Rat L6 Cells Research Articles

Identification of Flavonoids-based HITS to Inhibit GSK3β Activity: An <i>In Silico</i> and <i>In Vitro</i> Analysis

Background: Glycogen synthase kinase-3 (GSK-3) is a protein kinase with two isoforms, alpha and beta. GSK-3β, specifically, is found in nervous tissue and has been linked to the development of various human diseases. Aim: This study aimed to investigate the potential of flavonoids as inhibitors of GSK-3β using in silico and in vitro analyses. Methods: Schrodinger Maestro software was used for in silico molecular docking simulations to assess the interaction between flavonoids (ligands) and GSK-3β (target protein). In vitro cytotoxicity studies were performed on Rat L6 skeletal muscle cells to determine the safety of the selected flavonoids. Additionally, the neuroprotective effects of the flavonoids were evaluated using an oxygen-glucose deprivation model on SHSY 5Y neuroblastoma cells. Results: The in silico analysis revealed that all the studied flavonoids interacted with valine 135, a key amino acid for GSK-3β inhibition. Furthermore, the in vitro studies showed that the selected flavonoids were safe up to a concentration of 30 μM in rat L6 cells. Among the tested flavonoids, hesperetin, a flavanone, demonstrated the most potent neuroprotective effect in the SHSY 5Y cell line, with an IC50 value of 13.30 μM. Conclusion: Hesperetin, a flavanone, emerged as the most promising candidate among the tested flavonoids for further investigation as a potential GSK-3β inhibitor with neuroprotective properties.

Anemoside B4 Exerts Hypoglycemic Effect by Regulating the Expression of GLUT4 in HFD/STZ Rats.

Anemoside B4 (B4) is a saponin that is extracted from Pulsatilla chinensis (Bge.), and Regel exhibited anti-inflammatory, antioxidant, antiviral, and immunomodulatory activities. However, its hypoglycemic activity in diabetes mellitus has not been evaluated. Here, we explored the effect of B4 on hyperglycemia and studied its underlying mechanism of lowering blood glucose based on hyperglycemic rats in vivo and L6 skeletal muscle cells (L6) in vitro. The rats were fed a high-fat diet (HFD) for one month, combined with an intraperitoneal injection of 60 mg/kg streptozotocin (STZ) to construct the animal model, and the drug was administrated for two weeks. Blood glucose was detected and the proteins and mRNA were expressed. Our study showed that B4 significantly diminished fasting blood glucose (FBG) and improved glucose metabolism. In addition, B4 facilitated glucose utilization in L6 cells. B4 could enhance the expression of glucose transporter 4 (GLUT4) in rat skeletal muscle and L6 cells. Mechanistically, B4 elevated the inhibition of the phosphatidylinositol 3-kinase (PI3K)/protein kinase B (AKT) signaling pathways. Furthermore, we confirmed the effect of B4 on glucose uptake involved in the enhancement of GLUT4 expression in part due to PI3K/AKT signaling by using a small molecule inhibitor assay and constructing a GLUT4 promoter plasmid. Taken together, our study found that B4 ameliorates hyperglycemia through the PI3K/AKT pathway and promotes GLUT4 initiation, showing a new perspective of B4 as a potential agent against diabetes.

Binding of a Naja naja venom acidic phospholipase A2 cognate complex to membrane-bound vimentin of rat L6 cells: Implications in cobra venom-induced cytotoxicity

An acidic phospholipase A2 enzyme (NnPLA2-I) interacts with three finger toxins (cytotoxin and neurotoxin) from Naja naja venom to form cognate complexes to enhance its cytotoxicity towards rat L6 myogenic cells. The cytotoxicity was further enhanced in presence of trace quantity of venom nerve growth factor. The purified rat myoblast cell membrane protein showing interaction with NnPLA2-I was identified as vimentin by LC-MS/MS analysis. The ELISA, immunoblot and spectrofluorometric analyses showed greater binding of NnPLA2-I cognate complex to vimentin as compared to the binding of individual NnPLA2-I. The immunofluorescence and confocal microscopy studies evidenced the internalization of NnPLA2-I to partially differentiated myoblasts post binding with vimentin in a time-dependent manner. Pre-incubation of polyvalent antivenom with NnPLA2-I cognate complex demonstrated better neutralization of cytotoxicity towards L6 cells as compared to exogenous addition of polyvalent antivenom 60–240 min post treatment of L6 cells with cognate complex suggesting clinical advantage of early antivenom treatment to prevent cobra venom-induced cytotoxicity. The in silico analysis showed that 19–22 residues, inclusive of Asp48 residue, of NnPLA2-I preferentially binds with the rod domain (99–189 and 261–335 regions) of vimentin with a predicted free binding energy (ΔG) and dissociation constant (KD) values of −12.86 kcal/mol and 3.67 × 10−10 M, respectively; however, NnPLA2-I cognate complex showed greater binding with the same regions of vimentin indicating the pathophysiological significance of cognate complex in cobra venom-induced cytotoxicity.

