| Objective:In recent years, the incidence of type2diabetes mellitus presents the fast growth trend, and the death toll caused by diabetes mellitus ranks only after cardiac-cerebral vascular diseases and malignant tumor. Diabetes related complications such as coronary heart disease, diabetic nephropathy, diabetic retinopathy and diabetic peripheral neuropathy are the main reasons for death and disability, which threatened human health seriously. Because insulin resistance is an important pathogenesis of type2diabetes mellitus in early stage and there should be many years of insulin resistance before the onset of type2diabetes mellitus, it is the key point that taking measures to improve insulin sensitivity in insulin resistance stage to treat type2diabetes mellitus and prevent diabetes and related complications.The main methods to treat diabetes mellitus are oral medications, insulin injection, diet control and etc. Although those methods have good curative effect to improve blood sugar content and insulin sensitivity, there are some toxic and side effects. But over time, the damage to islet β cells cannot be avoided, and the quality of life in diet will be greatly reduced. Therefore, looking for a therapy with good curative effect, stability and non-toxic side effects is a major subject in front of the medical professionals.Acupuncture has been more and more widely used in preventing and controlling insulin resistance related diseases with obvious curative effect, which is gradually confirmed in clinical researches. In order to further explore the mechanisms of acupuncture treatment on insulin resistance, this project focused on "" electropuncture on insulin resistance model of Wistar rats after high-fat diet, under the guidance of the theory of traditional Chinese medicine as "preventive treatment of disease", by observing SIRT1activating PGC-1α and its downstream related genes and proteins expression in skeletal muscle, a insulin target organ, by deacetylation. Priority observations were made on mitochondria biosynthesis and oxidative related gene and protein expression and the relations between these changes and increased insulin sensitivity. This project will further reveal the function of "" electroacupuncture on insulin resistance in mitochondrial glucose and fatty acid utilization to provide the theoretical support and scientific basis for clinical use of acupuncture treatment on insulin resistance and related diseases in the future.Methods:Forty-eight male Wistar rats at8weeks of age were randomly divided into4groups:normal group (n=12), model group (n=12), electroacupuncture group (EA, n=12) and resveratrol group (n=12). For those in normal group, the basal feed (total heat is3.8kcal/g, including70%carbohydrate,20%protein and10%fat) was given; while high-fat diet (total heat is5.4kcal/g, including38.5%carbohydrate,15%protein and46.5%fat) for8weeks to establish the rat model with insulin resistance. After8weeks’ feeding,2Wistar rats of each group were randomly selected to detect whole body insulin sensitivity which was presented with glucose infusion rate (GIR) by high insulin-glucose clamps. During treatment, all the rats were fed with basal feed and free access to food and water. For EA group, Zusanli (ST36) and Fenglong (ST40) were inserted perpendicularly with3-5mm, while Guanyuan(CV4) was inserted obliquely towards processus xiphoideus and Zhongwan(CV12) towards symphysis pubis with3-5mm. After twirling the needles for lmin, HANS LH202H electric acupuncture therapeutic apparatus was used with continuous wave, frequency of2Hz and intensity of1mA for10min, once per day, five times per week for8weeks. One pair of electrodes on the homolateral Zusanli (ST36) and Fenglong (ST40) and the other on Guanyuan(CV4) and Zhongwan(CV12). Purpose-made clothes were used to fix the rats during the treatment. For resveratrol group, the resveratrol (100mg/kg) was given by intragastric dministration on10:00am everyday for8weeks. And then, insulin sensitivity in skeletal muscle was detected. The expression of SIRT1mRNA, PGC-1αmRNA and NRF-1mRNA were detected with real-time PCR, protein expression of PGC-1α and NRF-1with western blot, and the activity of CPT-1, MCAD and LCAD were detected with colorimetric method. All the data were given statistic analysis.Results:1. Basic biochemical information of the rats in each group:fasting blood glucose levels of the rats in each group has no obvious difference. Fasting plasma insulin level in model group was significantly higher than that in normal group (p<0.05), an average glucose infusion rate GIR60～120(～20%, P<0.05) was significantly lower than normal group, suggesting that rats in the model group has a definite system insulin resistance. After8weeks treatment with EA or resveratrol intervention, fasting plasma insulin levels significantly reduced, GIR60～120increased significantly (p<0.05), suggesting both EA and resveratrol could improve insulin sensitivity induced by the high-fat diet system. In addition, the fasting blood lipids such as TG and FFA in model group were higher than that in normal group (p<0.05). TG and FFA significantly reduced after EA, tending to normal level. Compared with model group, there was significant difference (p<0.05); while there was no obvious effect after the intervention of resveratrol.2. The expression of SIRT1mRNA in skeletal muscle of the rats in ach group:the expression level of quadriceps nucleus SIRT1mRNA in EA group increased significantly after8weeks treatment. Compared with model group, there was significant difference (P<0.05). The expression level of quadriceps nucleus SIRT1mRNA in resveratrol group rats increased significantly. Compared with model group, there was significant difference (P<0.05). There was no statistically significant difference between resveratrol group and EA group.3. The protein expression of PGC-la in skeletal muscle of the rats in each group:compared with normal group, the protein expression of PGC-1α decreased in the skeletal muscle of model group with significant difference significance (P<0.01); protein expression of PGC-la in the skeletal muscle of resveratrol and EA groups increased more significantly than that in model group with extremely significant (P<0.01); The protein expression of PGC-1α in skeletal muscle between EA and resveratrol group has no obvious difference.4. The protein expression of NRF-1in skeletal muscle of the rats in each group:compared with normal group, the expression of skeletal muscle protein NRF-1in model group decreased with statistically significant (P<0.05); the expression of NRF-1in resveratrol and EA groups were higher than that in model group with significant difference (P<0.05); there was no significant difference between EA and resveratrol group.5. The expression of PGC-lamRNA in skeletal muscle of the rats in each group:compared with normal group, the expression of skeletal muscle PGC-1αmRNA in model group decreased with significant difference (P<0.05); the expression of PGC-1αmRNA in resveratrol and EA groups were higher than that in model group with significant difference (P<0.05); there was no significant difference between EA and resveratrol group.6. The expression of NRF-1mRNA in skeletal muscle of the rats in each group:compared with normal group, the expression of skeletal muscle NRF-1mRNA in model group decreased with significant difference (P<0.05); the expression of NRF-1mRNA in resveratrol and EA groups were higher than that in model group with significant difference (P<0.05); there was no significant difference between EA and resveratrol group.7. The activity detection of CPT-1, MCAD and LCAD in mitochondria of the rats in each group:after8weeks’ treatment, the activity of SS mitochondrial CPT-1, MCAD and LCAD in the rats skeletal muscle of EA group increased significantly with significant difference compared with that of model group (P<0.05). the activity of SS mitochondrial CPT-1, MCAD and LCAD in the rats skeletal muscle of resveratrol group increased significantly with significant difference compared with that of model group (P<0.05). There was no significant difference between EA and resveratrol group.Conclusion:1. Electroacupuncture may increase the expression and activity of SIRT1in insulin target organ and activate PGC-1α via deacetylation.2. Electroacupuncture may reduce or control insulin resistance by inducing the proteins and enzymes related with mitochondria biosynthesis and oxidative and enhance the oxidizability of mitochondria following the activation of SIRT1/PGC-1α.3. Electroacupuncture may improve insulin resistance in skeletal muscle of the rats fed with high fat diet by correcting the balance between lipid transport and fatty acid oxidation in SS mitochondria of skeletal muscle so as to reduce the ectopic deposition of lipids. |
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