OBJECTIVES: To investigate the protective effects of dexmedetomidine (DEX) against heat stress (HS)-induced oncosis in human skeletal muscle cells (HSKMCs) and its underlying mechanisms. METHODS: A HSKMC model of HS-induced oncosis were established by 43 ℃ water bath for 4 h, and the effects of treatments with 30 μmol/L DEX, ML385 (a Nrf2 inhibitor) +DEX, si-Nrf2+HS, and si-Nrf2+DEX prior to modeling on cell viability was assessed using CCK-8 assay. Oncosis characteristics were evaluated using transmission electron microscopy and Annexin V-FITC/PI flow cytometry. The oxidative stress markers (GSH, GSH-Px, MDA, SOD and ROS), mitochondrial membrane potential, energy metabolism, and inflammatory cytokines (TNF-α, IL-6 and IL-1β) in the cells were quantified using standard kits, and the expressions of porimin, caspase-3 and Nrf2 pathway proteins were analyzed using Western blotting and qRT-PCR. RESULTS: HS induced typical oncotic features in HSKMCs including organelle swelling and cytoplasmic vacuolization. DEX pretreatment significantly attenuated these changes, reduced Annexin V⁺/PI⁺ cell ratio and cellular porimin expression, and lowered the levels of ROS and MDA while restoring GSH and SOD levels. DEX pretreatment also significantly increased the mitochondrial membrane potential and ATP level, upregulated the expressions of Nrf2, p-Nrf2, HO-1 and NQO1, and suppressed the expressions of TNF-α, IL-6 and IL-1β. The protective effects of DEX were obviously attenuated by interventions with ML385 or si-Nrf2. CONCLUSIONS DEX mitigates HS-induced HSKMC oncosis by activating the Nrf2/HO-1 pathway to relieve oxidative stress, mitochondrial dysfunction, and inflammatory responses.
Humans ; Dexmedetomidine/pharmacology* ; NF-E2-Related Factor 2/metabolism* ; Oxidative Stress/drug effects* ; Heat-Shock Response/drug effects* ; Signal Transduction/drug effects* ; Membrane Potential, Mitochondrial ; Muscle, Skeletal/cytology* ; Heme Oxygenase-1/metabolism* ; Apoptosis/drug effects*

The purpose of this study is to construct a muscle-specific synthetic promoter library, screen out muscle-specific promoters with high activity, analyze the relationship between element composition and activity of highly active promoters, and provide a theoretical basis for artificial synthesis of promoters. In this study, 19 promoter fragments derived from muscle-specific elements, conserved elements, and viral regulatory sequences were selected and randomLy connected to construct a muscle-specific synthetic promoter library. The luciferase plasmids pCMV-Luc and pSPs-Luc were constructed and transfected into the myoblast cell line C2C12. The activities of the synthesized promoters were evaluated by the luciferase activity assay. Two non-muscle-derived cell lines HeLa and 3T3 were used to verify the muscle specificity of the highly active promoters. The sequences of promoters with high activity, good muscle specificity, and correct sequences were analyzed to explore the relationship between the element composition and activity of promoters. We successfully constructed a muscle-specific promoter library and screened out 321 effective synthetic promoter plasmids. Among them, the activity of SP-301 promoter was 5.63 times that of CMV. The 15 promoters with high activity were muscle-specific. In the promoters with high activity and correct sequences, there was a relationship between their element composition and activity. Muscle-specific elements accounted for a high proportion in the promoters, while they had weak correlations with the promoter activity, being tissue-specific determinants. Viral elements accounted for no less than 20% in highly active promoters, which may be the key elements for the promoter activity. The content of conserved elements was proportional to the promoter activity. This study lays a theoretical foundation for the synthesis of tissue-specific efficient promoters and provides a new idea for the construction and application of in-situ gene delivery systems.
Promoter Regions, Genetic ; Humans ; Animals ; Mice ; Gene Library ; Cell Line ; Transfection ; HeLa Cells ; Luciferases/metabolism* ; Muscle, Skeletal/metabolism* ; Plasmids/genetics* ; Myoblasts/cytology*

