Search

The interplay between skeletal muscle and bone is primarily mechanical; however, biochemical crosstalk by secreted mediators has recently gained increased attention. The aim of this study was to investigate metabolic effects of conditioned medium from osteoblasts (OB-CM) on myotubes and vice versa. Human skeletal muscle cells incubated with OB-CM showed increased glucose uptake and oxidation, and mRNA expression of the glucose transporter (GLUT) 1, while fatty acid uptake and oxidation, and mRNA expression of the fatty acid transporter CD36 were decreased. This was supported by proteomic analysis, where expression of proteins involved in glucose uptake, glycolytic pathways, and the TCA cycle were enhanced, and expression of several proteins involved in fatty acid metabolism were reduced. Similar effects on energy metabolism were observed in human bone marrow stromal cells differentiated to osteoblastic cells incubated with conditioned medium from myotubes (SKM-CM), with increased glucose uptake and reduced oleic acid uptake. Proteomic analyses of the two conditioned media revealed many common proteins. Thus, our data may indicate a shift in fuel preference from fatty acid to glucose metabolism in both cell types, induced by conditioned media from the opposite cell type, possibly indicating a more general pattern in communication between these tissues. [ABSTRACT FROM AUTHOR]

Obesity and physical inactivity have a profound impact on skeletal muscle metabolism. In the present work, we have investigated differences in protein expression and energy metabolism in primary human skeletal muscle cells established from lean donors (BMI<25 kg/m2) and individuals with obesity (BMI>30 kg/m2). Furthermore, we have studied the effect of fatty acid pretreatment on energy metabolism in myotubes from these donor groups. Alterations in protein expression were investigated using proteomic analysis, and energy metabolism was studied using radiolabeled substrates. Gene Ontology enrichment analysis showed that glycolytic, apoptotic, and hypoxia pathways were upregulated, whereas the pentose phosphate pathway was downregulated in myotubes from donors with obesity compared to myotubes from lean donors. Moreover, fatty acid, glucose, and amino acid uptake were increased in myotubes from individuals with obesity. However, fatty acid oxidation was reduced, glucose oxidation was increased in myotubes from subjects with obesity compared to cells from lean. Pretreatment of myotubes with palmitic acid (PA) or eicosapentaenoic acid (EPA) for 24 h increased glucose oxidation and oleic acid uptake. EPA pretreatment increased the glucose and fatty acid uptake and reduced leucine fractional oxidation in myotubes from donors with obesity. In conclusion, these results suggest that myotubes from individuals with obesity showed increased fatty acid, glucose, and amino acid uptake compared to cells from lean donors. Furthermore, myotubes from individuals with obesity had reduced fatty acid oxidative capacity, increased glucose oxidation, and a higher glycolytic reserve capacity compared to cells from lean donors. Fatty acid pretreatment enhances glucose metabolism, and EPA reduces oleic acid and leucine fractional oxidation in myotubes from donor with obesity, suggesting increased metabolic flexibility after EPA treatment. [ABSTRACT FROM AUTHOR]

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

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]

Comparing energy metabolism in human skeletal muscle and primary skeletal muscle cells in obesity, while focusing on glucose and fatty acid metabolism, shows many common changes. Insulin‐mediated glucose uptake in skeletal muscle and primary myotubes is decreased by obesity, whereas differences in basal glucose metabolism are inconsistent among studies. With respect to fatty acid metabolism, there is an increased uptake and storage of fatty acids and a reduced complete lipolysis, suggesting alterations in lipid turnover. In addition, fatty acid oxidation is decreased, probably at the level of complete oxidation, as β‐oxidation may be enhanced in obesity, which indicates mitochondrial dysfunction. Metabolic changes in skeletal muscle with obesity promote metabolic inflexibility, ectopic lipid accumulation, and formation of toxic lipid intermediates. Skeletal muscle also acts as an endocrine organ, secreting myokines that participate in interorgan cross talk. This review highlights interventions and some possible targets for treatment through action on skeletal muscle energy metabolism. Effects of exercise in vivo on obesity have been compared with simulation of endurance exercise in vitro on myotubes (electrical pulse stimulation). Possible pharmaceutical targets, including signaling pathways and drug candidates that could modify lipid storage and turnover or increase mitochondrial function or cellular energy expenditure through adaptive thermogenic mechanisms, are discussed. [ABSTRACT FROM AUTHOR]

Copyright of Applied Physiology, Nutrition & Metabolism is the property of Canadian Science Publishing and its content may not be copied or emailed to multiple sites or posted to a listserv without the copyright holder's express written permission. However, users may print, download, or email articles for individual use. This abstract may be abridged. No warranty is given about the accuracy of the copy. Users should refer to the original published version of the material for the full abstract. (Copyright applies to all Abstracts.)

