Liraglutide ameliorates palmitic acid-induced insulin resistance in L6 skeletal muscle cells by regulating Sestrin2 / 中华内分泌代谢杂志

Objective:To investigate the role of stress-inducible protein Sestrin2 (Sesn2) in the improvement of insulin resistance in rat L6 skeletal muscle cells treated with liraglutide.Methods:The establishment of insulin resistance model of rat L6 skeletal muscle cells was induced by palmitate. The experimental cells were divided into control group(Con group), palmitate 0.6 mmol/L treatment group(PA group), palmitate 0.6 mmol/L+ liraglutide 10 nmol/L treatment group(PA+ Lir10 group), palmitate 0.6 mmol/L+ liraglutide 100 nmol/L treatment group(PA+ Lir100 group), and palmitate 0.6 mmol/L+ liraglutide 1 000 nmol/L treatment group(PA+ Lir1000 group). The cell counting kit 8(CCK8) method was used to detect the cell activity in each group. Western blotting was used to detect the expression levels of glucose transporter 4(GLUT4), protein kinase B(Akt), phosphorylated protein kinase B(p-Akt), and Sesn2 protein in L6 cells. L6 cells were transfected with siRNA to inhibit the expression of Sesn2. The cells were treated with palmitate and liraglutide. Western blotting was used to detect the expression levels of Sesn2, Akt, p-Akt, and GLUT4 protein in L6 cells.Results:Compared with Con group, the cell survival rate, p-Akt/Akt ratio, Sesn2, and GLUT4 protein expression in PA group decreased significantly( P<0.05). After liraglutide intervention, the cell activity, p-Akt/Akt ratio, Sesn2, and GLUT4 protein expression of PA+ Lir100 and PA+ Lir1000 groups was increased( P<0.05). After inhibiting the expression of Sesn2, p-Akt/Akt ratio and GLUT4 protein in transfected si-Sesn2 and treated with 0.6 mmol/L palmitate group(PA+ si-Sesn2 group) and transfected si-Sesn2 and treated with 0.6 mmol/L palmitate+ liraglutide 100 nmol/L group (Lir100+ PA+ si-Sesn2 group) were significantly lower than those in transfection negative group (si-Con group; P<0.05). Even after liraglutide intervention, compared with PA+ si-Sesn2 group, p-Akt/Akt ratio and GLUT4 protein expression level were not significantly increased in Lir100+ PA+ si-Sesn2 group ( P>0.05). Conclusions:Palmitate could induce the decrease of p-Akt/Akt ratio and GLUT4 protein expression in L6 cells. Liraglutide upregulates the expression of Sesn2, which leads to the increase of p-Akt/Akt ratio and GLUT4 protein expression and contributes to the improvement of insulin resistance.

Cloning and expression of the growth hormone gene and its effect on the proliferation of skeletal muscle cells of Guizhou cattle / 生物工程学报

The aim of this study was to investigate the expression of growth hormone (GH) gene on skeletal muscle cell proliferation of Guizhou cattle. The coding sequence of cattle GH gene was amplified by reverse transcription PCR, cloned into the pUCM-T vector and then used to construct the GH gene overexpression vector pEGFP-N3-GH. The expression of the GH gene in skeletal muscle-related tissues (psoas major and longissimus dorsi) of Guizhou cattle was determined by real-time fluorescent quantitative PCR (RT-qPCR). This was followed by culturing and identification of the bovine primary skeletal muscle cells. Subsequently, we introduced the GH gene overexpression vector into the cells to investigate its effect on the proliferation of bovine skeletal muscle cells and the expression of insulin like growth factor 1 and 2 genes related to skeletal muscle growth and development. RT-qPCR results showed that the expression level of GH gene was higher in the psoas major than in the longissimus dorsi of Guizhou cattle, and the expression level in the psoas major of Guanling cattle and Weining cattle was significantly higher than in the longissimus dorsi (P<0.05). The transfection and proliferation results showed that pEGFP-N3-GH significantly increased the expression of GH, IGF-1, and IGF-2 genes in skeletal muscle cells compared to pEGFP-N3 (PP<0.05), and that overexpression of the GH gene also significantly increased the proliferation rate of skeletal muscle cells at the four periods examined (PP<0.01). Our results suggest that GH gene can promote the proliferation of skeletal muscle cells of Guizhou cattle and exerts a positive regulatory effect. This lays the foundation for further exploring the mechanism by which the GH gene affects the growth and development of Guizhou cattle.

