| Objective In this study,we chose high-fat diet to induce insulin-resistant obese rats.The electro-acupuncture and dietary calorie control were used as the intervention method.Taking the hypothalamic-pituitary-adrenal axis as the breakthrough point,we compared the effects of electro-acupuncture and calorie control on the prevention and treatment of in obese rats with insulin resistance.The possible mechanism of electro-acupuncture combined with calorie control on insulin resistance and obesity was explored from the negative feedback function of HPA axis and the influence of fat browning.Methods1.100 Wistar male rats were fed for one week and randomly divided into normal group(n = 20)and model group(n = 80).The normal group was fed with standard diet with a caloric coefficient of 3.8 kcal/g,and the model group was fed with high fat diet with a caloric coefficient of 5.4 kcal/g.After fed for eight weeks,five rats were randomly selected from each group.The glucose infusion rate(GIR)was measured by high insulin-positive glucose clamp technique.The insulin sensitivity was evaluated.At the same time,the weight of the rats and the length from nose to anus were measured.Lee's values were calculated for obesity.After the rats were successfully building,5 rats were randomly selected from each group and performed microdialysis cannula on the third ventricle was.Seven days after microdialysis catheterization,the cerebrospinal fluid of dialysis was collected.Collecting the cerebrospinal fluid before and after electro-acupuncture on both “Zusanli(ST36)” and "Fenglong(ST40)" for 30 minutes.Collecting its dialysis solution,the hemodialysis of CRH,Glu and Glu in the dialysate were measured by Elisa and high performance liquid chromatography(HPLC).The contents of CRH,Glu and GABA in dialysis solution were measured by Elisa and high performance liquid chromatography(HPLC).2.After the end of the 8th week,40 rats were randomly chosen from the model group that the weight increased by more than 20% than normal group.Then,the 40 rats were divided into model group,caloric control group,electro-acupuncture group,electro-acupuncture + caloric control group(N=10),and 10 rats in the normal group were selected and treated for 8 weeks.The normal group was fed with standard diet,and the model group continued to be fed with high fat diet.The calorie control group was fed with 30%calorie control.The electro-acupuncture group was treated with high fat diet and electro-acupuncture at the same time.The electro-acupuncture + caloric control group was fed with 30% calorie control and electro-acupuncture.For the intervention of electro-acupuncture,the four points of Zusanli,Fenglong,Guanyuan,Zhongwan were selected to needle,and the needles were connected to the Hans instrument(2Hz/1m A,10min),three times a week for 8weeks;For the intervention of calorie restriction,the food intake was measured in the model group every day,and 70% of the daily intake of the rats in the model group was adjusted to the calorie control group.In the phase of intervention,the weight was recorded every two weeks.At the end of 6th week,intraperitoneal glucose tolerance test(IPGTT)and insulin tolerance(IPITT)were monitored.At the end of the treatment,the changes of lipid factors TG/TC/FFA and HPA axis hormone CRH/ACTH/CORT were detected by elisa and biochemical methods.The expression and location of the protein CRH and GR in the hypothalamus was observed by Western blotting and immunofluorescence double labeling.The morphologic change in the white adipose tissue(WAT)was observed by HE staining.The m RNA levels of GR and UCP1 in WAT were observed by real-time fluorescence quantitative technique.Results1.Evaluation of obese rats model with insulin resistance Compared with the normal group,the body weight and Lee's index of the model group were significantly higher than those of the normal group(P<0.05),and the glucose infusion rate was significantly less than that of the normal group(P<0.01),but there was no significant difference between the two groups for the length from nose to Anal(P>0.05).2.Effects of electro-acupuncture on hypothalamic neuroendocrine factors in obese rats with insulin resistance by microdialysis technique Before the electro-acupuncture,cerebrospinal fluid CRH in model group was significantly higher than the normal group(P<0.01),but after electro-acupuncture for 30 mins,the model group CRH levels were significantly lower than before(P<0.01),while there was no significant change between the normal group before and after the electro-acupuncture(P>0.05).Before the electro-acupuncture,cerebrospinal fluid Glu and Glu/GABA in model group was significantly higher than the normal group(P<0.01),but GABA levels were lower than the control group(P<0.05).After 30 mins electro-acupuncture,the level of Glu in model group decreased significantly(P<0.01),while there was significantly difference about GABA(P>0.05).3.Effects of electro-acupuncture combined with calorie control on body weight,glucose tolerance,insulin tolerance,TG/TC/FFA and HPH axis related hormone CRH/ACTH/CORT in obese rats with insulin resistance After the intervention of electro-acupuncture and calorie control,the body weight of the normal group and the model group increased at a relatively stable growth rate,while the gain rates of the three intervention groups were significantly slowed down,and the decreased degree of weight was significantly higher in the calorie control group than the EA group.What's more,the body weight of EA + calorie control group slowly decreased starting from the second week,and there was no significant difference between the intervention group and the normal weight group