An in-vitro investigation in rat L6 and mouse C2C12 cell lines into the skeletal muscle toxicity observed in the mouse of Trypanosoma Cruzi whole cell inhibitor compounds

An in-vitro investigation in rat L6 and mouse C2C12 cell lines into the skeletal muscle toxicity observed in the mouse of Trypanosoma Cruzi whole cell inhibitor compounds

The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.

Using differentiated rat L6 cells, we studied the direct effect of 3,5,3'-triiodo-l-thyronine (T3) and 3,5-diiodo-l-thyronine (T2) on the response to insulin in presence of fatty acids with a varying degree of saturation. We found that T3 and T2 both invert the response to insulin by modulating Akt Ser473 phosphorylation in the presence of palmitate and oleate. Both hormones prevented palmitate-induced insulin resistance, whereas increased insulin sensitivity in the presence of oleate was reduced, with normalization to (or, in the case of T3, even below) control levels. Both hormones effectively reduced intracellular acylcarnitine concentrations. Interestingly, insulin sensitization was lowered by incubation of the myotubes with relevant concentrations of palmitoylcarnitines (C16) and increased by oleylcarnitines and linoleylcarnitines (C18:1 and C18:2, respectively). The efficiency of mitochondrial respiration decreased in the order palmitate-oleate-linoleate; in the presence of palmitate, only T3 increased ATP synthesis-independent cellular respiration and mitochondrial respiratory complex activities. Both hormones modulated gene expression and enzyme activities related to insulin sensitivity, glucose metabolism, and lipid handling. Although T2 and T3 differentially regulated the expression of relevant genes involved in glucose metabolism, they equally stimulated related metabolic activities. T2 and T3 differentially modulated mitochondrial fatty acid uptake and oxidation in the presence of each fatty acid. The results show that T2 and T3 both invert the fatty acid-induced response to insulin but through different mechanisms, and that the outcome depends on the degree of saturation of the fatty acids and their derived acylcarnitines.-Giacco, A., delli Paoli, G., Senese, R., Cioffi, F., Silvestri, E., Moreno, M., Ruoppolo, M., Caterino, M., Costanzo, M., Lombardi, A., Goglia, F., Lanni, A., de Lange, P. The saturation degree of fatty acids and their derived acylcarnitines determines the direct effect of metabolically active thyroid hormones on insulin sensitivity in skeletal muscle cells.

IL6 and LIF mRNA expression in skeletal muscle is regulated by AMPK and the transcription factors NFYC, ZBTB14, and SP1.

Adenosine monophosphate-activated protein kinase (AMPK) controls glucose and lipid metabolism and modulates inflammatory responses to maintain metabolic and inflammatory homeostasis during low cellular energy levels. The AMPK activator 5-aminoimidazole-4-carboxamide-1-β-4-ribofuranoside (AICAR) interferes with inflammatory pathways in skeletal muscle, but the mechanisms are undefined. We hypothesized that AMPK activation reduces cytokine mRNA levels by blocking transcription through one or several transcription factors. Three skeletal muscle models were used to study AMPK effects on cytokine mRNA: human skeletal muscle strips obtained from healthy men incubated in vitro, primary human muscle cells, and rat L6 cells. In all three skeletal muscle systems, AICAR acutely reduced cytokine mRNA levels. In L6 myotubes treated with the transcriptional blocker actinomycin D, AICAR addition did not further reduce Il6 or leukemia inhibitory factor ( Lif) mRNA, suggesting that AICAR modulates cytokine expression through regulating transcription rather than mRNA stability. A cross-species bioinformatic approach identified novel transcription factors that may regulate LIF and IL6 mRNA. The involvement of these transcription factors was studied after targeted gene-silencing by siRNA. siRNA silencing of the transcription factors nuclear transcription factor Y subunit c ( Nfyc), specificity protein 1 ( Sp1), and zinc finger and BTB domain containing 14 ( Zbtb14), or AMPK α1/α2 subunits, increased constitutive levels of Il6 and Lif. Our results identify novel candidates in the regulation of skeletal muscle cytokine expression and identify AMPK, Nfyc, Sp1, and Zbtb14 as novel regulators of immunometabolic signals from skeletal muscle.