Heat stroke induces oncosis rather than apoptosis in skeletal muscle cells / 解放军医学杂志

Objective To investigate the oncosis of skeletal muscle cells during heatstroke, and the mechanism severe heatstroke associated with rhabdomyolysis. Methods The human skeletal muscle cells (HSKMC) were divided into control group and heat shock group. The control group was incubated at 37 ℃, 5% CO2 cell incubator, while the heat shock group was exposed at 43 ℃, 5% CO2 cell incubator for 2 hours, followed by incubating at 37 ℃, 5% CO2 cell incubator. At incubation point of 0, 6, 12, and 24 hours, the CCK-8 method was used to detect cell viability; the LDH method was used to detect cytotoxicity; the transmission electron microscope was used to detect cell ultrastructure changes; Annexin -FITC/PI double staining method was used to detect double-positive cell rate; Western blotting method was used to detect porimin and caspase-3 protein expression. Results Compared with the control group, HSKMC cell viability decreased with cytotoxicity increased at 0 h after rewarming in a time-dependent manner (P0.05). Conclusion Heat stroke-induced oncosis rather than apoptosis of skeletal muscle cells.

Effects of Dandelion Extract on the Proliferation of Rat Skeletal Muscle Cells and the Inhibition of a Lipopolysaccharide-Induced Inflammatory Reaction / 中华医学杂志(英文版)

<p><b>Background</b>Dandelion is commonly used in traditional Chinese medicine with several active compounds found in extracts. It has a variety of pharmacological effects, such as a reduction in swelling and inflammation, and detoxification. The mechanism by which dandelion extract inhibits the inflammatory response in skeletal muscle cells remains unknown; therefore, the aim of this study was to investigate the effects of dandelion extract root on the proliferation of skeletal muscle cells and the alleviation of lipopolysaccharide (LPS)-induced inflammatory response in vitro.</p><p><b>Methods</b>Rat skeletal muscle cells were isolated from Sprague-Dawley rat and cultured in vitro which were cultured in basal medium, or medium containing LPS or dandelion extract. Cell counting kit-8 (CCK-8) was employed to measure cell proliferation; meanwhile, the optimal concentration of dandelion extract and treatment time were selected. Crystal violet staining was used to detect the proliferation of muscle cells. Western blotting analysis was used to detect the levels of inflammatory factors, myogenic factor, and p-AKT protein expression.</p><p><b>Results</b>The optimal concentration and treatment time of dandelion extract for the following study were 5 mg/ml and 4 days, respectively. Dandelion extract was found to increase proliferation of rat skeletal muscle cells (t = 3.145, P < 0.05), with the highest effect observed at 5 mg/ml. LPS was found to decrease proliferation of skeletal muscle cells (t = -131.959, P < 0.001), and dandelion extract could against this affection (t = 19.466, P < 0.01). LPS could induce expression of inflammatory factors, including interleukin (IL)-1β, IL-6 and tumor necrosis factor (TNF)-α (IL-1β: t = 9.118, P < 0.01; IL-6: t = 4.346, P < 0.05; TNF-α: t = 15.806, P < 0.05), and dandelion extract was shown to reduce LPS-induced expression of IL-1β, IL-6 and TNF-α (IL-1β: t = -2.823, P < 0.05; IL-6: t = -3.348, P < 0.01; and TNF-α: t = -3.710, P < 0.01). Furthermore, LPS was also shown to decrease expression of myogenic factor, including myod1 and myogenin (MyoD1: t = 4.039, P < 0.05 and myogenin: t = 3.300, P < 0.01), but dandelion extract was shown to against this effect of LPS (MyoD1: t = -3.160, P < 0.05 and myogenin: t = -3.207, P < 0.01). And then, LPS was found to increase expression of p-AKT protein (p-AKT/AKT: t = 4.432, P < 0.05). Moreover, expression of p-AKT protein was found to decrease, with 5 mg/ml of dandelion extract (p-AKT/AKT: t = -3.618, P < 0.05).</p><p><b>Conclusions</b>The findings indicate that dandelion extract plays an important role in skeletal muscle cells viability regulation, promote cells proliferation by increasing level of p-AKT protein expression, and reduce LPS-induced expression of inflammatory factors, inhibiting the inflammatory response of rat skeletal muscle cells.</p>

The effect of heat stress on the permeability, cytoskeleton and cell cycle of human skeletal muscle cell / 中华急诊医学杂志

Objective To study the effect of heat stress on the permeability,cytoskeleton and cell cycle of human skeletal muscle cell (HSKMC).Methods The HSKMC membrane permeability was detected by calcium ion inflow with flow cytometer,the cytoskeleton was stained by CBB 250,and the cell cycle was determined by flow cytometer.Results After 1 h of heat stress on human HSKMC cells under different temperature gradient,the median level of calcium ion was 91.63 in 43 ℃ heat stress group compared with 22.98 in 37 ℃ control group.As temperature increased,thicker and shorter cytoskeleton and stress fiber were shown under the high power lens of microscope.The DNA expression of skeleton cells at G0/G1 stage was 44.13-62.98 in groups under heat stress.Compared with normal control group,DNA expression was much higher in heat stress group,when HSKMC was cultured under 37 ℃ temperature for another 18 h,it kept decreasing DNA expression to a similar level as control group.Conclusions Heat stress can cause calcium iron inflow resulting in intracellular calcium overload,and affect the cytoskeleton leading to loss of normal web ordered arrangement and increased gap in HSKMC cells,which give rise to blocking cell cycle into G0/G1 stage.

