Effect of Uncaria tomentosa aqueous extract on the response to palmitate-induced lipotoxicity in cultured skeletal muscle cells.

BACKGROUND: Type 2 diabetes mellitus (T2DM) is frequently associated with dyslipidemia, which corresponds to the increase in the triglycerides and fatty acid concentrations in tissues, such as the skeletal muscle. Also, T2DM molecular mechanism involves increasing in reactive oxygen species (ROS) production and oxidative stress. The use of herbal medicines such as Uncaria tomentosa (Ut) has been proposed as an auxiliary treatment for patients with T2DM. In this study, it was evaluated the effect of Ut aqueous extract on cell viability and ROS production, in skeletal myoblasts from C2C12 lineage exposed to the free fatty acid palmitate (PA). METHODS: Cells were incubated with PA in different concentrations ranging from 10 to 1000 µM, for 24 or 48 h, for cytotoxicity assay. Cell death, DNA fragmentation and ROS production assays were performed in cell cultures incubated with PA for 24 h, in the pre (preventive condition) or post treatment (therapeutic condition) with 250 µg/ml Ut aqueous extract, for 2 or 6 h. Cell death was evaluated by MTT method or flow cytometry. ROS generation was measured by fluorescence spectroscopy using the DCFDA probe. RESULTS: Cell viability was reduced to approximately 44% after the incubation with PA for 24 h from the concentration of 500 µM. In the incubation of cells with 500 µM PA and Ut extract for 6 h, in both conditions (preventive or therapeutic), it was observed an increase of 27 and 70% in cell viability respectively, in comparison to the cultures incubated with only PA. Also, the incubation of cultures with 500 µM PA, for 24 h, increased 20-fold the ROS formation, while the treatment with Ut extract, for 6 h, both in the preventive or therapeutic conditions, promoted decrease of 21 and 55%, respectively. CONCLUSION: The Ut extract was efficient in promoting cell protection against PA lipotoxicity and ROS generation, potentially preventing oxidative stress in C2C12 skeletal muscle cells. Since T2DM molecular mechanism involves oxidative stress condition and it is often associated with dyslipidemia and fatty acid accumulation in muscle tissue, these results open perspectives for the use of Ut as an auxiliary strategy for T2DM management.

Differential Fibrotic Response of Muscle Fibroblasts, Myoblasts, and Myotubes to Cholic and Deoxycholic Acids.

Fibrosis is a condition characterized by an increase in the components of the extracellular matrix (ECM). In skeletal muscle, the cells that participate in the synthesis of ECM are fibroblasts, myoblasts, and myotubes. These cells respond to soluble factors that increase ECM. Fibrosis is a phenomenon that develops in conditions of chronic inflammation, extensive lesions, or chronic diseases. A pathological condition with muscle weakness and increased bile acids (BA) in the blood is cholestatic chronic liver diseases (CCLD). Skeletal muscle expresses the membrane receptor for BA called TGR5. To date, muscle fibrosis in CCLD has not been evaluated. This study aims to assess whether BA can induce a fibrotic condition in muscle fibroblasts, myoblasts, and myotubes. The cells were incubated with deoxycholic (DCA) and cholic (CA) acids, and fibronectin protein levels were evaluated by Western blot. In muscle fibroblasts, both DCA and CA induced an increase in fibronectin protein levels. The same response was found in fibroblasts when activating TGR5 with the specific receptor agonist (INT-777). Interestingly, DCA reduced fibronectin protein levels in both myoblasts and myotubes, while CA did not show changes in fibronectin protein levels in myoblasts and myotubes. These results suggest that DCA and CA can induce a fibrotic phenotype in muscle-derived fibroblasts. On the other hand, DCA decreased the fibronectin in myoblasts and myotubes, whereas CA did not show any effect in these cell populations. Our results show that BA has different effects depending on the cell population to be analyzed.

Development of new 1, 3-dihydroxyacridone derivatives as Akt pathway inhibitors in skeletal muscle cells.

