G. Hege Thoresen

Eicosapentaenoic acid (20:5 n-3) increases fatty acid and glucose uptake in cultured human skeletal muscle cells.

This study was conducted to evaluate the chronic effects of eicosapentaenoic acid (EPA) on fatty acid and glucose metabolism in human skeletal muscle cells. Uptake of [14C]oleate was increased >2-fold after preincubation of myotubes with 0.6 mM EPA for 24 h, and incorporation into various lipid classes showed that cellular triacylgycerol (TAG) and phospholipids were increased 2- to 3-fold compared with control cells. After exposure to oleic acid (OA), TAG was increased 2-fold. Insulin (100 nM) further increased the incorporation of [14C]oleate into all lipid classes for EPA-treated myotubes. Fatty acid β-oxidation was unchanged, and complete oxidation (CO2) decreased in EPA-treated cells. Basal glucose transport and oxidation (CO2) were increased 2-fold after EPA, and insulin (100 nM) stimulated glucose transport and oxidation similarly in control and EPA-treated myotubes, whereas these responses to insulin were abolished after OA treatment. Lower concentrations of EPA (0.1 mM) also increased fatty acid and glucose uptake. CD36/FAT (fatty acid transporter) mRNA expression was increased after EPA and OA treatment compared with control cells. Moreover, GLUT1 expression was increased 2.5-fold by EPA, whereas GLUT4 expression was unchanged, and activities of the mitogen-activated protein kinase p38 and extracellular signal-regulated kinase were decreased after treatment with OA compared with EPA. Together, our data show that chronic exposure of myotubes to EPA promotes increased uptake and oxidation of glucose despite a markedly increased fatty acid uptake and synthesis of complex lipids.

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.

The Role of PPARα Activation in Liver and Muscle

PPARα is one of three members of the soluble nuclear receptor family called peroxisome proliferator-activated receptor (PPAR). It is a sensor for changes in levels of fatty acids and their derivatives that responds to ligand binding with PPAR target gene transcription, inasmuch as it can influence physiological homeostasis, including lipid and carbohydrate metabolism in various tissues. In this paper we summarize the involvement of PPARα in the metabolically active tissues liver and skeletal muscle and provide an overview of the risks and benefits of ligand activation of PPARα, with particular consideration to interspecies differences.

Cytoplasmic retention of peroxide‐activated ERK provides survival in primary cultures of rat hepatocytes

Reactive oxygen species (ROS) are implicated in tissue damage causing primary hepatic dysfunction following ischemia/reperfusion injury and during inflammatory liver diseases. A potential role of extracellular signal‐regulated kinase (ERK) as a mediator of survival signals during oxidative stress was investigated in primary cultures of hepatocytes exposed to ROS. Hydrogen peroxide (H2O2) induced a dose‐dependent activation of ERK, which was dependent on MEK activation. The ERK activation pattern was transient compared with the ERK activation seen after stimulation with epidermal growth factor (EGF). Nuclear accumulation of ERK was found after EGF stimulation, but not after H2O2 exposure. A slow import/rapid export mechanism was excluded through the use of leptomycin B, an inhibitor of nuclear export sequence–dependent nuclear export. Reduced survival of hepatocytes during ROS exposure was observed when ERK activation was inhibited. Ribosomal S6 kinase (RSK), a cytoplasmic ERK substrate involved in cell survival, was activated and located in the nucleus of H2O2‐exposed hepatocytes. The activation was abolished when ERK was inhibited with U0126. In conclusion, our results indicate that activity of ERK in the cytoplasm is important for survival during oxidative stress in hepatocytes and that RSK is activated downstream of ERK. Supplementary material for this article can be found on the HEPATOLOGY website (http://www.interscience.wiley.com/jpages/0270‐9139/suppmat/index.html). (HEPATOLOGY 2005;42:200–207.)

Response to transforming growth factor α (TGFα) and epidermal growth factor (EGF) in hepatocytes: Lower EGF receptor affinity of TGFα is associated with more sustained activation of p42/p44 mitogen-activated protein kinase and greater efficacy in stimulation of DNA synthesis

The epidermal growth factor (EGF) receptor mediates the effects of both EGF and transforming growth factor α (TGFα). Recent data suggested that EGF acts as a partial agonist/antagonist in hepatocytes, TGFα exerting a larger maximal stimulation of DNA synthesis than EGF. To further study the mechanisms involved in mediating the different effects of EGF and TGFα, we have examined receptor binding of the two growth factors and their action on the p42/p44 mitogen‐activated protein (MAP) kinase activity in hepatocytes. Single‐ligand concentration curves and competition experiments showed that the binding affinity to a common population of surface binding sites was about 20‐fold lower for TGFα than for EGF. MAP kinase activity responded to EGF and TGFα with different kinetics. While the two agents produced almost identical acute (5 min) stimulation (peak about fivefold), TGFα produced a more sustained MAP kinase activity than EGF. The difference between EGF and TGFα was still detectable 24 h after growth factor addition. The results show that in hepatocytes a lower receptor affinity of TGFα, as compared to EGF, is associated with a more sustained activation of the MAP kinase and a greater efficacy in the stimulation of DNA synthesis. This suggests that differential interaction of these two agents with the EGF receptor results in differences in the downstream events elicited at a given level of receptor occupancy. The data also are compatible with a role of a prolonged MAP kinase activity in the mitogenic effects of EGF and TGFα. J. Cell. Physiol. 175:10–18, 1998.

