| Objectives Obesity is one of the major public problems affecting human health.In obesity,increased circulating lipid levels and changes in intracellular signaling can cause insulin resistance and lead to liver steatosis.Silent mating type information regulation 2tomolog l（SIRT1）and adenosine-activated protein kinase（AMPK）have been shown to correlate and play important roles in the regulation of lipid homeostasis and insulin sensitivity.SIRT1 is histonedeacetylases Ⅲ,which was initially thought to be involved in physiological functions such as extending biological life span and limiting calorie intake.In recent years,more and more studies have revealed that SIRT1 plays a key role in sugar and fat metabolism,and SIRT1 activators such as resveratrol have been used as anti-aging and antioxidant products to improve energy metabolism.AMPK,as an important kinase regulating energy homeostasis,is one of the central regulators of eukaryotic cell and organism metabolism,responsible for regulating the energy input and output of cells,maintaining the smooth operation of cell physiological activities,and mainly regulating protein metabolism,lipid metabolism and carbohydrate metabolism.Therefore,in this study,the insulin resistance obesity model induced by high fat diet was taken as the research object,and the intervention was conducted by calorie control,electroacupuncture and electroacupuncture combined with calorie control.The regulatory effect of SIRT1 and AMPK on liver lipid metabolism was taken as the entry point.To observe the effects of different intervention methods on body weight,Lee’s index,lipid metabolism,insulin sensitivity,liver histomorphology and protein expression of SIRT1,AMPK and phosphorylated adenosine-activated protein kinase（p-AMPK）in the liver of insulin resistant obese rats.Furthermore,the effects of electroacupuncture and calorie control on lipid metabolism and SIRT1/AMPK expression in liver of insulin-resistant obese rats were analyzed and evaluated.Methods1.Modeling: Seventy five-week-old SPF Wistar male rats were selected according to random number table method,15 rats were fed with ordinary diet,and 55 rats were fed with high fat diet to induce insulin resistance obesity model.After feeding for 10 weeks,the weight and Lee’s index of rats were used to evaluate the obesity model.Three rats in the normal diet group and 12 rats in the high fat diet group were randomly selected for hyperinsulinemic euglycemic clamp to measure glucose infusion rate（GIR）and evaluate insulin sensitivity.Meeting both criteria is considered successful.Grouping: 10 rats fed with ordinary diet were randomly selected and divided into normal group,and the rats successfully modeled were randomly selected and divided into model group,Calorie control group,electroacupuncture group and electroacupuncture combined with Calorie control group,with 10 rats in each group.2.Intervention methods: Normal group: ordinary feed;Model group:high-fat feed;Caloric control group: high-fat diet was fed alone and 30% caloric restriction was given every day;Electroacupuncture group: high-fat feed,electroacupuncture intervention;electroacupuncture combined with Calorie control group:electroacupuncture and Calorie control intervention.Electroacupuncture intervention methods: acupoint selection: "Zusanli"（ST36）,"Fenglong"（ST40）,"Guanyuan"（CV4）,"Zhongwan"（CV12）;Use 0.25×25 mm disposable millimeter needle,connect HANS LH202 H electroacupuncture instrument electrode,parameter setting:2 Hz,1 m A,continuous wave.Electroacupuncture for 15 minutes was administered 3 times a week for 8 weeks.3.Indicators: Body weight and anal nose length of all rats were measured at 0,2,4,6 and 8 weeks after intervention,and Lee’s index was calculated.Intraperitoneal glucose tolerance（IPGTT）and insulin tolerance（IPITT）were measured in all rats at the 6th week of intervention.After treatment,3 rats in each group were randomly selected for hyperinsulinemic euglycemic clamp to detect GIR,and the contents of Insulin,total cholesterol（TC）,triglyceride（TG）and free fatty acid（FFA）in the remaining rats were determined by enzymed-linked immunosorbent assay（ELISA）.The changes of liver tissue structure were detected by hematoxylin-eosin staining（HE）,and the expressions of SIRT1,AMPK and p-AMPK proteins in liver tissues were detected by western blotting（WB）and immunohistochemistry（IHC）.Results 1.Effects of high fat feeding for 8 weeks on body weight,Lee’s index and GIR of rats: After 8 weeks feeding,compared with ordinary feeding group,body weight and Lee’s index of rats in high fat feeding group increased（P<0.01）,and GIR decreased（P<0.01）.2.Effects of electroacupuncture combined with calorie control on body weight,obesity degree,lipid metabolism and insulin sensitivity of insulin-resistant obese rats:（1）Comparison of weight of rats in each group: Compared with normal group,weight of model group increased from 0 weeks to 8 weeks of intervention（P<0.01）.Compared with the model group,at the 4th week of intervention,the body weight of the calorie control group and the electroacupuncture combined with calorie control group began to differ（P<0.05,P<0.01）;at the 6th week of intervention,the weight of the electroacupuncture group began to differ（P<0.01）,and the weight loss of the electroacupuncture combined with calorie control group was more significant than that of the caloric control group and electroacupuncture group（P<0.05,P<0.01）;at the 8th week of intervention,the body weight of the three intervention groups was significantly reduced（P<0.01）,and the weight loss of the electroacupuncture combined with calorie control group was more significant than that of the caloric control group and electroacupuncture group（P<0.01）;there was no statistical significance in the weight loss between the caloric control group and electroacupuncture group（P>0.05）.