until the 8th weeks(P>0.05).After injection of glucose and insulin,the blood glucose levels respectively at 30 min,60min,90 min and 120 min in model group were significantly higher than the normal group,and the under area of the glucose tolerance curve(IPGTT-AUC)and insulin tolerance curve(IPITT-AUC)were also significantly higher than the normal group(P <0.01).The glucose levels in the three intervention groups at above time points was lower than the model group,and the IPGTT-AUC and IPITT-AUC were significantly lower than the model group(P <0.01).What's more,the deceased degree in EA + calorie control group was significantly higher than the calorie control group(P <0.05).Compared with the normal group,the serum levels of TG,TC and FFA in the model group were significantly increased(P <0.01).Compared with the model group,the levels of TG,TC and FFA the three intervention group were significantly decreased,and the effect of EA group and EA + calorie control group was more obvious than the calorie control group,the EA + calorie control group was the best among them(P <0.05).Compared with the normal group,the levels of HPA-related hormones CRH,ACTH and CORT in the model group were significantly higher(P<0.01).Compared with the model group,the HPA axis-related hormones were significantly decreased in the three intervention groups,and the effects of EA group and EA + calorie control group were better than that the calorie control group.The effect of EA + calorie control group was better than that of EA or calorie control alone(P <0.05).4.Effect of electro-acupuncture combined with calorie Control on hypothalamic CRH and GR Protein in obese rats with insulin resistance Observed by the Western blot: Compared with the normal group,the expression of CRH protein in model group was significantly increased(P<0.01),while GR protein expression were significantly decreased(P <0.01);Compared with the model group,the CRH protein levels in the three intervention groups were significantly decreased,while the GR levels were significantly increased(P <0.01),and the effect in the EA +calorie control group was better than EA or calorie control alone(P <0.05).Observed by the immunofluorescence: In the normal group,CRH positive cells with red fluorescence and GR positive cells with green fluorescence,mainly distributed in the cytoplasm of large cell neurons in PVN,occasionally in the nucleus,and,mainly distributed in the cytoplasm,a few found in the nucleus,and most of the CRH and GR existed co-localization.Compared with the normal group,the CRH positive cells in the model group were mainly distributed in the nucleus,and the number of cells was significantly higher than that of the normal group,the intensity of fluorescence was enhanced,while the GR positive cells were mainly distributed in the nucleus,the number of positive cells was significantly decreased than that of the normal group,and the ratio of CRH/GR cells was significantly higher than that of the normal group.The number of CRH-positive cells in the three intervention groups was significantly decreased than the model group,which located in the nucleus and cytoplasm,but the number of GR cells was significantly upregulated than that in the model group,and CRH/GR ratio was significantly lower than the model group,especially in the EA + calorie control group.5.Effect of electro-acupuncture combined with calorie Control on white adipose tissue GR and UCP1 m RNA in obese rats with insulin resistance Observed by the HE staining: Compared with the normal group,the cell diameter of adipocytes increased obviously and the number of adipocytes in the model group was significantly decreased under the same horizon(P<0.05),the cell nucleus was extruded into crescent or oblong.Compared with the model group,the number of fat cells in the three intervention group increased significantly,and the diameter reduced.What's more,the effect of EA +calorie control group is better than its alone(P<0.05).Conclusions1.The obese rats with insulin resistance can be successfully induced by high fat diet for 8 weeks,which was confirmed by the hyperinsulinemiceuglycemic-clamp.High fat diet can cause neuroendocrine dysfunction,and electro-acupuncture has a positive effect on the neuroendocrine factor CRH/Glu/GABA in obese rats with insulin resistance,which was confirmed by the real-time monitoring of the third ventricle microdialysis.2.Electro-acupuncture and calorie control can delay the growth rate of weight in rats,improve glucose tolerance and insulin tolerance,down-regulate blood lipid levels and HPA-related hormone levels.Electro-acupuncture combined with calorie control could antagonize the excitability of HPA axis,improve glucose and lipid metabolism disorders and enhance insulin sensitivity on the obese rats with insulin resistance,that the effect was significantly better than its alone.3.Electro-acupuncture combined with calorie control can significantly upregulate the expression of GR in the hypothalamic paraventricular nucleus of obese rats with insulin resistance and down-regulate the expression of CRH neurons in PVN.It showed that EA combined with calorie control by regulating GR expression through the hypothalamus GC-GR/CRH pathways improved HPA axis negative feedback function,inhibited the HPA axis' excitatory against stress injury.4.Electro-acupuncture combined with calorie control can significantly reduce the expression of GR in white adipose tissue,and increase the expression of UCP1,indicating that EA combined with calorie control can effectively promote white fat browning.And its mechanism may be related to the function of HPA axis. |
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