Role of GDF11 in skeletal muscle cell insulin resistance induced by palmitate / 中国药理学通报

Aim To investigate the role of GDF11 in palmitate induced skeletal muscle insulin resistance.Methods The C2C12 cells were sorted into control group, GDF11 intervention group, palmitate group and GDF11 combined with palmitate group.Cell viability was measured by CCK-8, and the glucose uptake was determined by 2NBDG.The mRNA level of myotube marker genes(desmin,myogenin), insulin mediate glucose uptake related genes(GLUT-4,IRS-1) and PGC-1α were tested by RT-PCR.The protein expression of PGC-1α was detected by western blot.Results GDF11 had little effect on cell viability of skeletal muscle cells.Compared with control group, the glucose uptake and the expression of GLUT-4,IRS-1,PGC-1α were significantly decreased by palmitate intervention.Compared with palmitate group, the glucose uptake and the expression of GLUT-4,IRS-1,PGC-1α were not significantly changed by GDF11.Conclusion Palmitate can induce skeletal muscle cell insulin resistance, but GDF11 may not significantly improve the skeletal muscle cell insulin resistance.

Metformin ameliorates PA-induced skeletal muscle insulin resistance by suppressing SREBP-1c / 中国药理学通报

Aim Metformin has been the first-line oral agent for the treatment of type 2 diabetes. The results from preliminary studies suggested that sterol regulatory element binding protein-1c( SREBP-1c) inhibited the transcription of insulin receptor substrate-1 ( IRS-1), which plays a key role in PA-induced skeletal muscle insulin resistance. In the current study, we investiga-ted the role and mechanism of SREBP-1c in metformin ameliorating PA-induced skeletal muscle insulin resist-ance. Methods L6 cells were treated with metformin (1,10 mmol·L - 1 ) for 24h in 500 μmol·L - 1 PA-in-duced insulin-resistant state and then harvested for pro-tein and glucose uptake assay. Glucose uptake was performed by 2-NBDG method. The protein expression of SREBP-1c, FAS, p-IRS-1 ( Tyr608 / 612), IRS-1, p-AKT ( Ser473 ) and AKT was detected by western blot. The effects of metformin on SREBP-1c and IRS-1 gene transcription were assessed by a dual-luciferase reporter assay. CHIP assay was performed to examine the binding of SREBP-1c protein to the IRS-1 promoter region by metformin treatment. Results PA treatment decreased glucose uptake in L6 myotubes. The protein expression of SREBP-1c and its downstream molecule FAS was increased significantly after exposure to PA. By contrast, the proteins related to insulin signaling pathway including IRS-1, p-IRS-1( Tyr608 / 612) and p-AKT ( Ser473) / AKT were decreased significantly. Metformin increased glucose uptake in a dose-depend-ent manner compared to PA-cultured L6 cells. The SREBP-1c and FAS protein levels were decreased by metformin treatment. Correspondingly, p-IRS-1 (Tyr608 / 612), IRS-1, p-AKT(Ser473) / AKT protein levels were increased significantly. The results from dual-luciferase reporter assay indicated metformin sup-pressed SREBP-1c promoter activity and enhanced IRS-1 promoter. The results from CHIP assay showed that metformin decreased binding of SREBP-1c protein to the IRS-1 promoter region (about 30% ). Conclu-sion Metformin can improve PA-induced muscular in-sulin resistance by suppressing SREBP-1c.

Effect of overexpression of LYRM1 gene on reactive oxygen species in skeletal muscle cells / 中华实用儿科临床杂志

Objective To explore the effect of LYRM1 overexpression on production of reactive oxygen species (ROS) in skeletal muscle cells.Methods Rat myoblasts(L6) were transfected with either an empty vector or a LYRM1 expression vector.Cells were screened and the expression of LYRM1 protein in cells was identified.L6 cells were incubated in culture solution with H2-DCFDA after they were differentiated.Then fluorescence intensity of ROS in L6 was observed by fluorescence microscope,and the content of ROS was determined by flow cytometry.Results The relative fluorescence intensity of ROS in L6 overexpressing LYRM1 was 24.8933 ± 4.4574,while that in contrast cells was 13.1512 ± 0.7347,the difference between them was significant(t =24.12,P =0.00).Conclusions Overexpression LYRM1 can increase the production of ROS in skeletal muscle cells.LYRM1 overexpression may be influence the mitochondrial function and induce the mitochondrial damage of skeletal muscle cells.

