human skeletal muscle cells. Furthermore, the effect of the LXR agonist was additive to acute effects of insulin on palmitate uptake and metabolism. Consistently, activation of LXRs induced the expression of relevant genes: fatty acid translocase (CD36/FAT), glucose transporters (GLUT1 and -4), sterol regulatory element-binding protein-1c, peroxisome proliferator-activated receptor-gamma, carnitine palmitoyltransferase-1, and uncoupling protein 2 and 3. Interestingly, in response to activation of LXRs, myotubes from patients with type 2 diabetes showed an elevated uptake and incorporation of palmitate into complex lipids but an absence of palmitate oxidation to CO(2). These results provide evidence for a functional role of LXRs in both lipid and glucose metabolism and energy uncoupling in human myotubes. Furthermore, these data suggest that increased intramyocellular lipid content in type 2 diabetic patients may involve an altered response to activation of components in the LXR pathway. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]