Emodin ameliorates high-fat-diet induced insulin resistance in rats by reducing lipid accumulation in skeletal muscle

Emodin, an anthraquinone derivative isolated from root and rhizome of Rheum palmatum, has been reported to have promising anti-diabetic activity. The present study was to explore the possible mechanism of emodin to ameliorate insulin resistance. Insulin resistance was induced by feeding a high fat diet to Sprague-Dawley rats. The blood glucose and lipid profiles in serum were measured by an enzymatic method, and a hyperinsulinaemic-euglycaemic clamp was used to evaluate insulin resistance. L6 cells were cultured and treated with palmitic acid and emodin. The lipid content was assayed in the soleus muscle and L6 cells by Oil Red O staining. Western blot, qRT-PCR, and immunohistochemical staining were used to detect the following in the rat soleus muscle and L6 cells: protein levels, mRNA levels of FATP1, FATP4, transporter fatty acid translocase (FAT/CD36), and plasma membrane-associated fatty acid protein (FABPpm). We found that blood glucose, triglyceride (TG), total cholesterol (TC) and low-density lipoprotein cholesterol (LDL-C) were significantly decreased in the emodin group. Oil Red O staining and the level of TG in skeletal muscle and L6 cells confirmed that lipid deposition decreased after treatment with emodin. Furthermore, the protein levels and mRNA levels of FATP1 in skeletal muscle and in L6 cells of rats were significantly decreased, yet the protein levels and mRNA levels of FATP4, FAT/CD36 and FABPpm did not drop off significantly. The study suggest that emodin ameliorates insulin resistance by reducing FATP1-mediated skeletal muscle lipid accumulation in rats fed a high fat diet.

Quercetin Protects Rat L6 Myocytes from Antimycin A-Induced Mitochondrial Dysfunction

Objective: Mitochondrial dysfunction is often associated with various disorders such as diabetes, Alzheimer’s etc. Reactive oxygen species (ROS), aging, and reduction of mitochondrial biogenesis contribute to mitochondrial dysfunction. Antimycin-A (AMA) damages the mitochondria through inhibition of mitochondrial electron transport. The present study sought to investigate effects of quercetin on rat L6 cells and whether quercetin protects mitochondria against oxidative damage caused by AMA. Methods: Rat L6 myocytes were used for the study. Effects of quercetin on Antimycin-A induced mitochondrial dysfunction was studied using cytotoxicity, ATP levels, mitochondrial superoxide production and NDUFB8 mRNA expression. Results: In this study, exposure of L6 myocytes to AMA induced an increase cell death, decreased ATP content, followed by a decrease in mitochondrial superoxide, and decreased expression of NDUFB8. We found that quercetin protected myocytes from antimycin-A (AMA) induced L6 cell death as evidenced from increased lactate dehydrogenase (LDH) leakage into extracellular medium, protected ATP production, prevented increase in oxidative stress and restored levels of NDUFB8 mRNA expression implying improved mitochondrial function. Conclusion: These results suggest that the quercetin showed protective effect against AMA-induced mitochondrial dysfunction by increasing ATP production, decreasing oxidative stress, and restoring mitochondrial function.