It is now well established that inflammation plays an important role in the development of numerous chronic metabolic diseases including insulin resistance (IR) and type 2 diabetes (T2DM). Skeletal muscle is responsible for 75% of total insulin-dependent glucose uptake; consequently, skeletal muscle IR is considered to be the primary defect of systemic IR development. Our pre- vious study has shown that rutaecarpine (Rut) can benefit blood lipid profile, mitigate inflammation, and improve kidney, liver, pan- creas pathology status of T2DM rats. However, the effects of Rut on inflammatory cytokines in the development of IR-skeletal muscle cells have not been studied. Thus, our objective was to investigate effects of Rut on inflammatory cytokines interleukiri (IL)-1, IL-6 and tumor necrosis factor (TNF)-α in insulin resistant primary skeletal muscle cells (IR-PSMC). Primary cultures of skeletal muscle cells were prepared from 5 neonate SD rats, and the primary rat skeletal muscle cells were identified by cell morphology, effect of ru- taecarpine on cell proliferation by MTT assay. IR-PSMC cells were induced by palmitic acid (PA), the glucose concentration was measured by glucose oxidase and peroxidase (GOD-POD) method. The effects of Rut on inflammatory cytokines IL-1, IL-6 and TNF-α in IR-PSMC cells were tested by enzyme-linked immunosorbent assay (ELISA) kit. The results show that the primary skeletal muscle cells from neonatal rat cultured for 2-4 days, parallel alignment regularly, and cultured for 7 days, cells fused and myotube formed. It was shown that Rut in concentration 0-180. 0 μmol x L(-1) possessed no cytotoxic effect towards cultured primary skeletal muscle cells. However, after 24 h exposure to 0.6 mmol x L(-1) PA, primary skeletal muscle cells were able to induce a state of insulin resistance. The results obtained indicated significant decrease (P < 0.05 to P < 0.001) IL-1, IL-6 and TNF-α production by cultured IR-PSMC cells when incubating 24 hours with Rut, beginning from 20 to 180.0 μmol x L(-1). IL-1, IL-6 and TNF-α in the Rut treated groups were dose-dependently decreased compared with that in the IR-PSMC control group. Our results demonstrated that the Rut promoted glucose consumption and improved insulin resistance possibly through suppression of inflammatory cytokines in the IR-PSMC cells.
Animals ; Cell Proliferation ; drug effects ; Cytokines ; metabolism ; Female ; Glucose ; metabolism ; Indole Alkaloids ; pharmacology ; Inflammation ; metabolism ; Insulin Resistance ; Male ; Muscle, Skeletal ; cytology ; drug effects ; metabolism ; Quinazolines ; pharmacology ; Rats

OBJECTIVESTo evaluate the expression profile of myoD microRNA-29 (miR-29) family in L6 myoblast differentiated to myotube of L6 myotube treated by glucose and insulin, and to further probe the molecular mechanism of myoD regulating the expression of miR-29 clusters.

METHODSThe expression of myoD and miR-29 family was detected by using real-time PCR and Western blot analysis. The potential promoter and transcription factors binding sites of miR-29 clusters were predicted by Promoter scan and transcriptional factor search. The promoter sequence of miR-29b1-a and miR-29b2-c cluster was cloned into a luciferase reporter plasmid and the regulatory effect of myoD was analyzed by using dual luciferase reporter assay. Electrophoretic mobility shift assay was further conducted to indicate the binding of myoD on specific sequence. Moreover, overexpression of myoD was achieved by a recombinant adenovirus system (Ad-myoD). L6 cells were infected with Ad-myoD and real-time PCR was conducted to analyze the expression of miR-29b and miR-29c.

RESULTSThe expression levels of myoD, miR-29a, miR-29b, and miR-29c were increased in L6 myoblast differentiated to myotube. The expression of myoD, miR-29b, and miR-29c was up-regulated in L6 myotube treated with glucose and insulin, but miR-29a depicted no significant change. Dual luciferase reporter gene assay showed that myoD functioned as a positive regulator of miR-29b2-c expression and myoD could bind to the specific sequence located at the promoter region of miR-29b2-c cluster. Enforced expression of myoD led to a marked increase of miR-29b and miR-29c levels in L6 cells.

