Remodelling of oxidative energy metabolism by galactose improves glucose handling and metabolic switching in human skeletal muscle cells

Cultured human myotubes have a low mitochondrial oxidative potential. This study aims to remodel energy metabolism in myotubes by replacing glucose with galactose during growth and differentiation to ultimately examine the consequences for fatty acid and glucose metabolism. Exposure to galactose showed an increased [14C]oleic acid oxidation, whereas cellular uptake of oleic acid uptake was unchanged. On the other hand, both cellular uptake and oxidation of [14C]glucose increased in myotubes exposed to galactose. In the presence of the mitochondrial uncoupler carbonylcyanide p-trifluormethoxy-phenylhydrazone (FCCP) the reserve capacity for glucose oxidation was increased in cells grown with galactose. Staining and live imaging of the cells showed that myotubes exposed to galactose had a significant increase in mitochondrial and neutral lipid content. Suppressibility of fatty acid oxidation by acute addition of glucose was increased compared to cells grown in presence of glucose. In summary, we show that cells grown in galactose were more oxidative, had increased oxidative capacity and higher mitochondrial content, and showed an increased glucose handling. Interestingly, cells exposed to galactose showed an increased suppressibility of fatty acid metabolism. Thus, galactose improved glucose metabolism and metabolic switching of myotubes, representing a cell model that may be valuable for metabolic studies related to insulin resistance and disorders involving mitochondrial impairments

Are cultured human myotubes far from home?

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

Electrical Pulse Stimulation of Cultured Human Skeletal Muscle Cells as an In Vitro Model of Exercise

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 MitoTracker®Red 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.This work was funded by the University of Oslo, Oslo University College, the Norwegian Diabetes Foundation, the Freia Chocolade Fabriks Medical Foundation and the Anders Jahre’s Foundation. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Treatment of human skeletal muscle cells with inhibitors of diacylglycerol acyltransferases 1 and 2 to explore isozyme-specific roles on lipid metabolism.

Diacylglycerol acyltransferases (DGAT) 1 and 2 catalyse the final step in triacylglycerol (TAG) synthesis, the esterification of fatty acyl-CoA to diacylglycerol. Despite catalysing the same reaction and being present in the same cell types, they exhibit different functions on lipid metabolism in various tissues. Yet, their roles in skeletal muscle remain poorly defined. In this study, we investigated how selective inhibitors of DGAT1 and DGAT2 affected lipid metabolism in human primary skeletal muscle cells. The results showed that DGAT1 was dominant in human skeletal muscle cells utilizing fatty acids (FAs) derived from various sources, both exogenously supplied FA, de novo synthesised FA, or FA derived from lipolysis, to generate TAG, as well as being involved in de novo synthesis of TAG. On the other hand, DGAT2 seemed to be specialised for de novo synthesis of TAG from glycerol-3-posphate only. Interestingly, DGAT activities were also important for regulating FA oxidation, indicating a key role in balancing FAs between storage in TAG and efficient utilization through oxidation. Finally, we observed that inhibition of DGAT enzymes could potentially alter glucose–FA interactions in skeletal muscle. In summary, treatment with DGAT1 or DGAT2 specific inhibitors resulted in different responses on lipid metabolism in human myotubes, indicating that the two enzymes play distinct roles in TAG metabolism in skeletal muscle

Effect of Phenolic Compounds from Elderflowers on Glucose- and Fatty Acid Uptake in Human Myotubes and HepG2-Cells

Type 2 diabetes (T2D) is manifested by progressive metabolic impairments in tissues such as skeletal muscle and liver, and these tissues become less responsive to insulin, leading to hyperglycemia. In the present study, stimulation of glucose and oleic acid uptake by elderflower extracts, constituents and metabolites were tested in vitro using the HepG2 hepatocellular liver carcinoma cell line and human skeletal muscle cells. Among the crude extracts, the 96% EtOH extract showed the highest increase in glucose and oleic acid uptake in human skeletal muscle cells and HepG2-cells. The flavonoids and phenolic acids contained therein were potent stimulators of glucose and fatty acid uptake in a dose-dependent manner. Most of the phenolic constituents and several of the metabolites showed high antioxidant activity and showed considerably higher α-amylase and α-glucosidase inhibition than acarbose. Elderflower might therefore be valuable as a functional food against diabetes

Novel methods for cold exposure of skeletal muscle in vivo and in vitro show temperature-dependent myokine production

