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Objective: To study insulin resistance (insulin resistance, IR) state the function of skeletal muscle mitochondria. Hyperinsulinemic - euglycemic clamp experiments evaluate insulin sensitivity based on the extraction of the skeletal muscle mitochondria, the determination of respiratory function and fatty acid β-oxidation state determination, in order to evaluate the skeletal muscle mitochondrial dysfunction and IR whether there is a close relationship. Method: 1. Establish IR 1 diabetes mellitus (type 1 diabetes mellitus, TIDM) rat model of type 2 diabetes (type 2 diabetes mellitus, T2DM) 3, while the establishment of normal controls (normal control, NC) group. Healthy male Sprague-Dawley (SD) rats were randomly divided into 10 NC group, IR group 10 T1DM group of 15, T2DM group 15. NC group and T1DM group rats fed with normal diet, IR group and T2DM group rats fed a high sugar high fat diet. 60mg/kg of streptozotocin 8 weeks after injection of the the T1DM group of rat peritoneal NC rats by intraperitoneal injection of an equal volume of citrate buffer; T2DM rats intraperitoneal injection of 30mg/kg of streptozotocin factors create a the model IR rats by intraperitoneal injection of citrate buffer volume. 2 after the success of the model, the rats of the four groups of rats were hyperinsulinemic - euglycemic clamp experiments clamp process 60-120 minutes average glucose infusion rate (glucose infusion rate, GIR) to reflect insulin sensitivity. Take rat soleus muscle approximately 1g mitochondrial separation, purification, all operations are carried out under strict state at 4 ℃. Clark oxygen electrode measured mitochondrial respiration rate in state Ⅲ Ⅳ state respiration rate of fresh extracted mitochondrial respiratory function measured ratio of the two is the mitochondrial respiratory control ratio (as respiratory control rate, RCR). Determination of mitochondrial fatty acid beta-oxidation state, on the one hand by protein immunoblotting (western blot) to detect mitochondrial carnitine acyl transferase - Ⅰ (carnitine palmitoyl transferase-Ⅰ, CPT-Ⅰ) of the protein, on the other hand application 14C-labeled isotope method for determination of the activity of CPT-Ⅰ. Results: 1. 8 weeks 4 rats weight (kg) for the NC group, 0.384 ± 0.08, IR group, 0.56 ± 0.04, the T1DM 0.444 ± 0.03, T2DM group, 0.52 ± 0.05, which IR group and T2DM rats body weight compared with NC group and the the T1DM group were significantly increased (P lt; 0.05). 4 groups of rats after modeling fasting glucose (fasting blood glucose, FBG) (mmol / L), respectively, for the NC group, 4.03 ± 0.93, IR group, 4.49 ± 0.71, T1DM group 20.90 ± 5.15, T2DM group 13.36 ± 4.56, which T1DM group the mode standard and T2DM group FBG reach into and compared with the NC group and IR group were significantly higher (P lt; 0.05). 2.4 rats GIR (mg · kg-1 · min-1) were the NC group 12.12 ± 2.70, IR group, 5.09 ± 0.94, 12.85 ± 2.43 T1DM, the T2DM group 5.99 ± 1.79. Except NC group with T1DM group, no significant difference in the IR group with T2DM rats GIR, the 2 groups GIR any remaining differences were statistically significant (P lt; 0.05), the IR group GIR significantly lower than NC group and T1DM group, the T2DM group GIR significantly lower than the NC group and T1DM group. 3.4 rat skeletal muscle mitochondria RCR NC group, 4.11 ± 0.30, the IR group 3.28 ± 0.25, 3.81 ± 1.17 T1DM, the T2DM group 3.49 ± 0.27. IR group and RCR T2DM group was significantly lower than the NC group (P lt; 0.05) the IR group RCR was significantly lower than T1DM group (P lt; 0.05). CPT-Ⅰ protein expression by Western blot was used to detect skeletal muscle mitochondria, relative gray ratio by the software scans quantitative protein bands were NC group, 0.43 ± 0.19, the IR group 0.18 ± 0.05, T1DM group 0.244 ± 0.02, T2DM group 0.22 ± 0.17. IR group and T2DM group CPT-Ⅰ protein expression were lower than the NC group (P lt; 0.05). 4 groups between skeletal muscle mitochondrial activity of CPT-Ⅰ no significant differences (P gt; 0.05). Conclusion: IR state impaired skeletal muscle mitochondrial respiratory function, reduced CPT-Ⅰ protein, there is a close relationship between skeletal muscle mitochondrial dysfunction and IR. 2 high glucose levels in skeletal muscle mitochondrial respiratory function and fatty acid β-oxidation state no obvious abnormalities.
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