Role Of The Endocannabinoid Receptor In The Regulation Of Hypoxia Training Modulated Glycolipid Disorders In Obese Mice

BackgroundObesity is a metabolic disease caused by excessive energy intake.Obesity are often accompanied by systemic glucose and lipid metabolism disorders,which are characterized by hypothalamic energy balance dysregulation;accelerated liver fat synthesis,oxidative decomposition dysfunction,skeletal muscle glucose absorption,fatty acid oxidation imbalance,and secretion dysfunction in adipose cells.Balanced diet or?and?exercise intervention is currently the most cost-effective methods to lose weight.The principle mainly includes restriction of energy intake and increase in energy consumption.These traditional methods for weight loss have significant effects but accompanied with evident problems:increased appetite and reduced basal metabolic rate after weight loss,which plays an important role in the rebound of body weight.In addition,recent studies have shown that the basal level of lipid metabolites in obese people is closely related to the effect of weight loss,including endocannabinoids.The Endocannabinoid System?ECS?is a complete system that includes cannabinoid receptors,ligands,cannabinoid synthase and degrading enzymes.ECS plays an important role in triggering energy metabolism and glycolipid metabolism disorders.However,it is not clear to what extent the expression of ECS in energy metabolic organs of obese mice changes after weight loss.Therefore,the first part of the study explored the changes in ECS composition of brain,liver,visceral fat and skeletal muscle on the basis of exploring the change of energy balance after traditional dietary weight loss.Hypoxia exposure can significantly reduce energy intake and increase energy expenditure,so it is used in the field of weight loss.Researchers have combined hypoxia exposure with aerobic training to explore whether it is possible to achieve both effective weight loss and prevention of excessive energy intake and reduced energy expenditure.Therefore,the second part of this study will comprehensively observe the effects of hypoxia training on macroscopic energy metabolism and micro glycolipid metabolism in obese mice and analyze the possible role of ECS in these regulation processes to provide theoretical basis for weight loss practice through hypoxia training.Purposes1)To explore the changes in energy metabolism and components of the endocannabinoid system in obese mice after dietary weight loss intervention or hypoxia training intervention;2)To explore the effects of hypoxia training on energy balance and glycolipid metabolism disorders and to explore the possible role of the endocannabinoid system in these regulatory processes.Methods81 healthy male SPF C57BL/6J mice with 4 weeks old were randomly divided into normal chow diet?NCD,n=18,fed on standard normal diet?and high-fat diet induction group?High-fat diet,HFD,n=63,fed on D12492 high-fat diet,fat provides 60%calories,Research Diets?.Obesity modeling was assessed 10 weeks after induction.Experiment 1:9 mice randomly selected from normal diet group were recorded as normal chow diet?NCD?;18 mice randomly selected from obese group were randomly divided into two groups:high-fat diet control group?High-fat diet control,HFD-C?and Switching diet to balanced diet group?SWD?.Intervention began at the week of 12:NCD group and HFD-C group maintained their original diet program.The SWD group was converted from the original high-fat diet to the standard normal diet?HFD?NCD?and the intervention lasted for 6 weeks.Testing indicators include:1)body weight,body fat,body fat percentage;blood glucose?glucometer?,TG,HDL/LDL-C?ELISA?,liver histological staining?HE and oil red O staining?;2)serum and Brain endocannabinoid content?high performance liquid chromatography?and cannabinoid receptor,synthetase and metabolic enzyme transcriptional expression in the brain,liver,epididymal fat and gastrocnemius?RT-PCR?;3)serum leptin,insulin,ghrelin,Glucagon,GIP,GLP-1 levels?suspension chips?;4)Energy metabolism indicators:VO₂,VCO₂,RER,TEE,activity and food intake?PhenoMaste system?Experiment 2:27 individuals were randomly selected from obese mice and randomly divided into 3 groups:obese control group?OB?,hypoxia training group?HT?and hypoxia training+CB1 receptor agonist group?HT+WIN?,9 per group.All mice still fed on a high-fat diet.From the 13th week,each group began a 4-week intervention:1)hypoxic training:hypoxia exposure combined with aerobic training intervention,HT group and HT+WIN group performed this intervention,while OB group keep quiet.Hypoxia exposure:mice were placed in an animal gas metabolism analyzer with an oxygen pressure of 110 mmHg and an air oxygen content of 14.7%from 08:30 to 16:30,8 h/d,6 d/w.Moderate intensity aerobic training:starting at 20:00 every day,40⁷0%VO₂max,zero slope,14 m/min,840 m/d,6 d/w.2)Injection of CB1 receptor agonist and placebo:HT+WIN group was administered by intraperitoneal injection of WIN55212-2,and HT and OB groups were given placebo injection under the same conditions?containing 0.5%DMSO and containing 1%Tween 80 in physiological saline solution?.Administered at a dose of 1 mg/kg and 10 ml/kg,6 d/w.The drug is prepared before use.In addition to the indicators listed in"Experiment 1",the test indicators include glucose tolerance and insulin resistance test,serum ALT?ELISA?,serum and liver FFA?ELISA?,liver CB1,SREBP-1,ATGL,CPT-1,PPAR?