CONCLUSIONMyoD might act as a crucial regulator of myogenesis and glucose metabolism in muscle through regulating the expression of miR-29b2-c.

Animals ; Cell Differentiation ; drug effects ; physiology ; Cell Line ; Gene Expression Regulation ; drug effects ; physiology ; Glucose ; pharmacology ; Hypoglycemic Agents ; pharmacology ; Insulin ; pharmacology ; Mice ; MicroRNAs ; biosynthesis ; genetics ; Multigene Family ; physiology ; Muscle Fibers, Skeletal ; cytology ; metabolism ; MyoD Protein ; genetics ; metabolism ; Myoblasts ; cytology ; metabolism ; Sweetening Agents ; pharmacology

OBJECTIVETo explore the effect of glucose and/or acanthopanacis senticosi administration supplement on AMP-activated protein kinase (AMPK) activation and its change in different phase after exercise in muscle cell of rat.

METHODS128 rats were divided into exercise control groups (C groups), exercise and glucose administration groups (G groups), exercise and acanthopanacis senticosi administration groups (A groups), exercise and glucose and acanthopanacis senticosi administration groups (GA groups). The glucose and acanthopanacis senticosi supplement were completed by intragastric administration in half hour after exercise. All rats were killed in different designed time points before or after glycogen depletion exercise (0 h, 4 h and 12 h respectively) and finally divided into 16 groups (n = 8). The values of AMPK in soleus muscle were analyzed by Western blot.

RESULTS(1) After exercise, the protein content of AMPK quickly increased and reached the peak (209.23 +/- 21.32) then gradually decreased. (2) Acanthopanacis senticosi administration markedly increased the protein content of AMPK in the 0 h and 4 h points after glycogen consumption training (225.11 +/- 20.58 and 186.31 +/- 15.26 vs 195.19 +/- 13.31 and 157.11 +/- 16.43) without any difference after 12 h. (3) Glucose administration had no significant effect on AMPK activation. (4) Both glycogen and acanthopanacis senticosi were supplied simultaneously that had enhanced the AMPK content in 4 h and 12 h point (217.96 +/- 19.25 and 191.86 +/- 14.69). However, the AMPK content in GA group was lower than that in the C groups at 12 h point (121.89 +/- 15.23 vs 137.92 +/- 16.01).

CONCLUSIONExercise could markedly activate the AMPK protein in muscle cell and acanthopanacis senticosi administration augmented such activation. Glucose administration had no significant effect on AMPK activation.

AMP-Activated Protein Kinases ; metabolism ; Animals ; Drugs, Chinese Herbal ; pharmacology ; Eleutherococcus ; Glucose ; pharmacology ; Male ; Muscle Cells ; drug effects ; enzymology ; Muscle, Skeletal ; cytology ; drug effects ; enzymology ; Physical Conditioning, Animal ; Rats ; Rats, Sprague-Dawley

Myostatin (Mstn) is a member of the transforming growth factor-beta superfamily that functions as a negative regulator of skeletal muscle growth and differentiation in mammals. The transcriptional regulation of Mstn is controlled by multiple genes including MEF2, which raise the importance of identifying the binding sites of MEF2 on myostatin promoter region and mechanisms underlying. In this study, we investigated the transcriptional regulation of MEF2 on porcine Mstn promoter activity in C2C12 cells. Sequence analysis of the 1 969 bp porcine Mstn promoter region revealed that it contained three potential MEF2 motifs. Using a serial deletion strategy, we tested the activity of several promoter fragments by luciferase assay. Overexpression of MEF2C, but not MEF2A increased Mstn promoter activity in all the promoter fragments with MEF2 motifs by two to six folds, in both C2C12 myoblasts and myotubes. When we transfected exogenous MEF2C, Mstn mRNA level was also upregulated in C2C12 cells, but the protein level was only significantly increased in myotubes. Thus, we propose that MEF2C could modulate and restrain myogenesis by Mstn activation and Mstn-dependent gene processing in porcine. Our research also provided potential targets and an effective molecule to regulate Mstn expression and gave a new way to explore the functional performance of Mstn.
Animals ; Cells, Cultured ; Gene Expression Regulation ; MEF2 Transcription Factors ; Mice ; Muscle, Skeletal ; metabolism ; Myoblasts ; cytology ; Myogenic Regulatory Factors ; genetics ; physiology ; Myostatin ; genetics ; physiology ; Promoter Regions, Genetic ; Swine