Proteins secreted from skeletal muscle serving a signalling role have been termed myokines. Many of the myokines are exercise factors, produced and released in response to muscle activity. Cold exposures affecting muscle may occur in recreational, occupational and therapeutic settings. Whether muscle temperature independently affects myokine profile, is still to be elucidated. We hypothesized that manipulating muscle temperature by means of external cooling would change myokine production and release. In the present study we have established new models for cold exposure of muscle in vivo and in vitro where rat hind limb or cultured human myotubes were cooled to 18 °C. After a recovery period, muscle tissue, cells and culture media were harvested for further analysis by qPCR and immunoassays. Expression of several myokine genes were significantly increased after cold exposure in both models: in rat muscle, mRNA levels of CCL2 (p = 0.04), VEGFA (p = 0.02), CXCL1 (p = 0.02) and RBM3 (p = 0.02) increased while mRNA levels of IL-6 (p = 0.03) were decreased; in human myotubes, mRNA levels of IL6 (p = 0.01), CXCL8 (p = 0.04), VEGFA (p = 0.03) and CXCL1 (p < 0.01) were significantly increased, as well as intracellular protein levels of IL-8 (CXCL8 gene product; p < 0.01). The corresponding effect on myokine secretion was not observed, on the contrary, IL-8 (p = 0.02) and VEGF (VEGFA gene product) p < 0.01) concentrations in culture media were reduced after cold exposure in vitro. In conclusion, cold exposure of muscle in vivo and in vitro had an effect on the production and release of several known exercise-related myokines. Myokine expression at the level of mRNA and protein was increased by cold exposure, whereas secretion tended to be decreased

Lack of the lysosomal membrane protein, GLMP, in mice results in metabolic dysregulation in liver

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

Enhanced Glucose Uptake in Human Liver Cells and Inhibition of Carbohydrate Hydrolyzing Enzymes by Nordic Berry Extracts

A Western lifestyle with low physical activity and a diet rich in sugar, fat and processed food contribute to higher incidences of diabetes and obesity. Enhanced glucose uptake in human liver cells was observed after treatment with phenolic extracts from different Nordic berries. All berry extracts showed higher inhibition against α-amylase and α-glucosidase than the anti-diabetic agent acarbose. Total phenolic content and phenolic profiles in addition to antioxidant activities, were also investigated. The berries were extracted with 80% methanol on an accelerated solvent extraction system (ASE) and then purified by C-18 solid phase extraction (SPE). Among the ASE methanol extracts, black chokeberry, crowberry and elderberry extracts showed high stimulation of glucose uptake in HepG2 cells and also considerable inhibitory effect towards carbohydrate hydrolyzing enzymes. SPE extracts with higher concentrations of phenolics, resulted in increased glucose uptake and enhanced inhibition of α-amylase and α-glucosidase compared to the ASE extracts. Crowberry and cloudberry were the most potent 15-lipoxygenase inhibitors, while bog whortleberry and lingonberry were the most active xanthine oxidase inhibitors. These results increase the value of these berries as a component of a healthy Nordic diet and have a potential benefit against diabetes

Novel methods for cold exposure of skeletal muscle in vivo and in vitro show temperature-dependent myokine production

Proteins secreted from skeletal muscle serving a signalling role have been termed myokines. Many of the myokines are exercise factors, produced and released in response to muscle activity. Cold exposures affecting muscle may occur in recreational, occupational and therapeutic settings. Whether muscle temperature independently affects myokine profile, is still to be elucidated. We hypothesized that manipulating muscle temperature by means of external cooling would change myokine production and release. In the present study we have established new models for cold exposure of muscle in vivo and in vitro where rat hind limb or cultured human myotubes were cooled to 18 °C. After a recovery period, muscle tissue, cells and culture media were harvested for further analysis by qPCR and immunoassays. Expression of several myokine genes were significantly increased after cold exposure in both models: in rat muscle, mRNA levels of CCL2 (p = 0.04), VEGFA (p = 0.02), CXCL1 (p = 0.02) and RBM3 (p = 0.02) increased while mRNA levels of IL-6 (p = 0.03) were decreased; in human myotubes, mRNA levels of IL6 (p = 0.01), CXCL8 (p = 0.04), VEGFA (p = 0.03) and CXCL1 (p < 0.01) were significantly increased, as well as intracellular protein levels of IL-8 (CXCL8 gene product; p < 0.01). The corresponding effect on myokine secretion was not observed, on the contrary, IL-8 (p = 0.02) and VEGF (VEGFA gene product) p < 0.01) concentrations in culture media were reduced after cold exposure in vitro. In conclusion, cold exposure of muscle in vivo and in vitro had an effect on the production and release of several known exercise-related myokines. Myokine expression at the level of mRNA and protein was increased by cold exposure, whereas secretion tended to be decreased