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]

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]

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]

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]

Non-shivering thermogenesis (NST) mediated by uncoupling protein 1 (UCP1) in brown adipose tissue (BAT) can be activated via the adrenergic system in response to cold or diet, contributing to both thermal and energy homeostasis. Other mechanisms, including metabolism of skeletal muscle, may also be involved in NST. However, relative contribution of these energy dissipating pathways and their adaptability remain a matter of long-standing controversy. We used warm-acclimated (30 °C) mice to characterize the effect of an up to 7-day cold acclimation (6 °C; CA) on thermoregulatory thermogenesis, comparing inbred mice with a genetic background conferring resistance (A/J) or susceptibility (C57BL/6 J) to obesity. Both warm-acclimated C57BL/6 J and A/J mice exhibited similar cold endurance, assessed as a capability to maintain core body temperature during acute exposure to cold, which improved in response to CA, resulting in comparable cold endurance and similar induction of UCP1 protein in BAT of mice of both genotypes. Despite this, adrenergic NST in BAT was induced only in C57BL/6 J, not in A/J mice subjected to CA. Cold tolerance phenotype of A/J mice subjected to CA was not based on increased shivering, improved insulation, or changes in physical activity. On the contrary, lipidomic, proteomic and gene expression analyses along with palmitoyl carnitine oxidation and cytochrome c oxidase activity revealed induction of lipid oxidation exclusively in skeletal muscle of A/J mice subjected to CA. These changes appear to be related to skeletal muscle NST, mediated by sarcolipin-induced uncoupling of sarco(endo)plasmic reticulum calcium ATPase pump activity and accentuated by changes in mitochondrial respiratory chain supercomplexes assembly. Our results suggest that NST in skeletal muscle could be adaptively augmented in the face of insufficient adrenergic NST in BAT, depending on the genetic background of the mice. It may provide both protection from cold and resistance to obesity, more effectively than BAT. [Display omitted] • Cold acclimation led to cold resistance and similar UCP1 level in B6 and A/J mice. • Adrenergic non-shivering thermogenesis (NST) in BAT was induced only in B6 mice. • Shivering did not compensate for the lack of induction of BAT NST in A/J mice. • Adaptive NST in skeletal muscle replaced defective BAT activation in A/J mice. • Muscle NST could contribute to obesity resistance of A/J mice. [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]

Objective: The purpose of the study was to determine the effects of passaging on retention of donor phenotypic characteristics in primary human myotubes.Methods: Primary muscle cultures and serial passaged myotubes from physically active, sedentary lean, and individuals with type 2 diabetes were established. Maximal ATP synthesis capacity (ATPmax) and resting ATP flux (ATPase) in vivo were measured by (31) P magnetic resonance spectroscopy, type-I fibers and intramyocelluar lipid (IMCL) in vastus lateralis tissue were determined using immunohistochemistry techniques, and oxidative phosphorylation complexes (OXPHOS) were measured by Western immunoblotting. Similar in vitro measures for lipid and type-I fibers were made in myotubes, along with mitochondrial content measured by MitoTracker.Results: Passage 4 and 5 measures for myotubes correlated positively with in vivo measurements for percent type-I fibers (P4: R(2)  = 0.39, p = 0.02; P5: R(2)  = 0.48, p = 0.01), ATPmax (P4: R(2)  = 0.30, p = 0.03; P5: R(2)  = 0.22, p = 0.05), and OXPHOS (P4: R(2)  = 0.44, p = 0.04; P5: R(2)  = 0.59, p = 0.006). No correlations were observed for IMCL. However, passage 4 measures for myotubes correlated with passage 5 measures for percent type-I fibers (R(2)  = 0.49, p = 0.01), IMCL (R(2)  = 0.80, p < 0.001), and mitochondrial content (R(2)  = 0.26, p = 0.03).Conclusions: Myotubes through the first two passages following immunopurification (referred to as passage 4 and 5) reflect the mitochondrial and type-I fiber content in vivo phenotype of the donor. [ABSTRACT FROM AUTHOR]

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]

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]

Objective This study investigated the relationship between in vitro lipid content in myotubes and in vivo whole body phenotypes of the donors such as insulin sensitivity, intramyocellular lipids (IMCL), physical activity, and oxidative capacity. Design and Methods Six physically active donors were compared to six sedentary lean and six T2DM. Lipid content was measured in tissues and myotubes by immunohistochemistry. Ceramides, triacylglycerols, and diacylglycerols (DAGs) were measured by LC-MS-MS and GC-FID. Insulin sensitivity was measured by hyperinsulinemic-euglycemic clamp (80 mU min−1 m−2), maximal mitochondrial capacity (ATPmax) by 31P-MRS, physical fitness by VO2max and physical activity level (PAL) by accelerometers. Results Myotubes cultured from physically active donors had higher lipid content (0.047 ± 0.003 vs. 0.032 ± 0.001 and 0.033 ± 0.001AU; P < 0.001) than myotubes from lean and T2DM donors. Lipid content in myotubes was not associated with IMCL in muscle tissue but importantly, correlated with in vivo measures of ATPmax ( r = 0.74; P < 0.001), insulin sensitivity ( r = 0.54; P < 0.05), type-I fibers ( r = 0.50; P < 0.05), and PAL ( r = 0.92; P < 0.0001). DAGs and ceramides in myotubes were inversely associated with insulin sensitivity ( r = −0.55, r = −0.73; P < 0.05) and ATPmax ( r = −0.74, r = −0.85; P < 0.01). Conclusions These results indicate that cultured human myotubes can be used in mechanistic studies to study the in vitro impact of interventions on phenotypes such as mitochondrial capacity, insulin sensitivity, and physical activity. [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]

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]

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]

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]

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]

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]

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]

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]