In the present work, four new compounds based on the privileged structure acridone were efficiently synthesized following simple operational techniques and biologically tested on proliferative skeletal muscle cells (C2C12) and rhabdomyosarcoma cells (RD) showing no significant changes in the number of dead or viable cells at 1 µM during 24 or 48 h of treatment. Of relevance, acridone derivatives 3a-3d at 0.5 µM for 24 h effectively inhibited Akt activation in C2C12, while at 1 µM only compounds 3a and 3b have effect. RD cells showed a different response pattern. These cells treated with 3a (0.5 µM), 3b (0.5 µM) or 3d (0.5 or 1 µM) for 24 h shown significant Akt inhibition. In addition, 3a-3d assayed at 1 µM for 48 h were highly successful in inhibiting Akt phosphorylation. Finally, based on molecular docking and molecular dynamics simulations, we rationalize the experimental results mentioned above and propose that 3-phosphoinositide-dependent kinase-1 (PDK1) could be one of the molecular targets of this new series of 1, 3-dihydroxyacridone derivatives. Biological and in silico studies revealed that 3b could be considered as the most promising prototype for the development of new antitumor agents.

RNA-Seq-Based Analysis of Cortisol-Induced Differential Gene Expression Associated with Piscirickettsia salmonis Infection in Rainbow Trout (Oncorhynchus mykiss) Myotubes.

Salmonid rickettsial septicemia (SRS) is the major infectious disease of the Chilean salmonid aquaculture industry caused by Piscirickettsia salmonis. Intensive farming conditions generate stress and increased susceptibility to diseases, being skeletal muscle mainly affected. However, the interplay between pathogen infection and stress in muscle is poorly understood. In this study, we perform an RNA-seq analysis on rainbow trout myotubes that are pretreated for 3 h with cortisol (100 ng/mL) and then infected with P. salmonis strain LF-89 for 8 h (MOI 50). Twelve libraries are constructed from RNA samples (n = 3 per group) and sequenced on Illumina HiSeq 4000. A total of 704,979,454 high-quality reads are obtained, with 70.25% mapped against the reference genome. In silico DETs include 175 total genes-124 are upregulated and 51 are downregulated. GO enrichment analysis reveals highly impacted biological processes related to apoptosis, negative regulation of cell proliferation, and innate immune response. These results are validated by RT-qPCR of nine candidate transcripts. Furthermore, cortisol pretreatment significantly stimulated bacterial gene expression of ahpC and 23s compared to infection. In conclusion, for the first time, we describe a transcriptomic response of trout myotubes infected with P. salmonis by inducing apoptosis, downregulating cell proliferation, and intrinsic immune-like response that is differentially regulated by cortisol.

Complexin-2 redistributes to the membrane of muscle cells in response to insulin and contributes to GLUT4 translocation.

Insulin stimulates glucose uptake in muscle cells by rapidly redistributing vesicles containing GLUT4 glucose transporters from intracellular compartments to the plasma membrane (PM). GLUT4 vesicle fusion requires the formation of SNARE complexes between vesicular VAMP and PM syntaxin4 and SNAP23. SNARE accessory proteins usually regulate vesicle fusion processes. Complexins aide in neuro-secretory vesicle-membrane fusion by stabilizing trans-SNARE complexes but their participation in GLUT4 vesicle fusion is unknown. We report that complexin-2 is expressed and homogeneously distributed in L6 rat skeletal muscle cells. Upon insulin stimulation, a cohort of complexin-2 redistributes to the PM. Complexin-2 knockdown markedly inhibited GLUT4 translocation without affecting proximal insulin signalling of Akt/PKB phosphorylation and actin fiber remodelling. Similarly, complexin-2 overexpression decreased maximal GLUT4 translocation suggesting that the concentration of complexin-2 is finely tuned to vesicle fusion. These findings reveal an insulin-dependent regulation of GLUT4 insertion into the PM involving complexin-2.

Regulation of Lin28a-miRNA let-7b-5p pathway in skeletal muscle cells by peroxisome proliferator-activated receptor delta.

