Metabolic switching of human skeletal muscle cells in vitro

In this review we will focus on external factors that may modify energy metabolism in human skeletal muscle cells (myotubes) and the ability of the myotubes to switch between lipid and glucose oxidation. We describe the metabolic parameters suppressibility, adaptability and substrate-regulated flexibility, and show the influence of nutrients such as fatty acids and glucose (chronic hyperglycemia), and some pharmacological agents modifying nuclear receptors (PPAR and LXR), on these parameters in human myotubes. Possible cellular mechanisms for changes in these parameters will also be highlighted.The present work was funded by University of Oslo, The European Nutrigenomics Organisation (NuGO), The Norwegian Diabetes Foundation, AstraZeneca, Freia Chocolade Fabriks Medical Foundation, and The Anders Jahre’s Foundatio

Overexpression of PGC-1α Increases Fatty Acid Oxidative Capacity of Human Skeletal Muscle Cells

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 fiber-type gene marker (MHCIIa) was decreased. Compared to skeletal muscle in vivo, PGC-1α overexpression increased expression of several genes, which were downregulated 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

Increase in Mitochondrial content after Electrical Pulse Stimulation is dependent on duration of stimulation

Increase in Mitochondrial content after Electrical Pulse Stimulation is dependent on duration of stimulation Daniel Conde B.S.1, Jeffrey D. Covington Ph.D.2, Cecilia Gamboa3 George A. King Ph.D.1, Arild C. Rustan Ph.D.4, Sudip Bajpeyi Ph.D.1. 1 Department of Kinesiology, University of Texas at El Paso, TX; 2Louisiana State University Health Sciences Center, New Orleans, LA; 3Clinical Laboratory Sciences, University of Texas at El Paso; 4Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo, Oslo, Norway. Skeletal Muscle Metabolism Laboratory; Kinesiology; University of Texas at El Paso; El Paso, TX Category: Masters Advisor / Mentor: Bajpeyi, Sudip ([email protected]) ABSTRACT We have previously shown that human skeletal muscle myotubes cultured in vitro, retain in vivo characteristics of the donors. Recent studies indicate that electrical pulse stimulation (EPS) can be used as an exercise mimetic in a cell culture model, and could be beneficial to understand molecular mechanisms underlying exercise training. Purpose: The purpose of this study was to compare acute, moderate and long duration EPS treatments on mitochondrial and lipid content in cultured myotubes. Methods: EPS stimulation was applied to human myotubes cultured from sedentary donors under three conditions: Acute (bipolar pulses of 100 Hz for 200 ms every 5th second; 30V for 60 min) and chronic stimulation (single bipolar pulses of 2 ms; 30V, 1Hz continuously for 24 h or 48 h). Mitochondrial and lipid contents were measured by primary antibody for complex IV and bodipy green dye, respectively, using immunohistochemistry techniques. Fluoroskan ascent microplate reader was used to quantify fluorescence signals. OXPHOS proteins were measured using western immunoblotting. Results: There was no change in lipid or mitochondrial content as assessed by immunohistochemistry after acute EPS stimulation. Chronic stimulation resulted in a significant increase in the mitochondrial content after 24 h (from 0.183 ± 0.02 AU to 0.350 ± 0.03 AU; p=0.008) and 48 h (from 0.290 ± 0.01 AU to 0.337 ± 0.01 AU; p=0.02) of continuous EPS stimulation. OXPHOS proteins increased after 48 h of EPS. There was also a significant increase in lipid content after 48 h of EPS stimulation (from 0.210 ± 0.01 AU to 0.256 ± 0.01 AU; p=0.02). Conclusion: These findings suggest that 48 h of chronic EPS results in an increase in both mitochondrial and lipid contents in human myotubes. The concomitant increase in lipid and mitochondrial content after exercise mimetic EPS stimulation supports the elevated level of intramyocellular lipid and mitochondrial content evident in endurance trained athletes

24 hours of Electrical Pulse Stimulation upregulates GLUT4 and AMPK protein content in human myotubes

Electrical pulse stimulation (EPS), an in vitro exercise mimetic, has been shown to increase mitochondrial and lipid content in cultured human myotubes. We have recently shown that myotubes retain certain in vivo characteristics of the donors. Purpose: We aimed to examine the EPS-induced adaptations in relation to Glucose Transporter Type 4 (GLUT4) and 5’Adenosine Monophosphate-activated Protein Kinase (AMPK) content using human myotubes. Additionally, we examined if the duration of EPS as well as cell harvest times (immediate vs. 24hrs. after the cessation of EPS) plays a role in EPS induced changes in GLUT4 and AMPK content. Methods: EPS was applied to myotubes 24 and 48 hr. (single bipolar pulses of 1 Hz for 2 ms; 30V) and were harvested at two different time points: immediately after (early harvest) and 24hr after (late harvest) the end of stimulation. Total GLUT4 and AMPK content were measured by western immunoblotting. Results: GLUT4 content was ~ 1.7 fold higher after 24 hr. early harvest and ~2.1 fold higher after 48 hr. late harvest stimulation. Total AMPK content was ~3.2 fold higher after 24 hr. early harvest stimulation and ~1.4 fold higher after 48 hr. late harvest stimulation. There was a ~0.6 fold decrease in AMPK after 24 hr. late harvest stimulation. Conclusion: These findings suggest that 24Hr of EPS stimulation upregulates GLUT4 and AMPK protein content. Duration and harvesting time (a reflection of post-exercise recovery) with regards to EPS treatment is a key factor leading to GLUT4 and AMPK content adaptations to exercise in human myotubes

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

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

Remodeling Lipid Metabolism and Improving Insulin Responsiveness in Human Primary Myotubes

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, (14)C 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 CO(2) (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

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

Substrate oxidation in primary human skeletal muscle cells is influenced by donor age

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