Diabetes mellitus is a complex metabolic disorder characterized by chronic hyperglycemia due to absolute or relative lack of insulin. Though great efforts have been made to investigate the pathogenesis of diabetes, the underlying mechanism behind the development of diabetes and its complications remains unexplored. Cumulative evidence has linked mitochondrial modification to the pathogenesis of diabetes and its complications and they are also observed in various tissues affected by diabetes. Proteomics is an attractive tool for the study of diabetes since it allows researchers to compare normal and diabetic samples by identifying and quantifying the differentially expressed proteins in tissues, cells or organelles. Great progress has already been made in mitochondrial proteomics to elucidate the role of mitochondria in the pathogenesis of diabetes and its complications. Further studies on the changes of mitochondrial protein specifically post-translational modifications during the diabetic state using proteomic tools, would provide more information to better understand diabetes.
Adipose Tissue ; metabolism ; Diabetes Complications ; Diabetes Mellitus ; metabolism ; pathology ; Humans ; Insulin ; metabolism ; Insulin-Secreting Cells ; cytology ; metabolism ; Liver ; metabolism ; Mitochondria ; metabolism ; Muscle, Skeletal ; metabolism ; Proteome ; metabolism ; Proteomics

Gene transfer of basic fibroblast growth factor (bFGF) has been shown to induce significant endothelial migration and angiogenesis in ischemic disease models. Here, we investigate what factors are secreted from skeletal muscle cells (SkMCs) transfected with bFGF gene and whether they participate in endothelial cell migration. We constructed replication-defective adenovirus vectors containing the human bFGF gene (Ad/bFGF) or a control LacZ gene (Ad/LacZ) and obtained conditioned media, bFGF-CM and LacZ-CM, from SkMCs infected by Ad/bFGF or Ad/LacZ, respectively. Cell migration significantly increased in HUVECs incubated with bFGF-CM compared to cells incubated with LacZ-CM. Interestingly, HUVEC migration in response to bFGF-CM was only partially blocked by the addition of bFGF-neutralizing antibody, suggesting that bFGF-CM contains other factors that stimulate endothelial cell migration. Several proteins, matrix metalloproteinase-1 (MMP-1), plasminogen activator inhibitor-1 (PAI-1), and cathepsin L, increased in bFGF-CM compared to LacZ-CM; based on 1-dimensional gel electrophoresis and mass spectrometry. Their increased mRNA and protein levels were confirmed by RT-PCR and immunoblot analysis. The recombinant human bFGF protein induced MMP-1, PAI-1, and cathepsin L expression in SkMCs. Endothelial cell migration was reduced in groups treated with bFGF-CM containing neutralizing antibodies against MMP-1 or PAI-1. In particular, HUVECs treated with bFGF-CM containing cell-impermeable cathepsin L inhibitor showed the most significant decrease in cell migration. Cathepsin L protein directly promotes endothelial cell migration through the JNK pathway. These results indicate that cathepsin L released from SkMCs transfected with the bFGF gene can promote endothelial cell migration.
Antibodies, Neutralizing/immunology ; Cathepsin L/genetics/*metabolism ; *Cell Movement ; Cells, Cultured ; Comet Assay ; Dependovirus/genetics ; Endothelial Cells/cytology/*metabolism ; Fibroblast Growth Factor 2/genetics/immunology/*metabolism ; Gene Transfer Techniques ; Humans ; Immunoblotting ; JNK Mitogen-Activated Protein Kinases ; Lac Operon/genetics ; Mass Spectrometry ; Matrix Metalloproteinase 1/biosynthesis/genetics ; Muscle, Skeletal/*metabolism ; Neovascularization, Physiologic ; Plasminogen Activator Inhibitor 1/biosynthesis/genetics ; RNA, Messenger/biosynthesis ; Reverse Transcriptase Polymerase Chain Reaction