Lin28a/miRNA let-7b-5p pathway has emerged as a key regulators of energy homeostasis in the skeletal muscle. However, the mechanism through which this pathway is regulated in the skeletal muscle has remained unclear. We have found that 8 wk of aerobic training (Tr) markedly decreased let-7b-5p expression in murine skeletal muscle, whereas high-fat diet (Hfd) increased its expression. Conversely, Lin28a expression, a well-known inhibitor of let-7b-5p, was induced by Tr and decreased by Hfd. Similarly, in human muscle biopsies, Tr increased LIN28 expression and decreased let-7b-5p expression. Bioinformatics analysis of LIN28a DNA sequence revealed that its enrichment in peroxisome proliferator-activated receptor delta (PPARδ) binding sites, which is a well-known metabolic regulator of exercise. Treatment of primary mouse skeletal muscle cells or C2C12 cells with PPARδ activators GW501516 and AICAR increased Lin28a expression. Lin28a and let-7b-5p expression was also regulated by PPARδ coregulators. While PPARγ coactivator-1α (PGC1α) increased Lin28a expression, corepressor NCoR1 decreased its expression. Furthermore, PGC1α markedly reduced the let-7b-5p expression. PGC1α-mediated induction of Lin28a expression was blocked by the PPARδ inhibitor GSK0660. In agreement, Lin28a expression was downregulated in PPARδ knocked-down cells leading to increased let-7b-5p expression. Finally, we show that modulation of the Lin28a-let-7b-5p pathway in muscle cells leads to changes in mitochondrial metabolism in PGC1α dependent fashion. In summary, we demonstrate that Lin28a-let-7b-5p is a direct target of PPARδ in the skeletal muscle, where it impacts mitochondrial respiration.

Myotube Protein Content Associates with Intracellular L-Glutamine Levels.

BACKGROUND/AIMS: Skeletal mass loss is reported in several catabolic conditions and it has been associated with a reduced intracellular L-glutamine content. We investigated the association of intracellular L-glutamine concentration with the protein content in skeletal muscle cells. METHODS: We cultivated C2C12 myotubes in the absence or presence of 2 (reference condition), 8 or 16 mM L-glutamine for 48 hours, and the variations in the contents of amino acids and proteins measured. We used an inhibitor of L-glutamine synthesis (L-methionine sulfoximine - MSO) to promote a further reduction in intracellular L-glutamine levels. Amino acids contents in cells and media were measured using LC-MS/MS. We measured changes in phosphorylated Akt, RP-S6, and 4E-BP1contents in the absence or presence of insulin by western blotting. RESULTS: Reduced intracellular L-glutamine concentration was associated with decreased protein content and increased protein breakdown. Low intracellular glutamine levels were also associated with decreased p-Akt contents in the presence of insulin. A further decrease in intracellular L-glutamine caused by glutamine synthetase inhibitor reduced protein content and levels of amino acids generated from glutamine metabolism and increased bAib still further. Cells exposed to high medium glutamine levels did not have any change in protein content but exhibited increased contents of the amino acids derived from L-glutamine metabolism. CONCLUSION: Intracellular L-glutamine levels per se play a role in the control of protein content in skeletal muscle myotubes.

Long-chain acyl-CoA synthetase 6 regulates lipid synthesis and mitochondrial oxidative capacity in human and rat skeletal muscle.

KEY POINTS: Long-chain acyl-CoA synthetase 6 (ACSL6) mRNA is present in human and rat skeletal muscle, and is modulated by nutritional status: exercise and fasting decrease ACSL6 mRNA, whereas acute lipid ingestion increase its expression. ACSL6 genic inhibition in rat primary myotubes decreased lipid accumulation, as well as activated the higher mitochondrial oxidative capacity programme and fatty acid oxidation through the AMPK/PGC1-α pathway. ACSL6 overexpression in human primary myotubes increased phospholipid species and decreased oxidative metabolism. ABSTRACT: Long-chain acyl-CoA synthetases (ACSL 1 to 6) are key enzymes regulating the partitioning of acyl-CoA species toward different metabolic fates such as lipid synthesis or ß-oxidation. Despite our understanding of ecotopic lipid accumulation in skeletal muscle being associated with metabolic diseases such as obesity and type II diabetes, the role of specific ACSL isoforms in lipid synthesis remains unclear. In the present study, we describe for the first time the presence of ACSL6 mRNA in human skeletal muscle and the role that ACSL6 plays in lipid synthesis in both rodent and human skeletal muscle. ACSL6 mRNA was observed to be up-regulated by acute high-fat meal ingestion in both rodents and humans. In rats, we also demonstrated that fasting and chronic aerobic training negatively modulated the ACSL6 mRNA and other genes of lipid synthesis. Similar results were obtained following ACSL6 knockdown in rat myotubes, which was associated with a decreased accumulation of TAGs and lipid droplets. Under the same knockdown condition, we further demonstrate an increase in fatty acid content, p-AMPK, mitochondrial content, mitochondrial respiratory rates and palmitate oxidation. These results were associated with increased PGC-1α, UCP2 and UCP3 mRNA and decreased reactive oxygen species production. In human myotubes, ACSL6 overexpression reduced palmitate oxidation and PGC-1α mRNA. In conclusion, ACSL6 drives acyl-CoA toward lipid synthesis and its downregulation improves mitochondrial biogenesis, respiratory capacity and lipid oxidation. These outcomes are associated with the activation of the AMPK/PGC1-α pathway.