/?/?and PGC-1?protein expression levels?Western blot?.Statistical analysisThe experimental results are expressed in the form of Mean±standard error?Mean±SEM?.Statistical analysis was performed using a two-tailed independent Student's t test to compare differences between the two groups;one-way ANOVA combined with Tukey's post hoc test was used to compare differences between the three groups.Results1)At 10 weeks of high-fat diet,the percentage of body fat?P<0.01?,fasting blood glucose?P<0.05?,and low-density lipoprotein?P<0.01?in mice meeting for obesity was significantly higher than that of the control group;2)Changes in phenotypic index after balanced diet induced weight loss:compared with HFD-C group,weight?P<0.01?,visceral fat mass?P<0.01?,body fat percentage?P<0.01?,fasting blood glucose?P<0.05?and LDL?P<0.05?level in SWD group decreased significantly.Except for the percentage of body fat?P<0.05?and LDL?P<0.05?,the other indexes in the SWD group were not significantly different from those in the NCD group;3)Changes in appetite regulation factors,energy intake and energy expenditure after balanced diet induced weight loss:Compared with HFD-C group,serum leptin?P<0.05?and insulin?P<0.05?levels decreased significantly and ghrelin?P<0.05?and GLP-1?P<0.05?levels were significantly increased in SWD group;and brain and serum 2-AG?P<0.05 and P<0.05?and AEA levels?P<0.01 and P<0.05?were significant decreased in SWD group.We also found the level of ghrelin in the SWD group was significantly higher than the NCD group?P<0.05?.There was no significant difference in food intake between the two groups,but the total energy consumption level?TEE?in the SWD group decreased significantly when compared to NCD group?P<0.05?;4)Cannabinoid receptor and metabolic enzyme expression after balanced diet induced weight loss:Compared with NCD group,SWD group showed significantly higer expression of ECS components in brain?DAGL?,P<0.05?,liver?CB1,DAGL?,NAPE-PLD,FAAH;all comparisons,P<0.05?and gastrocnemius muscle tissue?CB1,P<0.05?;5)Changes in phenotypic index after hypoxia training:Compared with OB group,body weight?P<0.01?and body fat?P<0.05?decreased significantly in HT group;6)Changes in appetite factors,energy intake and energy expenditure after hypoxia training:compared with OB group,serum leptin?P<0.05?,glucagon?P<0.05?and GIP?P<0.05?level was significantly decreased in HT group,while there was no significant difference in the levels of food intake and total energy expenditure?TEE?between the two groups;7)Changes in liver ECS expression after hypoxia training:Compared with OB group,the expression of CB1?P<0.05?and DAGL??P<0.05?decreased significantly,while the expression of MAGL increased significantly?P<0.01?in the liver of HT group.The2-AG content of the liver?P<0.05?also decreased significantly after hypoxia training.Intraperitoneal injection of CB1 receptor agonists hindered most of these regulatory processes;8)Changes in glucose metabolism in obese mice after hypoxia training:Compared with OB group,hypoxia training significantly reduced fasting blood glucose?P<0.01?and fasting insulin?P<0.05?levels in obese mice of HT group.And the area under the curves?AUC?of GTT?P<0.05?and ITT?P<0.05?test was significantly reduced in HT group;9)Changes in liver lipid metabolism in obese mice after hypoxia training:Compared with OB group,liver index?P<0.01?,serum ALT?P<0.01?and liver fat content were significantly decreased in HT group.Protein expression of CB1?P<0.05?and SREBP-1?P<0.01?was decreased significantly and expression of ATGL?P<0.05?and CPT-1?P<0.05?was elevated significantly,while PPAR?expression tends to decrease that close to significant difference?P=0.052?in HT group.Intraperitoneal injection of CB1receptor agonist hindered the down-regulation of CB1?P<0.01?,SREBP-1?P<0.05?and PPAR??P<0.01?by hypoxia training.Main conclusions1)After balanced diet induced weight loss,the disorders of appetite factors in mice still exist.The energy intake level is not significantly different from that of normal mice,while the energy expenditure level is significantly decreased.Besides,there are higher expression levels of ECS components in brain,liver and skeletal muscle after dietary weight loss;2)Hypoxia training can significantly reduce body weight and body fat,but can also reduce the satiety appetite levels,while energy intake and energy expenditure levels are not changed significantly.In addition,hypoxia training significantly improves the overactivity of liver ECS components in obese mice;3)Hypoxia training can significantly improve glucose intolerance,insulin resistance and hepatic lipid metabolism disorders,the mechanism of which is partly mediated by the down-regulation of CB1?SREBP-1/PPAR?signal to attenuate liver fat synthesis.