Intervention Of Silibinin In Insulin Resistance In Skeletal Muscle Cells Induced By Palmitate

OBJECTIVE:To investigate the methods for proliferation and differentiation of C2C12myoblasts in vitro, and observe the characteristics of myoblasts and myotubes. To establish and optimize the proliferation and differentiation condition, model of insulin resistance induced by palmitate which is based on the fluorescent probe for glucose uptake, and study on effects of silibinin upon insulin resistance and potential mechanisms.METHODS:(1) Cells were cultured with DMEM containing10%fetal bovine serum, the proliferation status being detected by cell morphology through phase contrast microscopy, and then differentiated into myotubes in DMEM containing1%fetal bovine serum (v/v) or2%horse serum (v/v), being observed under the inverted phase contrast microscope for the alteration of their appearance. Cells were also obvserved by Fluorescence microscope using Hoechst33342for nuclear staining.(2)2-NBDG was utilized as the fluorescent probe for glucose uptake by skeletal muscle cells, the the most suitable concentration and exposure period of2-NBDG fluorescence were valuated through microplate reader and fluorescence microscope.(3) After incubation with palmitate for16hours, glucose uptake by skeletal muscle was investigated under insulin stimulation, insulin sensitivity of skeletal muscle was appraised, and insulin resistance was modeled.(4) To study the effects of silibinin on insulin resistance induced by palmitate and potential mechanisms, glucose uptake was tested, GLUT4expression and translocation were investigated, the expression of key components in insulin signaling pathway such as AKT, p-AKT(Ser473), p-IRS-1(Ser307) and related serine/threonine kinases such as p-JNK、p-IKKβ、p-PKC-0were examined through Western blotting. Meanwhile, cell vialibity, ROS and LDH were investigated through MTT method, fluorescence microplate and ELISA, respectively.RESULTS:(1) Normal myoblasts were spindle-shaped after adherent. One myotube were fused from multiple myoblasts, and myotubes showed polarity, arranged gradually along a direction and formed into myotubes. Multiple nuclears appeared by Hoechst staining. The amount of myotubes differentiated in DMEM supplemented with2%horse serum (v/v) was less than that in DMEM containing1%fetal bovine serum (v/v), however, they were thicker.(2) The most suitable concentration and exposure period for myotubes incubation were100μmol·L-1and30min, respectively.(3) Compared with contrl group, after incubated with0.75mM palmitate, glucose uptake of skeletal muscle was significantly lower and the expression and translocation of GLUT4were significantly reduced.(4) Compared with the model group, silibinin concentration dependently increased glucose uptake, the expression and translocation of GLUT4under insulin stimulation. Silibinin significantly increased p-Akt (Ser473) expression under insulin stimulation, which was reversed by Wortmannin, a selective PI3K inhibitor. Silibinin significantly reduced the phosphorylation of IRS-1(Ser307), which was increased by palmitate incubation. Meanwhile, silibinin markedly decreased the expression of p-JNK and p-IKKβ,which was significantly enhanced by palmitate. There was no significant change of p-PKC-0under palmite and silibinin incubation. Palmitate induced the increased LDH and ROS, and the declined cell viability, which was improved by silibinin.CONCLUSION:(1) Myoblasts proliferated fast in vitro, can be differentiated to cells characterizing matured myotubes, which was controllable and identifiable and can be used to establish model of insulin resistance. The amount of the myotubes differentiated in DMEM containing2%horse serum (v/v) was less than that in DMEM containing1%fetal bovine serum (v/v), however they were thicker. DMEM containing2%horse serum (v/v) was used to differentiate C2C12in the following study because of good view under microscope.(2)2-NBDG-can be taken by myotubes and stay inside the cells, not being metabolized, which can be used as an optical probe for glucose uptake. After16hours of incubation with0.75mM palmitate, insulin resistence was induced in C2C12myotubes.(4) Silibinin reduced oxidative stress, inhibited activation of protein kinase such as JNK and IKKβ,improved abnormality of insulin signaling pathway, promoted translocation of GLUT4from cytosol to membrane, increased glucose uptake thus improved insulin resistance induced by palmitate.