The morphological analysis of autophagy in primary skeletal muscle cells infected with Toxoplasma gondii.

Toxoplasma gondii is an obligate intracellular protozoan parasite, the causative agent of toxoplasmosis, one of the most widespread zoonoses in the world. During the host immune response, tissue cysts are formed, allowing the maintenance of the parasite within the host cell. Autophagy, a degradation process of cellular components, is critical for cellular homeostasis. Recently, it has been proposed that autophagy participates in host-pathogen interactions. Autophagic inducers (rapamycin or glucose plus serum deprivation) inhibited infection and parasite proliferation in a clinically relevant model of primary skeletal muscle cells (SkMC). The ultrastructural analysis showed in SkMC submitted to autophagic stimuli the presence of structures suggestive of autophagosomes close to the parasitophorous vacuole containing degraded parasites. Fluorescence microscopy results pointed out the increase in LC3 puncta in these cells after incubation with autophagic inducers. In the present study, SkMC autophagy controlled the proliferation of tachyzoites inside the cell, data reinforced by ultrastructural evidences and increased LC3 expression.

1α,25 dihydroxi-vitamin D3 modulates CDK4 and CDK6 expression and localization.

We recently reported that the vitamin D receptor (VDR) and p38 MAPK participate in pro-differentiation events triggered by 1α,25(OH)2-vitamin D3 [1,25D] in skeletal muscle cells. Specifically, our studies demonstrated that 1,25D promotes G0/G1 arrest of cells inducing cyclin D3 and cyclin dependent kinases inhibitors (CKIs) p21(Waf1/Cip1) and p27(Kip1) expression in a VDR and p38 MAPK dependent manner. In this work we present data indicating that cyclin-dependent kinases (CDKs) 4 and 6 also play a role in the mechanism by which 1,25D stimulates myogenesis. To investigate VDR involvement in hormone regulation of CDKs 4 and 6, we significantly reduced its expression by the use of a shRNA against mouse VDR, generating the skeletal muscle cell line C2C12-VDR. Investigation of changes in cellular cycle regulating proteins by immunoblotting showed that the VDR is involved in the 1,25D -induced CDKs 4 and 6 protein levels at 6 h of hormone treatment. CDK4 levels remains high during S phase peak and G0/G1 arrest while CDK6 expression decreases at 12 h and increases again al 24 h. The up-regulation of CDKs 4 and 6 by 1,25D (6 h) was abolished in C2C12 cells pre-treated with the ERK1/2 inhibitor, UO126. Moreover, CDKs 4 and 6 expression induced by the hormone nor was detected when α and ß isoforms of p38 MAPK were inhibited by compound SB203580. Confocal images show that there is not co-localization between VDR and CDKs at 6 h of hormone treatment, however CDK4 and VDR co-localizates in nucleus after 12 h of 1,25D exposure. Of relevance, at this time 1,25D promotes CDK6 localization in a peri-nuclear ring. Our data demonstrate that the VDR, ERK1/2 and p38 MAPK are involved in the control of CDKs 4 and 6 by 1,25D in skeletal muscle cells sustaining the operation of a VDR and MAPKs -dependent mechanism in hormone modulation of myogenesis.

Interaction and cystogenesis of Toxoplasma gondii within skeletal muscle cells in vitro

Infection by the protozoan parasite Toxoplasma gondii is widely prevalent in humans and animals. To prevent human infection, all meat should be well cooked before consumption, since the parasite is present in skeletal muscle. In this context, the use of skeletal muscle cells (SkMCs) as a cellular model opens up new approaches to investigate T. gondii-host cell interactions. Immunofluorescent detection of proteins that are stage-specific for bradyzoites indicated that complete cystogenesis of T. gondii in in vitro cultures of SkMCs occurs after 96 h of infection. Ultrastructural analysis showed that, after 48 h of interaction, there were alterations on the parasitophorous vacuole membrane, including greater thickness and increased electron density at the inner face of the membrane. The present study demonstrates the potential use of primary cultures of SkMCs to evaluate different molecular aspects of T. gondii invasion and cystogenesis and presents a promising in vitro model for the screening of drug activities toward tissue cysts and bradyzoites.