Study On The Role And Mechanism Of Globular Domain Of Adiponectin (gAd) In Improving Insulin Resistance Of The 3T3-L1 Adipocytes

Objective:The study was to the role and mechanism of Globular domain of adiponectin (gAd) in improving Insulin resistance of the 3T3-L1 adipocytes.Methods:Culturing and differentiating 3T3-L1 cells, use oil red O for lipids when the cells were matured. After dialysis of gAd, determine protein concentration by Coomassie brilliant blue. Insulin resistance model of 3T3-L1 adipocytes was prepared with PA. Intervention the adipocytes which have been generated IR with different concentrations of gAd (250 ng/mL,500 ng/mL,1000 ng/mL) for 5h. Blank control group, the cell culture medium glucose content was detected with the glucose oxidize method, the mRNA expression of IRS-1, P13K, PKB, GLUT-4, AMPK, CPT-Ⅰwere detected with real-time quantitative PCR method. Phosphorylation AMPK Thr-172 and IRS-1 was detected by Western blot. SPSS 17.0 statistical analysis software was used to establish a database and analysis of the data, which data were expressed as mean±standard deviation (x±s), and the test level was a= 0.05.Results:1. Different concentrations of PA (0.25mmol/L,0.5mmol/L, 1.Ommol/L) effect on 3T3-L1 adipocytes for 24h can inhibited glucose uptake. Compared with the control group, the rate of glucose uptake in all experimental groups decreased 5.25%,10.29%,14.54%. So 3T3-L1 adipocytes can generate IR by PA.1.0 mmol/L of PA can inhibit glucose uptake significantly.2. Different concentrations of gAd (250 ng/mL,500 ng/mL,1000 ng/mL) acting in 3T3-L1 cells which have generated IR, can stimulate glucose uptake significantly (F=35.499, r=-0.883, P=0.005), Compared with the control group,250ng/mL of gAd can besignificantly enhanced glucose uptake (p=0.000).3. Different concentrations of gAd gradually increased the mRNA expression levels of AMPK (F=359.374, r=0.986, P=0.000), CPT-I (F=180.234, r=0.973, P=0.000) and in a dose-dependent manner. The experimental group and control group were significantly different (P<0.01), between groups in all experimental groups had significant differences (P <0.01). The results of Western blot illustrate that AMPK Thr-172 phosphorylation levels increased gradually followed by the concentration of gAd (F=269.407, r=0.982, P=0.000).4. Affect of gAd on IR 3T3-L1 adipocyte models of insulin P13K pathwayCompared with the control group,250 ng/mL gAd group mRNA expression levels of GLUT-4 (P=0.02)increased than the control group, but the IRS-1, PI3K, PKB have no significant difference compared with the control group; 500 ng/mL gAd Group mRNA expression levels of IRS-1 (P=0.031), P13K (P=0.002), PKB (P=0.01), GLUT-4 (P=0.000) significantly increased compared with controls, and the mRNA expression levels of P13K (t=-3.66 P=0.022), PKB (t= 4.161 P=0.014), GLUT-4 (t=-15.039, P=0.000) significantly increased compared with 250 ng/ mL gAd; 1000 ng/mL gAd group mRNA expression levels of IRS-1 (P=0.031), P13K (P= 0.000), PKB (P=0.000), GLUT-4 (P=0.000), was significantly increased compared with controls (P＜0.01), and mRNA expression levels of GLUT-4 (t=10.483, P=0.000), significantly increased compared with 500 ng/mL gAd group. The results of Western blot illustrate that IRS-1 phosphorylation levels increased gradually followed by the concentration of gAd (F=360.345, r=0.968, P=0.000).Conclusions:1. GAd can promote glucose uptake of 3T3-L1 adipocyte model which have generated IR.2. The mechanism of gAd improve insulin resistance may be increase AMPK Thr-172 phosphorylation, activation of AMPK, thus lift the inhibition on CPT-Ⅰ, increase free fatty acid oxidation.3. GAd can improve insulin resistance by gradually increased the mRNA expression levels of IRS-1,PI3K,PKB,GLUT-4,and increase IRS-1 phosphorylation levels.