Fish Glucose Transporter (GLUT)-4 Differs from Rat GLUT4 in Its Traffic Characteristics but Can Translocate to the Cell Surface in Response to Insulin in Skeletal Muscle Cells

In mammals, glucose transporter (GLUT)-4 plays an important role in glucose homeostasis mediating insulin action to increase glucose uptake in insulin-responsive tissues. In the basal state, GLUT4 is located in intracellular compartments and upon insulin stimulation is recruited to the plasma membrane, allowing glucose entry into the cell. Compared with mammals, fish are less efficient restoring plasma glucose after dietary or exogenous glucose administration. Recently our group cloned a GLUT4-homolog in skeletal muscle from brown trout (btGLUT4) that differs in protein motifs believed to be important for endocytosis and sorting of mammalian GLUT4. To study the traffic of btGLUT4, we generated a stable L6 muscle cell line overexpressing myc-tagged btGLUT4 (btGLUT4myc). Insulin stimulated btGLUT4myc recruitment to the cell surface, although to a lesser extent than rat-GLUT4myc, and enhanced glucose uptake. Interestingly, btGLUT4myc showed a higher steady-state level at the cell surface under basal conditions than rat-GLUT4myc due to a higher rate of recycling of btGLUT4myc and not to a slower endocytic rate, compared with rat-GLUT4myc. Furthermore, unlike rat-GLUT4myc, btGLUT4myc had a diffuse distribution throughout the cytoplasm of L6 myoblasts. In primary brown trout skeletal muscle cells, insulin also promoted the translocation of endogenous btGLUT4 to the plasma membrane and enhanced glucose transport. Moreover, btGLUT4 exhibited a diffuse intracellular localization in unstimulated trout myocytes. Our data suggest that btGLUT4 is subjected to a different intracellular traffic from rat-GLUT4 and may explain the relative glucose intolerance observed in fish.

High pressure induced changes in beef muscle proteome: correlation with quality parameters

The relationship between pressure induced changes on individual proteins and selected quality parameters in bovine longissimus thoracis et lumborum (LTL) muscle was studied. Pressures ranging from 200 to 600 MPa at 20 °C were used. High pressure processing (HPP) at pressures above 200 MPa induced strong modifications of protein solubility, meat colour and water holding capacity (WHC). The protein profiles of non-treated and pressure treated meat were observed using two dimensional electrophoresis. Proteins showing significant differences in abundance among treatments were identified by mass spectrometry. Pressure levels above 200 MPa strongly modified bovine LTL proteome with main effects being insolubilisation of sarcoplasmic proteins and solubilisation of myofibrillar proteins. Sarcoplasmic proteins were more susceptible to HPP effects than myofibrillar. Individual protein changes were significantly correlated with protein solubility, L*, b* and WHC, providing further insights into the mechanistic processes underlying HPP influence on quality and providing the basis for the future development of protein markers to assess the quality of processed meats.

Pathophysiology of muscle dysfunction in COPD

Muscle dysfunction often occurs in patients with chronic obstructive pulmonary disease (COPD) and may involve both respiratory and locomotor (peripheral) muscles. The loss of strength and/or endurance in the former can lead to ventilatory insufficiency, whereas in the latter it limits exercise capacity and activities of daily life. Muscle dysfunction is the consequence of complex interactions between local and systemic factors, frequently coexisting in COPD patients. Pulmonary hyperinflation along with the increase in work of breathing that occur in COPD appear as the main contributing factors to respiratory muscle dysfunction. By contrast, deconditioning seems to play a key role in peripheral muscle dysfunction. However, additional systemic factors, including tobacco smoking, systemic inflammation, exercise, exacerbations, nutritional and gas exchange abnormalities, anabolic insufficiency, comorbidities and drugs, can also influence the function of both respiratory and peripheral muscles, by inducing modifications in their local microenvironment. Under all these circumstances, protein metabolism imbalance, oxidative stress, inflammatory events, as well as muscle injury may occur, determining the final structure and modulating the function of different muscle groups. Respiratory muscles show signs of injury as well as an increase in several elements involved in aerobic metabolism (proportion of type I fibers, capillary density, and aerobic enzyme activity) whereas limb muscles exhibit a loss of the same elements, injury, and a reduction in fiber size. In the present review we examine the current state of the art of the pathophysiology of muscle dysfunction in COPD.

Muscle transcriptomic profiles in pigs with divergent phenotypes for fatness traits

BACKGROUND: Selection for increasing intramuscular fat content would definitively improve the palatability and juiciness of pig meat as well as the sensorial and organoleptic properties of cured products. However, evidences obtained in human and model organisms suggest that high levels of intramuscular fat might alter muscle lipid and carbohydrate metabolism. We have analysed this issue by determining the transcriptomic profiles of Duroc pigs with divergent phenotypes for 13 fatness traits. The strong aptitude of Duroc pigs to have high levels of intramuscular fat makes them a valuable model to analyse the mechanisms that regulate muscle lipid metabolism, an issue with evident implications in the elucidation of the genetic basis of human metabolic diseases such as obesity and insulin resistance. RESULTS: Muscle gene expression profiles of 68 Duroc pigs belonging to two groups (HIGH and LOW) with extreme phenotypes for lipid deposition and composition traits have been analysed. Microarray and quantitative PCR analysis showed that genes related to fatty acid uptake, lipogenesis and triacylglycerol synthesis were upregulated in the muscle tissue of HIGH pigs, which are fatter and have higher amounts of intramuscular fat than their LOW counterparts. Paradoxically, lipolytic genes also showed increased mRNA levels in the HIGH group suggesting the existence of a cycle where triacylglycerols are continuously synthesized and degraded. Several genes related to the insulin-signalling pathway, that is usually impaired in obese humans, were also upregulated. Finally, genes related to antigen-processing and presentation were downregulated in the HIGH group. CONCLUSION: Our data suggest that selection for increasing intramuscular fat content in pigs would lead to a shift but not a disruption of the metabolic homeostasis of muscle cells. Future studies on the post-translational changes affecting protein activity or expression as well as information about protein location within the cell would be needed to to elucidate the effects of lipid deposition on muscle metabolism in pigs.

