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

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