Antidiabetic activity of heart wood of Pterocarpus marsupium Roxb. and analysis of phytoconstituents.

The crude powder, ethanolic extract and aqueous, chloroform, hexane and n-butanol soluble fractions of ethanolic extract of heart wood of P. marsupium showed marked improvement on oral glucose tolerance post sucrose load in normal rats. All these fractions except aqueous fraction showed improvement on oral glucose tolerance post sucrose load on streptozotocin (STZ)-induced diabetic rats. The crude powder, ethanolic extract and hexane and n-butanol fractions showed marked decline in blood glucose level on STZ-induced diabetic rats. The ethanolic extract (100 mg/kg body weight) when given to STZ-induced diabetic rats for 10 consecutive days declined blood glucose, improved OGTT and increased their serum insulin levels. The ethanolic extract also showed marked improvement on oral glucose tolerance on high fat-low dosed STZ-induced diabetic rats and neonatally STZ treated rats. The ethanolic extract of P. marsupium also showed marked antidyslipidemic effects on high fat diet fed Syrian golden hamsters. Altered renal and hepatic function markers and serum insulin levels of high fat diet fed-low dosed STZ-treated diabetic rats were also found towards normalization when these animals were treated with ethanolic extract of P. marsupium for 28 consecutive days. The four out of five phenolic C-glycosides isolated from n-butanol fraction of ethanolic extract of P. marsupium enhanced glucose uptake by skeletal muscle cells (C2C12) in a dose dependent manner. It may primarily be concluded that phenolic-C-glycosides present in P. marsupium heart wood are the phytoconstituents responsible for the antihyperglycemic activity and validate the claim of antidiabetic activity of heart wood of P. marsupium.

IGF-I Exerts an Anti-inflammatory Effect on Skeletal Muscle Cells through Down-regulation of TLR4 Signaling

Although exercise-induced growth factors such as Insulin-like growth factor-I (IGF-I) are known to affect various aspects of physiology in skeletal muscle cells, the molecular mechanism by which IGF-I modulates anti-inflammatory effects in these cells is presently unknown. Here, we showed that IGF-I stimulation suppresses the expression of toll-like receptor 4 (TLR4), a key innate immune receptor. A pharmacological inhibitor study further showed that PI3K/Akt signaling pathway is required for IGF-I-mediated negative regulation of TLR4 expression. Furthermore, IGF-I treatment reduced the expression of various NF-kappaB-target genes such as TNF-alpha and IL-6. Taken together, these findings indicate that the anti-inflammatory effect of exercise may be due, at least in part, to IGF-I-induced suppression of TLR4 and subsequent downregulation of the TLR4-dependent inflammatory signaling pathway.

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.

Cloning and expression of Par6A cDNA / 基础医学与临床

Objective Cloning and expression of Par6A.Methods Par6A cDNA was amplified from rat L6 skeletal muscle cells by RT-PCR and the cloning and expression vectors of Par6A were constructed.The expression vector was transfected into 293 cells.Furthermore,the function of Par6A was confirmed by Co-immunoprecipitation.Results Par6A cDNA with approximately 1 kb in length was successfully amplified,and the expression vector of pDsRed-Express-N1-Par6A was constructed.The red fluorescene was seen under fluorescent microscope after 293ET cells were transfected for 24 h using the pDsRed-Express-N1-Par6A vector.The expressed Par6A protein can interacte with PKC?.Conclusion We successfully cloned the Par6A cDNA from rat L6 skeletal muscle cells,which provided a reliable method to study the function of Par6A.

Differentiation of mesenchymal cells derived from mouse fetal liver into skeletal muscle-like cells in vitro / 中国病理生理杂志

AIM: To study whether mesenchymal stem cells (MSCs) from fetal liver can differentiate into skeletal muscle-like cells. METHODS: MSCs were isolated from C57BL/6J mouse fetal liver and were induced by 5-azacytidine and amphotericin B. Myf5 and myogenin were tested by RT-PCR. Desmin and ?-actin was examined by immunocytochemistry. RESULTS: RT-PCR showed that the treated cells expressed Myf5 and myogenin orderly from 6 hours to 72 hours, while the untreated cells did not. Immunocytochemistry confirmed that these cells were positive for desmin and ?-actin and the positive rate was higher in 7 days than that in 14 days. CONCLUSION: MSCs derived from fetal liver can be induced into skeletal muscle-like cells in vitro.

Dynamic Changes of Transcriptional Activities of Nuclei of Rat Liver and Skeletal Muscle Cells in vitro after Severe Burns / 第三军医大学学报

The dynamic changes of the transcriptional activities of the nuclei of the rat liver and skeletal muscle cells were observed in the 6th, 12th, 24th, 48th, and 72nd hours after the animals were inflicted with 30% TBSA third degree burns.It was found that the incorporation of [3H]-UTP into the liver cell nuclei in vitro increased significantly in the 6th and 12th postburn hours. The peak of elevation occurred in the 6th hour postburn (from 7408 ?690 cpm/50?g DNA of normal to 10175 ? 1227 cpm/50?g DNA, P