The present study was aimed to investigate the effects of hypoxia on iron metabolism of skeletal muscle. Rat L6 skeletal muscle cells were randomly divided into three groups which were exposed to hypoxia (1% O(2)) for 0, 12, 24 h, respectively. Iron isotope tracing method was used to determine iron uptake and release. Iron content of labile iron pool (LIP) was investigated by flow cytometry, and the expressions of transferrin receptor 1 (TfR1), divalent metal transporter 1 (DMT1), ferroportin 1 (FPN1), hypoxia-inducible transcription factor-1 (HIF-1) in L6 cells were observed by Western blot. The results showed that, compared with 0-hour hypoxia group, 12-hour hypoxia group exhibited significantly increased iron uptake and LIP (P < 0.05), as well as decreased iron release (P < 0.01). Not only iron uptake and release, but also LIP in 24-hour hypoxia group were significantly decreased, compared with those in 0- and 12-hour hypoxia groups (P < 0.01). The expressions of HIF-1, DMT1 (IRE), DMT1 (non-IRE) and TfR1 in 12-hour hypoxia group were significantly increased compared with those in 0-hour hypoxia group (P < 0.01). On the contrary, the expressions of DMT1 (IRE), DMT1 (non-IRE) and FPN1 in 24-hour hypoxia group were significantly decreased compared with those in the other two groups. However, TfR1 expression in 24-hour hypoxia was higher than those in 0- and 12-hour hypoxia groups (P < 0.05 or P < 0.01). These results suggest hypoxia plays an important role in iron metabolism of skeletal muscle cells. Moderate hypoxia can increase iron uptake and decrease iron release, resulting in higher LIP, but a prolonged hypoxia induces a disordered iron metabolism of skeletal cells.
Animals ; Cation Transport Proteins ; metabolism ; Cell Hypoxia ; Cell Line ; Hypoxia-Inducible Factor 1, alpha Subunit ; metabolism ; Iron ; metabolism ; Muscle, Skeletal ; cytology ; metabolism ; Oxygen ; physiology ; Rats ; Receptors, Transferrin ; metabolism ; Time Factors

An increasing body of evidence shows that the lipid droplet, a neutral lipid storage organelle, plays a role in lipid metabolism and energy homeostasis through its interaction with mitochondria. However, the cellular functions and molecular mechanisms of the interaction remain ambiguous. Here we present data from transmission electron microscopy, fluorescence imaging, and reconstitution assays, demonstrating that lipid droplets physically contact mitochondria in vivo and in vitro. Using a bimolecular fluorescence complementation assay in Saccharomyces cerevisiae, we generated an interactomic map of protein-protein contacts of lipid droplets with mitochondria and peroxisomes. The lipid droplet proteins Erg6 and Pet10 were found to be involved in 75% of the interactions detected. Interestingly, interactions between 3 pairs of lipid metabolic enzymes were detected. Collectively, these data demonstrate that lipid droplets make physical contacts with mitochondria and peroxisomes, and reveal specific molecular interactions that suggest active participation of lipid droplets in lipid metabolism in yeast.
Animals ; Cell Line ; Genetic Complementation Test ; Lipid Metabolism ; Lipids ; Microscopy, Electron, Transmission ; Microscopy, Fluorescence ; Mitochondria ; metabolism ; Muscle Cells ; metabolism ; Muscle, Skeletal ; cytology ; metabolism ; Oncogene Proteins ; genetics ; metabolism ; Peroxisomes ; metabolism ; Plasmids ; Protein Binding ; Protein Interaction Mapping ; methods ; Rats ; Recombinant Fusion Proteins ; genetics ; metabolism ; Saccharomyces cerevisiae ; Transcription Factors ; genetics ; metabolism ; Transformation, Genetic