Spontaneous stage differentiation of mouse-virulent Toxoplasma gondii RH parasites in skeletal muscle cells: an ultrastructural evaluation

Although the predilection for Toxoplasma gondii to form cysts in the nervous system and skeletal and heart muscles has been described for more than fifty years, skeletal muscle cells (SkMCs) have not been explored as a host cell type to study the Toxoplasma-host cell interaction and investigate the intracellular development of the parasite. Morphological aspects of the initial events in the Toxoplasma-SkMC interaction were analysed and suggest that there are different processes of protozoan adhesion and invasion and of the subsequent fate of the parasite inside the parasitophorous vacuole (PV). Using scanning electron microscopy,Toxoplasma tachyzoites from the mouse-virulent RH strain were found to be attached to SkMCs by the anterior or posterior region of the body, with or without expansion of the SkMC membrane. This suggests that different types of parasite internalization occurred. Asynchronous multiplication and differentiation of T. gondii were observed. Importantly, intracellular parasites were seen to display high amounts of amylopectin granules in their cytoplasm, indicating that tachyzoites of the RH strain were able to differentiate spontaneously into bradyzoites in SkMCs. This stage conversion occurred in approximately 3 percent of the PVs. This is particularly intriguing as tachyzoites of virulent Toxoplasma strains are not thought to be prone to cyst formation. We discuss whether biological differences in host cells are crucial to Toxoplasma stage conversion and suggest that important questions concerning the host cell type and its relevance in Toxoplasma differentiation are still unanswered.

Effect of Intravenous Immuoglobulin on Interleukin-6 Production in Human Skeletal Muscle Cell Line (HM4)

BACKGROUND: Intravenous immunoglobulin (IVIg) has been widely used in the management of patients with various autoimmune neurological diseases, however, its action mechanisms have not fully been elucidated yet. This study focused on the effects of IVIg on the production of interleukin-6 (IL-6), one of major proinflammatory cytokine, using a human skeletal muscle cell line (HM4). METHODS: After HM4 cells were cultured in Dulbecco's modified eagle's medium (DMEM) containing 5% fetal bovine serum for 24 h, the culture medium was changed with serum-free media. TNF-alpha (tumor necrosis factor-alpha, 100 ng/mL) and IVIg (5 mg/mL) were treated alone or in combination and cultured for various time. RT-PCR and ELISA kit were employed for mRNA expression and secretion of IL-6, respectively. RESULTS: Treatment with TNF-alpha or/and IVIg significantly induced IL-6 mRNA expression (p<0.001). Although IL-6 production was markedly increased by TNF-alpha (p<0.001), IVIg treatment alone or in combination with TNF-alpha had no effect on the production of IL-6 except at 6 h after the treatment. CONCLUSIONS: IVIg seems not to have a significant effect on IL-6 production as an action mechanism of its immunomodulatory capabilities, at least in the HM4 cell line.

The Expression of Cytokines and Chemokine mRNA by Human Skeletal Muscle Cell Line (SKM14)

BACKGROUND: Muscle is a target of immunological injury in several muscle diseases, such as idiopathic inflammatory myopathy. However, it is also a target for gene therapy. Therefore, it is important to understand the immunological capabilities of muscle cells. To assess as to whether muscle cells are actively involved in the inflamed muscle tissue, a human skeletal muscle cell line was tested for the expression of several cytokines and chemokine at the mRNA level. METHODS: A human skeletal muscle cell line (SKM14) had been developed by a retroviral vector encoding v-myc transfection into a 12-week-old human fetal skeletal muscle tissue characterized by the immunostaining of several musclespecific markers. Human skeletal myoblasts of this cell line were tested for their capacity to express different cytokines (IL-1beta, -6, -10, -12, -15, and TNF-alpha) and chemokine (IL-8) mRNA levels at the basal state and in the presence of TNF-alpha(10 ng/ml). RESULTS: The SKM14 cell line was confirmed to be able to express various cytokines constitutively (IL-6, -8, -12, -15, and TNF-alpha) and in the presence of TNF-alpha(IL-1beta, -6, -8, -10, -12, -15, and TNF-alpha). CONCLUSIONS: Our results suggest that muscle cells may play a role as immunocompetent cells.