Study of a multiprotein complex involved in excitation‐transcription coupling in skeletal cells (1164.9)

Pannexin‐1 (Panx1) releases ATP to the extracellular space in skeletal muscle cells in response to electrical stimulation. ATP is a relevant mediator between membrane depolarization and gene expression. We propose that this signaling pathway would require the proper coordination between the voltage sensor (dihydropyridine receptor, DHPR), pannexin1 channels (PnX1, ATP release conduit), nucleotide receptors, and several signaling molecules. The goal of this study was to assess protein‐protein interactions within the E‐T machinery in several models. Newborn derived myotubes, adult fibers, triad fractions from rat or mouse skeletal muscles and L6 cells overexpressing PnX1 have been used. In all the models co‐immunoprecipitation between DHPR, PnX1, P2Y2 receptor and dystrophin has been detected. Using 2D blue‐native SDS/PAGE we detected that DHPR, PnX1, P2Y2, caveolin3 and dystrophin belong to the same multiprotein complex. Using the proximity ligation assay associated to confocal microscopy, we observed co‐localization between DHPR, PnX1, P2Y2 and caveolin3.Unveiling the molecular actors involved in the regulation of gene expression could contribute to the understanding and treatment of skeletal muscle disorders such as muscular dystrophies and sarcopenia associated with aging.Grant Funding Source: Supported by Fondecyt‐1110467‐11100454, Conicyt‐79090021, FONDAP‐15010006, ACT‐1111

Epigallocatechin-3-O-gallate (EGCG) protects the insulin sensitivity in rat L6 muscle cells exposed to dexamethasone condition

The tea polyphenol epigallocatechin-3-O-gallate (EGCG) displays some antidiabetic effects; however the mechanisms are incompletely understood. In the present study, the investigation of the effects of EGCG on insulin resistance was performed in rat L6 cells treated with dexamethasone. We found that dexamethasone increased Ser307 phosphorylation of insulin receptor substrate-1 (IRS-1) and reduced phosphorylation of AMPK and Akt. Furthermore, glucose uptake and glucose transporter (GLUT4) translocation were inhibited by dexamethasone. However, the treatment of EGCG improved insulin-stimulated glucose uptake by increasing GLUT4 translocation to plasma membrane. Furthermore, we also demonstrated these EGCG effects essentially depended on the AMPK and Akt activation. Together, our data suggested that EGCG inhibited dexamethasone-induced insulin resistance through AMPK and PI3K/Akt pathway.

Preliminary quantitative profile of differential protein expression between rat L6 myoblasts and myotubes by stable isotope labeling with amino acids in cell culture

Defining the mechanisms governing myogenesis has advanced in recent years. Skeletal-muscle differentiation is a multi-step process controlled spatially and temporally by various factors at the transcription level. To explore those factors involved in myogenesis, stable isotope labeling with amino acids in cell culture (SILAC), coupled with high-accuracy mass spectrometry (LTQ-Orbitrap), was applied successfully. Rat L6 cell line is an excellent model system for studying muscle myogenesis in vitro. When mononucleate L6 myoblast cells reach confluence in culture plate, they could transform into multinucleate myotubes by serum starvation. By comparing protein expression of L6 myoblasts and terminally differentiated multinucleated myotubes, 1170 proteins were quantified and 379 proteins changed significantly in fully differentiated myotubes in contrast to myoblasts. These differentially expressed proteins are mainly involved in inter-or intracellular signaling, protein synthesis and degradation, protein folding, cell adhesion and extracellular matrix, cell structure and motility, metabolism, substance transportation, etc. These findings were supported by many previous studies on myogenic differentiation, of which many up-regulated proteins were found to be involved in promoting skeletal muscle differentiation for the first time in our study. In summary, our results provide new clues for understanding the mechanism of myogenesis.

Acetyl- l-carnitine inhibits TNF-α-induced insulin resistance via AMPK pathway in rat skeletal muscle cells

In this study, we demonstrated effects of acetyl- l-carnitine (ALC) on insulin resistance induced by tumor necrosis factor-α (TNF-α) in rat L6 cells. TNF-α downregulated insulin-stimulated glucose uptake and increased Serine 307 phosphorylation of insulin receptor substrate-1 (IRS-1). However, the treatment of ALC improved insulin-stimulated glucose uptake via AMP-activated protein kinase (AMPK) activation in a dose-dependent manner. Together, our data suggest that ALC inhibits TNF-α-induced insulin resistance through AMPK pathway in skeletal muscle cells.