（2）Comparison of Lee’s index of rats in each group: Compared with normal group,Lee’s index of rats in model group increased from 0weeks to 8 weeks of intervention（P<0.01）.Compared with the model group,at the 4th week of intervention,Lee’s index in the electroacupuncture combined with calorie control group began to differ（P<0.01）;at the 6th week of intervention,Lee’s index began to differ between the calorie control group and the electroacupuncture group（P<0.01）,and the decrease of Lee’s index in the electroacupuncture combined with calorie control group was more obvious than that in the calorie control group and the electroacupuncture group（P<0.05,P<0.01）;at the 8th week of intervention,Lee’s index in the three intervention groups decreased significantly（P<0.01）,and the decrease of Lee’s index in the electroacupuncture combined with calorie control group was more significant than that in the calorie control group and electroacupuncture group（P<0.05,P<0.01）,and there was no statistical significance in the difference between the calorie control group and electroacupuncture group（P>0.05）.（3）Comparison of serum lipids among all groups: After 8 weeks of intervention,compared with normal group,the contents of TC,TG and FFA in model group were increased（P<0.01）;compared with the model group,the contents of TC,TG and FFA in the three intervention groups decreased（P<0.01）,and the contents of TC,TG and FFA decreased more significantly in the electroacupuncture combined with calorie control group than in the calorie control group and electroacupuncture group（P<0.05,P<0.01）.（4）Comparison of insulin sensitivity indexes in each group:（1）Changes of IPGTT in all groups: Before glucose injection（0 min）,there was no significant difference in blood glucose between groups（P>0.05）.From the beginning of glucose injection to 30 th min,the blood glucose of rats in each group increased;from 30 th min to 120 th min,blood glucose was decreased in all groups.Compared with normal group,blood glucose in model group increased from 30 th min to 120 th min（P<0.01）.Compared with model group,blood glucose in the three intervention groups decreased at 30 th min（P<0.01）,and there was no significant difference in blood glucose among the three intervention groups（P>0.05）.From 60 th min to 120 th min,the decrease of blood glucose in the electroacupuncture combined with calorie control group was more significant than that in the caloric control group and electroacupuncture group（P<0.01,P<0.05）,and there was no significant difference in blood glucose between the caloric control group and electroacupuncture group（P>0.05）.（2）Changes in IPITT of rats in each group: Before insulin injection（0 min）,there was no significant difference in blood glucose between groups（P>0.05）.Within 60 min after insulin injection,blood glucose in all groups decreased.After 60 th min,blood glucose began to rise in each group.From 30 th min to 120 th min,the blood glucose in model group was higher than that in normal group.At 30 th min,blood glucose in the three intervention groups was lower than that in the model group（P<0.05,P<0.01）,but there was no significant difference in blood glucose among the three intervention groups（P>0.05）.From 60 th min to 90 th min,the blood glucose of the three intervention groups was lower than that of the model group（P < 0.05,P < 0.01）,the blood glucose of the electroacupuncture combined with calorie control group was lower than that of the caloric control group and electroacupuncture group（P<0.01,P<0.05）,and there was no statistical significance between the caloric control group and electroacupuncture group（P>0.05）.At 120 th min,the blood glucose of the three intervention groups was lower than that of the model group（P<0.01）,the blood glucose of the electroacupuncture combined with calorie control group was lower than that of the calorie control group（P<0.05）,and there was no significant difference in blood glucose between the calorie control group and the electroacupuncture group（P>0.05）.（3）Changes of GIR in each group: Before intervention,compared with normal group,GIR in model group decreased（P<0.01）;there was no significant difference in GIR between the model group and the three intervention groups（P>0.05）.After 8 weeks of intervention,compared with the normal group,the GIR of the model group decreased（P<0.01）;compared with the model group,the GIR of the three intervention groups increased（P<0.01,P<0.05）,and the GIR of the electroacupuncture combined with calorie control group increased more significantly than that of the calorie control group and the electroacupuncture group（P<0.05,P<0.01）.