ERK2 prohibits apoptosis-induced subcellular translocation of orphan nuclear receptor NGFI-B/TR3.

Transcription factor NGFI-B (neuronal growth factor-induced clone B), also called Nur77 or TR3, is an immediate early gene and an orphan member of the nuclear receptor family. The NGFI-B protein also has a function distinct from that of a transcription factor; it translocates to mitochondria to initiate apoptosis. Recently, it was demonstrated that NGFI-B interacts with Bcl-2 by inducing a conformational change in Bcl-2, converting it from protector to a killer (Lin, B., Kolluri, S. K., Lin, F., Liu, W., Han, Y. H., Cao, X., Dawson, M. I., Reed, J. C., and Zhang, X. K. (2004) Cell 116, 527–540). After exposing rat cerebellar granule neurons to glutamate (100 μm, 15 min), NGFI-B translocated to the mitochondria. Growth factors such as the epidermal growth factor activate the MAP kinase ERK, the activity of which may determine whether a cell survives or undergoes apoptosis. In the present study we found that the epidermal growth factor activated ERK2 in cerebellar granule neurons and that this activation prohibited glutamate-induced subcellular translocation of NGFI-B. Likewise, overexpressed active ERK2 resulted in a predominant nuclear localization of green fluorescent protein-tagged NGFI-B. Thus, activation of ERK2 may overcome apoptosis-induced subcellular translocation of NGFI-B. This finding represents a novel and rapid growth factor survival pathway that is independent of gene regulation.

Activation of p42/p44 mitogen-activated protein kinase by angiotensin II, vasopressin, norepinephrine, and prostaglandin F2α in hepatocytes is sustained, and like the effect of epidermal growth factor, mediated through pertussis toxin-sensitive mechanisms

Several agents that act through G‐protein‐coupled receptors and also stimulate phosphoinositide‐specific phospholipase C (PI‐PLC), including angiotensin II, vasopressin, norepinephrine, and prostaglandin (PG) F2α, activated the ERK1 (p44mapk) and ERK2 (p42mapk) members of the mitogen‐activated protein (MAP) kinase family in primary cultures of rat hepatocytes, measured as phosphorylation of myelin basic protein (MBP) by a partially purified enzyme, immunoblotting, and in‐gel assays. All these agonists induced a peak activation (two to threefold increase in MBP‐phosphorylation) at 3–5 min, followed by a brief decrease, and then a sustained elevation or a second increase of the MAP kinase activity that lasted for several hours. Although all the above agents also stimulated PI‐PLC, implicating a Gq‐dependent pathway, the elevations of the concentration of inositol (1,4,5)‐trisphosphate did not correlate well with the MAP kinase activity. Furthermore, pretreatment of the cells with pertussis toxin markedly reduced the MAP kinase activation by angiotensin II, vasopressin, norepinephrine, or PGF2α. In addition, hepatocytes pretreated with pertussis toxin showed a diminished MAP kinase response to epidermal growth factor (EGF). The results indicate that agonists acting via G‐protein‐coupled receptors have the ability to induce sustained activation of MAP kinase in hepatocytes, and suggest that Gi‐dependent mechanisms are required for full activation of the MAP kinase signal transduction pathway by G‐protein‐coupled receptors as well as the EGF receptor. J. Cell. Physiol. 175:348–358, 1998.

Prostaglandin E2 upregulates EGF-stimulated signaling in mitogenic pathways involving Akt and ERK in hepatocytes.

Prostaglandins (PGs) such as PGE2 enhance proliferation in many cells, apparently through several distinct mechanisms, including transactivation of the epidermal growth factor (EGF) receptor (EGFR) as well as EGFR‐independent pathways. In this study we found that in primary cultures of rat hepatocytes PGE2 did not induce phosphorylation of the EGFR, and the EGFR tyrosine kinase blockers gefitinib and AG1478 did not affect PGE2‐stimulated phosphorylation of ERK1/2. In contrast, PGE2 elicited EGFR phosphorylation and EGFR tyrosine kinase inhibitor‐sensitive ERK phosphorylation in MH1C1 hepatoma cells. These findings suggest that PGE2 elicits EGFR transactivation in MH1C1 cells but not in hepatocytes. Treatment of the hepatocytes with PGE2 at 3 h after plating amplified the stimulatory effect on DNA synthesis of EGF administered at 24 h and advanced and augmented the cyclin D1 expression in response to EGF in hepatocytes. The pretreatment of the hepatocytes with PGE2 resulted in an increase in the magnitude of EGF‐stimulated Akt phosphorylation and ERK1/2 phosphorylation and kinase activity, including an extended duration of the responses, particularly of ERK, to EGF in PGE2‐treated cells. Pertussis toxin abolished the ability of PGE2 to enhance the Akt and ERK responses to EGF. The results suggest that in hepatocytes, unlike MH1C1 hepatoma cells, PGE2 does not transactivate the EGFR, but instead acts in synergism with EGF by modulating mitogenic mechanisms downstream of the EGFR. These effects seem to be at least in part Gi protein‐mediated and include upregulation of signaling in the PI3K/Akt and the Ras/ERK pathways. J. Cell. Physiol. 214: 371–380, 2008.

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, 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.

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.