The Effect Of Conditioned Medium From Hypoxia-treated Adipocytes And Macrophages On Insulin Function In Skeletal Muscle Cells

Objective:To study the roles of obesity and hypoxia on insulin effect in skeletal muscle cells and to exlpore the mechanism of insulin resistance in obesity, GLUT4translocation and phosphorylation of insulin signaling molecules in skeletal muscle cells were determined under incubation with conditioned medium (CM) from adipocytes, macrophages or saturated fatty acid, respectively.Methods:1To prepare CM from adipocytes and macrophages cultured under normoxia and hypoxia (1%O2,5%CO2,94%N2) condition, respectively.2To determine GLUT4translocation under basal and insulin condition by absorptiometric analysis and to determinate phosphorylation of insulin signaling molecules under basal and insulin condition by Western Blot in skeletal muscle cells incubated with or without CM from adipocytes and macrophages.3To determine GLUT4translocation under basal and insulin condition with absorptiometric analysis and to determinate phosphorylation of insulin signaling molecules under basal and insulin condition by Western Blot in skeletal muscle cells with or without palmitate incubation.4To determine TNF-a level in CM from adipocytes and macrophages by ELISA.5To determine levels of adiponectin mRNA and TNF-a mRNA of adipocytes and level of TNF-a mRNA of macrophages under normoxia and hypoxia by Real-time PCR.6To observe the influence on macrophage chemotactic movement to adipocytes with transwell system.7To statistical analyze the data with SPSS software13.0.Results:1The GLUT1increased in adipocytes and macrophages under hypoxia treatment.2Compared to control group, GLUT4translocation in basal and insulin condition showed no significant difference in C2C12and L6skeletal muscle cells incubated with CM from normoxia-treated adipocytes. Compared to control group, GLUT4 translocation in basal condition increased significantly (p＜0.05, p＜0.01) and GLUT4translocation under insulin condition had no significant difference in C2C12and L6skeletal muscle cells incubated with CM from hypoxia-treated adipocytes. Compared to control group, the fold increase above basal of GLUT4translocation after insulin stimulation showed decrease tendency, but no significant difference in C2C12skeletal muscle cells, but decreased significantly in L6skeletal muscle cells (p＜0.05) incubated with CM from hypoxia-treated adipocytes.3Compared to control group, GLUT4translocation under basal and insulin condition showed increase tendency, but no significant difference in C2C12skeletal muscle cells incubated with CM from normoxia-treated macrophages. Compared to control group, the GLUT4translocation under basal condition increased significantly (p＜0.05), and the translocation under insulin condition had an increase tendency but no significant difference in skeletal muscle cells incubated with CM from hypoxia-treated macrophages. Compared to control group, the fold increase above basal of GLUT4translocation after insulin stimulation had a decrease tendency, but no significant difference in skeletal muscle cells incubated with CM from normoxia-treated and hypoxia-treated macrophages, respectively.4Compared to control group, the phosphorylation of Akt under basal condition had an increase tendency but no significant difference and the phosphorylation of Akt under insulin condition had no significant difference in C2C12skeletal muscle cells incubated with CM from normoxia-treated adipocytes. Compared to control group, the phosphorylation of Akt under basal condition had an increase tendency but no significant difference and the phosphorylation of Akt under insulin condition had no significant difference in C2C12skeletal muscle cells incubated with CM from hypoxia-treated adipocytes.5Compared to control group, the phosphorylation of Akt under basal condition had an increase tendency but no significant difference, and the phosphorylation of Akt under insulin condition had no significant difference in C2C12skeletal muscle cells incubated with CM from normoxia-treated and hypoxia treated macrophages.6Compared to control group, the GLUT4translocation under basal condition showed no significant difference and the GLUT4translocation under insulin condition showed a significant decrement in skeletal muscle cells with palmitate incubation. Compared to basal condition, the phosphorylation of Akt under insulin condition incresed in vehicle group. Compared to vehicle group, the phosphorylation of Akt under insulin condition had a significant decrement and the phosphorylation of S6K and IRS1S636/639under insulin condition had no significant difference in skeletal muscle cells with palmitate incubation.7TNF-a increased in CM from hypoxia-treated adipocytes and macrophages.8The mRNA of adiponectin decreased in hypoxia-treated adipocytes, the mRNA of TNF-a increased in hypoxia-treated adipocytes and macrophages.9Compared to nomorxia-treated group, macrophages migration increased in hypoxia-treated group.Conclusions1Adipocytes and macrophages response to hypoxia treatment. CM from adipocytes or macrophages can be made in this way to mimic the hypoxia state of adipose tissue in obesity.2The fold increase above basal of GLUT4translocation after insulin stimulation shows decrease tendency, but no significant difference in skeletal muscle cells incubated with CM from normoxia-treated adipocytes compared to control group. Incubation of CM from hypoxia-treated adipocytes causes insulin resistance in skeletal muscle cells.3The fold increase above basal of GLUT4translocation after insulin stimulation shows decrease tendency, but no significant difference in skeletal muscle cells incubated with CM from normoxia-treated and hypoxia-treated macrophages compared to control group.4Incubation with palmitate causes insulin resistance in a S6K-independent mechanism in skeletal muscle cells.5. Expression of adiponectin decreased and expression of TNF-a increased in hypoxia-treated adipocytes. Expression of TNF-a increased in hypoxia-treated macrophages. Adiponectin and TNF-a may involve in the mechanism of insulin resistance in skeletal muscle cells caused by CM from hypoxia-treated adipocytes.6. Adipocytes under hypoxia condition attract more macrophages, which may play a role in the process of obesity to whole body insulin resistance.