（4）Changes of INS in each group: After 8 weeks of intervention,compared with normal group,INS content in model group increased（P<0.01）;compared with the model group,the INS content in the three intervention groups decreased（P<0.01）,and the INS content in the electroacupuncture combined with calorie control group decreased more significantly than that in the calorie control group and electroacupuncture group（P<0.01,P<0.05）,and there was no statistical significance in the INS content between the calorie control group and electroacupuncture group（P>0.05）.3.Effects of electroacupuncture combined with calorie control on liver histomorphology and expression of SIRT1,AMPK and p-AMPK proteins in insulin resistant obese rats （1）Histomorphological comparison of liver in each group: The normal group had clear cord structure,regular morphology,large,round,centered nuclei,less heterochromatin and light staining,and clearly visible nucleoli.In model group,the cord structure of liver cells was disorganized,the liver cells were swollen and deformed,and many fat droplets of different sizes could be seen inside the cells.The cytoplasm was less and was squeezed around the cells,and the nucleus was squeezed to one side.In the caloric control group,the cords of hepatocytes were still fuzzy and the hepatocytes were swollen,and the hepatocytes were mainly composed of vesicular lipid droplets,but a few vesicular lipid droplets still existed.In the electroacupuncture group,the cords of liver cells were blurred,the liver cells were swollen,the cells contained more lipid droplets,and the nucleus was squeezed.Compared with model group,calorie control group and electroacupuncture group,the cords of liver cells in electroacupuncture combined with Calorie control group were clearer,cytoplasm was more uniform,and there were lipid droplets in cells.（2）Comparison of the protein expressions of SIRT1,AMPK and p-AMPK in liver of rats in each group detected by WB:（1）SIRT1 protein expression in each group: Compared with normal group,SIRT1 protein expression in model group was decreased（P<0.01）;compared with the model group,SIRT1 protein expression in the three intervention groups increased（P<0.01,P<0.05）,SIRT1 protein expression in the calorie control group increased more significantly than that in the electroacupuncture group（P< 0.05）,SIRT1 protein expression in the electroacupuncture combined with calorie control group increased more significantly than that in the calorie control group and electroacupuncture group（P<0.01）.（2）AMPK protein expression in all groups: There was no statistical significance among all groups（P>0.05）.（3）p-AMPK protein expression in each group: Compared with normal group,p-AMPK protein expression in model group decreased（P<0.01）;compared with the model group,the expression of p-AMPK protein in the three intervention groups increased（P<0.01）,the expression of p-AMPK protein in the calorie control group increased more significantly than that in the electroacupuncture group（P<0.01）,and the expression of p-AMPK protein in the electroacupuncture combined with calorie control group increased more significantly than that in the calorie control group and electroacupuncture group（P<0.05,P<0.01）.（3）Comparison of the expression of SIRT1,AMPK and p-AMPK protein in liver of rats in each group by IHC:（1）SIRT1 protein expression in each group: Compared with normal group,SIRT1 protein expression in model group was decreased（P< 0.01）;compared with the model group,the SIRT1 protein expression in the three intervention groups increased（P<0.01）,and the SIRT1 protein expression in the electroacupuncture combined with Calorie control group increased more significantly than that in the caloric control group and electroacupuncture group（P<0.05,P<0.01）.（2）AMPK protein expression among all groups: There was no statistical significance in AMPK protein expression among all groups（P>0.05）.（3）p-AMPK protein expression in each group: Compared with normal group,p-AMPK protein expression in model group decreased（P<0.01）;compared with the model group,the expression of p-AMPK protein in the three intervention groups increased（P < 0.01）,and the expression of p-AMPK protein in the electroacupuncture combined with Calorie control group increased more significantly than that in the calorie control group and electroacupuncture group（P<0.05,P<0.01）,and the expression of p-AMPK protein in the calorie control group was higher than that in the electroacupuncture group（P<0.01）.Conclusions1.Calorie control,electroacupuncture and electroacupuncture combined with calorie control can reduce the body weight and obesity degree of insulin resistant obese rats,reduce blood lipids,improve intraperitoneal glucose tolerance and insulin tolerance,up-regulate insulin sensitivity,reduce the accumulation of lipid droplets in the liver,and improve the liver tissue morphology.2.The regulation effect of electroacupuncture combined with calorie control on body weight and obesity degree,lipid metabolism,intraperitoneal glucose tolerance and insulin tolerance,insulin sensitivity,liver SIRT1 and p-AMPK protein expression was better than that of calorie control and electroacupuncture.3.The regulation effect of calorie control on SIRT1 and P-AMPK protein expression in liver was better than electroacupuncture alone.4.In conclusion,electroacupuncture combined with calorie control has a more significant effect on weight loss,lipid metabolism, insulin sensitivity,hyperinsulinemia and liver SIRT1 and P-AMPK protein expression. |
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