The use of established skeletal muscle cell lines to assess potential toxicity from embedded metal fragments

The use of novel materials on the modern battlefield, both in military munitions as well as in Improvised Explosive Devices, opens the possibility of wounds with embedded fragments whose health effects and toxicity characteristics have not been fully investigated, if at all. The costly and time-consuming nature of standard two-year lifespan studies prohibits the testing of many materials. In this report, we describe an in vitro system for rapidly assessing potential toxicity of metals and metal mixtures. Using rat L6 and mouse C2C12 skeletal muscle cells and tests for cellular viability, we have shown that two militarily relevant tungsten alloy mixtures (W/Ni/Co and W/Ni/Fe) significantly decreased the metabolic viability of rat L6 cells, whereas the viability of mouse C2C12 cells was not affected by W/Ni/Co and only slightly affected by W/Ni/Fe. In addition, viability assessed through lysosomal uptake of neutral red dye was not affected by either mixture in either cell line indicating that the mitochondria may be the target organelle of these unique metal mixtures. Development of this in vitro screening system may provide a procedure by which the potential toxicities of embedded metal fragments can be rapidly assessed.

CRM 1-mediated degradation and agonist-induced down-regulation of β-adrenergic receptor mRNAs

The β 1-adrenergic receptor (β 1-AR) mRNAs are post-transcriptionally regulated at the level of mRNA stability and undergo accelerated agonist-mediated degradation via interaction of its 3′ untranslated region (UTR) with RNA binding proteins, including the HuR nuclear protein. In a previous report [Kirigiti et al. (2001). Mol. Pharmacol. 60:1308–1324], we examined the agonist-mediated down-regulation of the rat β 1-AR mRNAs, endogenously expressed in the rat C6 cell line and ectopically expressed in transfectant hamster DDT 1MF2 and rat L6 cells. In this report, we determined that isoproterenol treatment of neonatal rat cortical neurons, an important cell type expressing β 1-ARs in the brain, results in significant decreases in β 1-AR mRNA stability, while treatment with leptomycin B, an inhibitor of the nuclear export receptor CRM 1, results in significant increases in β 1-AR mRNA stability and nuclear retention. UV-crosslinking/immunoprecipitation and glycerol gradient fractionation analyses indicate that the β 1-AR 3′ UTR recognize complexes composed of HuR and multiple proteins, including CRM 1. Cell-permeable peptides containing the leucine-rich nuclear export signal (NES) were used as inhibitors of CRM 1-mediated nuclear export. When DDT 1MF2 transfectants were treated with isoproterenol and peptide inhibitors, only the co-addition of the NES inhibitor reversed the isoproterenol-induced reduction of β 1-AR mRNA levels. Our results suggest that CRM 1-dependent NES-mediated mechanisms influence the degradation and agonist-mediated down-regulation of the β 1-AR mRNAs.

Multiple signalling pathways involved in β2‐adrenoceptor‐mediated glucose uptake in rat skeletal muscle cells

1. Beta-adrenoceptor (AR) agonists increase 2-deoxy-[3H]-D-glucose uptake (GU) via beta2-AR in rat L6 cells. The beta-AR agonists, zinterol (beta2-AR) and (-)-isoprenaline, increased cAMP accumulation in a concentration-dependent manner (pEC50=9.1+/-0.02 and 7.8+/-0.02). Cholera toxin (% max increase 141.8+/-2.5) and the cAMP analogues, 8-bromo-cAMP (8Br-cAMP) and dibutyryl cAMP (dbcAMP), also increased GU (196.8+/-13.5 and 196.4+/-17.3%). 2. The adenylate cyclase inhibitor, 2',5'-dideoxyadenosine (50 microM), significantly reduced cAMP accumulation to zinterol (100 nM) (109.7+35.0 to 21.6+4.5 pmol well(-1)), or forskolin (10 microM) (230.1+/-58.0 to 107.2+/-26.3 pmol well(-1)), and partially inhibited zinterol-stimulated GU (217+/-26.3 to 176.1+/-20.4%). The protein kinase A (PKA) inhibitor, 4-cyano-3-methylisoquinoline (100 nM), did not inhibit zinterol-stimulated GU. The PDE4 inhibitor, rolipram (10 microM), increased cAMP accumulation to zinterol or forskolin, and sensitised the GU response to zinterol, indicating a stimulatory role of cAMP in GU. 3. cAMP accumulation studies indicated that the beta2-AR was desensitised by prolonged stimulation with zinterol, but not forskolin, whereas GU responses to zinterol increased with time, suggesting that receptor desensitisation may be involved in GU. Receptor desensitisation was not reversed by inhibition of PKA or Gi. 4. PTX pretreatment (100 ng ml(-1)) inhibited insulin or zinterol-stimulated but not 8Br-cAMP or dbcAMP-stimulated GU. The PI3K inhibitor, LY294002 (1 microM), inhibited insulin- (174.9+/-5.9 to 142.7+/-2.7%) and zinterol- (166.9+/-7.6 to 141.1+/-8.1%) but not 8 Br-cAMP-stimulated GU. In contrast to insulin, zinterol did not cause phosphorylation of Akt. 5. The results suggest that GU in L6 cells involves three mechanisms: (1) an insulin-dependent pathway involving PI3K, (2) a beta2-AR-mediated pathway involving both cAMP and PI3K, and (3) a receptor-independent pathway suggested by cAMP analogues that increase GU independently of PI3K. PKA appears to negatively regulate beta2-AR-mediated GU.