Metabolic effects of insulin and IGFs on gilthead sea bream (Sparus aurata) muscle cells.

Primary cultures of gilthead sea bream myocytes were performed in order to examine the relative metabolic function of insulin compared with IGF-I and IGF-II (insulin-like growth factors, IGFs) at different stages in the cell culture. In these cells, the in vitro effects of insulin and IGFs on 2-deoxyglucose (2-DG) and L-alanine uptake were studied in both myocytes (day 4) and small myotubes (day 9). 2-DG uptake in gilthead sea bream muscle cells was increased in the presence of insulin and IGFs in a time dependent manner and along with muscle cell differentiation. On the contrary, L-alanine uptake was also stimulated by insulin and IGFs but showed an inverse pattern, being the uptake higher in small myocytes than in large myotubes. The results of preincubation with inhibitors (PD-98059, wortmannin, and cytochalasin B) on 2-DG uptake indicated that insulin and IGFs stimulate glucose uptake through the same mechanisms, and evidenced that mitogenesis activator protein kinase (MAPK) and PI3K-Akt transduction pathways mediate the metabolic function of these peptides. In the same way, we observed that GLUT4 protein synthesis was stimulated in the presence of insulin and IGFs in gilthead sea bream muscle cells in a different manner at days 4 or 9 of the culture. In summary we describe here, for the first time, the effects of insulin and IGFs on 2-DG and L-alanine uptake in primary culture of gilthead sea bream muscle cells. We show that both MAPK and PI3K-Akt transduction pathways are needed in order to control insulin and IGFs actions in these cells. Moreover, changes in glucose uptake can be explained by the action of the GLUT4 transporter, which is stimulated in the presence of insulin and IGFs throughout the cell culture.

Formoterol treatment downregulates the myostatin system in skeletal muscle of cachectic tumour-bearing rats

Cachexia is a common systemic manifestation. Additionally, myostatin is known to be a negative regulator of skeletal muscle development. The present study aimed to investigate whether formoterol down-regulates the myostatin system in skeletal muscle of tumour-bearing rats. Real-time PCR and Western blotting were used for the analysis. Results showed that rats bearing the Yoshida AH-130 ascites hepatoma, a cachexia-inducing tumour, exhibited marked muscle wasting that affected the mass of the muscles studied. The cachectic animals exhibited a significant increase in the mRNA levels of the myostatin receptor (ActIIB) in gastrocnemius muscles. Notably, the expression of the various forms of follistatin, a protein with the opposite effects to those of myostatin, was significantly reduced as a result of the implantation of the tumour. When the animals were treated with formoterol, a β-agonist with anti-cachectic potential, increases in skeletal muscle weights were observed. The β-agonist significantly increased levels of various follistatin isoforms and significantly decreased the expression levels of the myostatin receptor. In addition, formoterol treatment resulted in a significant decrease of the myostatin protein content of the gastrocnemius muscle. In conclusion, the results presented indicate that certain anabolic actions of formoterol on the skeletal muscle of cachectic animals may be mediated via the myostatin system.

Developmental and tissue-specific involvement of peroxisome proliferator-activated receptor-alpha in the control of mouse uncoupling protein-3 gene expression.

Uncoupling protein-3 (UCP3) is a member of the mitochondrial carrier family expressed preferentially in skeletal muscle and heart. It appears to be involved in metabolic handling of fatty acids in a way that minimizes excessive production of reactive oxygen species. Fatty acids are powerful regulators of UCP3 gene transcription. We have found that the role of peroxisome proliferator-activated receptor-α (PPARα) on the control of UCP3 gene expression depends on the tissue and developmental stage. In adults, UCP3 mRNA expression is unaltered in skeletal muscle from PPARα-null mice both in basal conditions and under the stimulus of starvation. In contrast, UCP3 mRNA is down-regulated in adult heart both in fed and fasted PPARα-null mice. This occurs despite the increased levels of free fatty acids caused by fasting in PPARα-null mice. In neonates, PPARα-null mice show impaired UCP3 mRNA expression in skeletal muscle in response to milk intake, and this is not a result of reduced free fatty acid levels. The murine UCP3 promoter is activated by fatty acids through either PPARα or PPARδ but not by PPARγ or retinoid X receptor alone. PPARδ-dependent activation could be a potential compensatory mechanism to ensure appropriate expression of UCP3 gene in adult skeletal muscle in the absence of PPARα. However, among transcripts from other PPARα and PPARδ target genes, only those acutely induced by milk intake in wild-type neonates were altered in muscle or heart from PPARα-null neonates. Thus, PPARα-dependent regulation is required for appropriate gene regulation of UCP3 as part of the subset of fatty-acid-responsive genes in neonatal muscle and heart.