Study Of Muscular Mitochondrial Function In Insulin Resistance Rats

Objective:To study the function of skeletal muscle mitochondria under the state of insulin resistance (IR). Extract the skeletal muscle mitochondria on basis of assessing insulin sensitivity by hyperinsulinemic euglycemic clamp experiment, make respiratory efficiency determination and fat acidβ-oxidation state determination to evlauate if there exists close relationship between the dysfunction of skeletal muscle mitochondria and IR.Methods:1. Establish three rat models as IR, type 1 diabetes mellitus (T1DM), type 2 diabetes mellitus (T2DM), meanwhile setting up normal control (NC) group. Randomly divide 50 healthy male Sprague-Dawley (SD) rats into NC group (10rats), IR group (10 rats), T1DM group (15rats), T2DM group (15 rats). Of which, NC group and T1DM group rats were fed with common diet, IR group and T2DM group rats were fed with high glucose and high lipid diet. After 8 weeks'feeding, inject 60mg/kg streptozotocin to the abdominal cavity of T1DM group rats and 30mg/kg streptozotocin to the abdominal cavity of T2DM group rats and establish relevant models. Meanwhile, inject 60mg/kg Citrate buffer to the abdominal cavity of T1DM group rats and 30mg/kg Citrate buffer to the abdominal cavity of T2DM group rats as a contrast.2. After successful modeling, make hyperinsulinemic euglycemic clamp experiment on the four groups of rats, the average glucose infusion rate (GIR) of 60 minutes to 120 minutes during the process of clamping is used to reflect the insulin sensitivity of different groups of rats. 3. Take 1g soleus muscle of rat to make separation and purification of mitochondria, all of which were made under the temperature of 4℃. Make respiratory efficiency determination on newly extracted fresh mitochondria by adopting Clark oxygen electrode method and obtain mitochondriaⅢstate respiration rate andⅣstate respiration rate, the ratio of the two is the respiratory control rate (RCR) of mitochondria.4. Determine fat acidβ-oxidation state of mitochondria. On the one hand, test the protein expression of carnitine palmitoyl transferase-Ⅰ(CPT-Ⅰ) of mitochondria through western blot, on the other, test CPT-Ⅰenzyme activity applying 14C marked isotope method.Results:1. The body (kg) weight of the rats that had been fed for 8 weeks were 0.38±0.08 for NC group,0.56±0.04 for IR group,0.44±0.03 for T1DM group, and 0.52±0.05 for T2DM group respectively, of which, the body weight of IR group and T2DM group rats increased obviously compared to that of T1DM group and NC group (P<0.05). The fasting blood glucose (FBG) (mmol/L) of the four groups rats after model establishment were 4.03±0.93 for NC group,4.49±0.71 for IR group,20.90±5.15 for T1DM group,13.36±4.56 for T2DM group, of which, that of T1DM group and T2DM group increased obviously compared to that of NC group and IR group (P<0.05), reminding the success of model establishment.2. The GIR (mg.kg-1.min-1) for the four groups of rats were 12.12±2.70 for NC group,5.09±0.94 for IR group,12.85±2.43 for T1DM group, and 5.99±1.79 for T2DM group respectively. Except the GIR difference for NC group and T1DM group rats, IR group and T2DM group rats had no statistical significance, the GIR difference for any of the rest two groups randomly divided among the four had statistical significance (P<0.05), the GIR for IR group was obviously lower than that of NC group and T1DM group, and the GIR for T2DM group was obviously lower than that of NC group and T1DM group.3. The RCR for the four groups of rats were 4.11±0.30 for NC group, 3.28±0.25 for IR group,3.81±1.17 for T1DM group, and 3.49±0.27 for T2DM group respectively. The RCR for IR group and T2DM group were obviously lower than that of NC group (P<0.05), and the RCR for IR group was obviously lower than that of T1DM group (P<0.05).4. Test protein expression of skeletal muscle mitochondria CPT-Ⅰthrough western blot, quantify the relative gray value of each protein band by software scanning, which were 0.43±0.19 for NC group,0.18±0.05 for IR group,0.24±0.02 for T1DM group, and 0.22±0.17 for T2DM group respectively. The relative gray value for NC group was obviously higher than that of IR group and T2DM group (P<0.05). The results of determination of the 14C marked enzyme activity of skeletal muscle mitochondria CPT-Ⅰ:the difference between the 4 groups had no statistical significance (P>0.05).Conclusions:1. The respiratory efficiency of skeletal muscle mitochondria under IR state was damaged, the CPT-Ⅰprotein expression decreased, there existed close relationship between dysfunction of skeletal muscle mitochondria and IR.2. The respiratory efficiency and fat acidβ-oxidation state of skeletal muscle mitochondria under high glucose state were not damaged.