Adenovirus Vectors Based on Human Adenovirus Type 19a Have High Potential for Human Muscle-Directed Gene Therapy

Until recently, adenovirus-based gene therapy has been almost exclusively based on human adenovirus serotype 5 (Ad5). The aim of this study was to systematically compare the efficiency of transduction of primary muscle cells from various species by two adenoviral vectors from subgroups C and D. Transduction of a panel of myoblasts demonstrated a striking specificity of an Ad19a-based replication-defective E1-deleted vector (Ad19aEGFP) for human cells, whereas the Ad5-based vector had high affinity for nonhuman primate myoblasts. Transgene expression correlated well with cell-associated vector genomes. Up to 6.59% of the initially applied Ad19aEGFP vector particles were taken up by human myoblasts, as compared with 0.1% of the corresponding Ad5 vector. Remarkably, Ad19aEGFP but not Ad5EGFP efficiently transduced differentiated human myotubes, an in vitro model for skeletal muscle transduction. Uptake of Ad19aEGFP vector particles in human myotubes was 12-fold more efficient than that of Ad5EGFP. Moreover, both vectors demonstrated an early block at the level of vector uptake in mouse myoblasts and rat L6 cells. Investigation of the underlying mechanism for binding and uptake of the two vectors by human myoblasts showed high susceptibility for Ad19a to neuraminidase and wheat germ agglutinin (WGA) lectin, whereas Ad5-mediated transduction was dependent on binding to the coxsackie-adenovirus receptor (CAR) and sensitive to soluble RGD peptide and heparin. Our study offers insights into species-dependent factors that determine Ad tropism and, moreover, provides a basis for application of the novel Ad19a-based vector for gene transfer into human skeletal muscle.

Quantification of hormone-induced atrophy of large myotubes from C2C12 and L6 cells: atrophy-inducible and atrophy-resistant C2C12 myotubes

Myofiber atrophy is the final outcome of muscle wasting induced by catabolic factors such as glucocorticoids and thyroid hormones. We set up an in vitro system to define the catabolic reaction based on myotube atrophy. Both mouse C(2)C(12) and rat L6 cells were used. C(2)C(12) myotube formation was improved by replacing horse serum with the serum substitute Ultroser G. A new method was developed to quantify size changes of large (0.5-1 mm) myotubes only, excluding remaining myoblasts and small myotubes. Dexamethasone reduced myotube size by 30% in L6 but not in C(2)C(12) myotubes. Expression of the glucocorticoid receptor was twofold higher in L6 myotubes than in C(2)C(12) myotubes. In both cell lines, 3,3',5-triiodo-l-thyronine (T(3)) did not induce a significant size reduction. Expression of the major T(3) receptor (T(3)Rbeta1) was higher in L6 myotubes. We investigated whether the changes in myotube size are related to changes in atrogin-1 expression, as this enzyme is thought to be a key factor in the initiation of muscle atrophy. Dexamethasone induced a twofold increase of atrogin-1 mRNA; again, only L6 myotubes were susceptible. Interestingly, atrogin-1 expression in Ultroser G-fused C(2)C(12) myotubes was lower than that in horse serum-fused myotubes. Furthermore, dexamethasone treatment increased atrogin-1 expression only in horse serum-fused myotubes but not in Ultroser G-fused myotubes. Ultroser G-induced fusion may result in atrophy-resistant C(2)C(12) myotubes. Therefore, C(2)C(12) myotubes offer an ideal system in which to study skeletal muscle atrophy because, depending on differentiation conditions, C(2)C(12) cells produce atrophy-inducible and atrophy-resistant myotubes.

A new mitochondrial RNA deletion fragment accelerated by oxidative stress in rat L6 cells

RNA deletions may be easier to detect and more extensive than DNA deletions. Two large deletion fragments (1120 and 7811 bp) of mitochondrial RNA were observed in rat L6 muscle cells. At the site of the 1120 bp deletion, the remaining RNA fragment was re-linked by a short additional section (GGTATGAAGCT). These kinds of deletions were accelerated by oxidative stress and were not observed in mitochondrial DNA.

A comparison of the actions of BIBN4096BS and CGRP8–37 on CGRP and adrenomedullin receptors expressed on SK‐N‐MC, L6, Col 29 and Rat 2 cells

1. The ability of the CGRP antagonist BIBN4096BS to antagonize CGRP and adrenomedullin has been investigated on cell lines endogenously expressing receptors of known composition. 2. On human SK-N-MC cells (expressing human calcitonin receptor-like receptor (CRLR) and receptor activity modifying protein 1 (RAMP1)), BIBN4096BS had a pA(2) of 9.95 although the slope of the Schild plot (1.37 +/- 0.16) was significantly greater than 1. 3. On rat L6 cells (expressing rat CRLR and RAMP1), BIBN4096BS had a pA(2) of 9.25 and a Schild slope of 0.89 +/- 0.05, significantly less than 1. 4. On human Colony (Col) 29 cells, CGRP(8-37) had a significantly lower pA(2) than on SK-N-MC cells (7.34 +/- 0.19 (n = 7) compared to 8.35 +/- 0.18, (n = 6)). BIBN4096BS had a pA(2) of 9.98 and a Schild plot slope of 0.86 +/- 0.19 that was not significantly different from 1. At concentrations in excess of 3 nM, it was less potent on Col 29 cells than on SK-N-MC cells. 5. On Rat 2 cells, expressing rat CRLR and RAMP2, BIBN4096BS was unable to antagonize adrenomedullin at concentrations up to 10 microM. CGRP(8-37) had a pA(2) of 6.72 against adrenomedullin. 6. BIBN4096BS shows selectivity for the human CRLR/RAMP1 combination compared to the rat counterpart. It can discriminate between the CRLR/RAMP1 receptor expressed on SK-N-MC cells and the CGRP-responsive receptor expressed by the Col 29 cells used in this study. Its slow kinetics may explain its apparent 'non-competitive' behaviour. At concentrations of up to 10 micro M, it has no antagonist actions at the adrenomedullin, CRLR/RAMP2 receptor, unlike CGRP(8-37).

Retargeting of viral glycoproteins into a non-exporting compartment during the myogenic differentiation of rat L6 cells.

We have analysed protein trafficking during the differentiation of rat L6 myoblasts into myotubes. Different proteins were found to lose different amounts of their processing by the Golgi apparatus during the myogenic differentiation, indicating that they were transported to this organelle with differing efficiencies. In order to investigate the destination of the nonprocessed glycoproteins we analysed the behaviour of vesicular stomatitis virus (VSV) and Semliki Forest virus glycoproteins in the presence of Brefeldin A, which returns the enzymes of the Golgi apparatus to the ER. Such experiments indicated that during myogenesis a fraction of both glycoproteins was shunted into a compartment that did not participate recycling with the Golgi apparatus. Immunofluorescence studies with the mutant VSV tsO45 G protein suggested that this compartment was diffusively distributed. We investigated whether the cytoplasmic tail had a role in the myogenic transport modulation by analysing the behaviour of recombinant VSV G proteins. Exchanging the cytoplasmic tail or the tail plus the membrane anchor had no effect, suggesting that the luminal portion was responsible for the diverted transport. Taken together, the results suggest that during the myogenesis of L6 myoblasts, varying fractions of different viral glycoproteins were sorted from the ER into a specific compartment that did not recycle with the Golgi apparatus.