| Background Obesity is a negative energy balanced metabolic disease caused by excessive energy intake and insufficient energy expenditure,often accompanied with glucose and lipid metabolism disorders in peripheral metabolic organs.Epidemiological studies have found that obesity can increase the risk of non-alcoholic fatty liver disease(NAFLD)and the prevalence of NAFLD among obese people in Asia’s countries is reaching 68.5% with a tendency to younger.Nowadays,exercise intervention combined with diet control is the most effective way to lose weight,but often accompanied by weight rebound.Recently,more and more studies have found that compared with simple exercise interventions,hypoxia training has a better effect on weight loss,which can effectively suppress appetite and increase energy expenditure.Studies have shown that weight loss can effectively improve obesity induced metabolic diseases.Additionally,studies have found that hypoxic training can regulate the expression of key genes in hepatic lipid metabolism,but there are few studies on hepatic lipid metabolism in obese mice,and the mechanism is still unclear.Therefore,the first part of this study verified the regulation of hypoxia training on hepatic lipid metabolism and explored the possible mechanism of hypoxia training on regulating hepatic lipid metabolism Obese people often have lower serum magnesium levels,which may even cause hypomagnesemia.Exercise can increase the consumption of body’s magnesium.Our previous results showed that serum magnesium concentration of obese adolescents affected the weight loss of hypoxia training.After hypoxic training intervention,the red blood cell magnesium level and femoral magnesium content of obese mice were significantly reduced,suggesting that hypoxic training exacerbated the magnesium loss in obese mice.Therefore,magnesium supplement is especially important for obese people who lose weight through exercise or hypoxia training.Given the widely used of exercise combination with nutrition in weight loss.The second part of this study will use hypoxia training combined with magnesium supplement intervention to verify the weight loss effect and its impact on metabolismrelated organs.Based on the metabolic phenotypes,to further explore the regulation of hypoxia training combined with magnesium supplement on hepatic lipid metabolism in obese mice.To provide the theoretical basis for the application of hypoxia training combined with nutrition intervention on weight loss for obese people,and especially for the improvement of liver lipid metabolism.Purpose(1)To verify the effect of hypoxia training on the regulation of liver lipid metabolism in obese mice,and explore the possible mechanism of hypoxia training to regulate liver lipid metabolism;(2)To investigate the effects of hypoxia training combined with magnesium supplement on weight loss and metabolic organ metabolism.In addition,to explore the possible mechanism of its regulation on hepatic lipid metabolism.Methods Research 1: Forty-four seven-week-old SPF-grade male healthy C57BL/6J mice were reared adaptively for one week and randomly divided into control group(Control,Con,n = 8,ordinary feed)and high-fat diet group(High fat diet-induced obesity,DIO,n = 36,feed containing 60% fat,D12492,Research Diets),mice had ad libitum access to food and water.After 8 weeks of high-fat diet intervention,mice were evaluated for obesity modeling.After the successful establishment of the obese model,36 mice were randomly divided into 4 groups: High fat diet-induced obesity group(DIO),Training group(T),Hypoxia group(H)and Hypoxia Training group(HT),9 mice in each group.Mice in each group were fed with high-fat diet.Subsequently,each group of mice began a 4-week hypoxic training intervention: 1)Group T: intermediate intensity running intervention on the treadmill,60% of maximum speed,15m/min,zero slope,six days per week.2)Group H: during 08: 30am-16: 30 pm,the mice were placed on a small animal metabolism analyzer with oxygen partial pressure of 110 mm Hg and oxygen content of 14.7%,six days per week.3)Group HT: training intervention combined with hypoxic environment intervention.No intervention in the DIO group,and the measurement indicators are shown in the technology roadmap 1.Research 2: Forty-eight SPF-grade male healthy C57BL/6J mice aged 7 weeks were reared adaptively for one week and randomly divided into a normal diet control group(Chow diet,CD,n = 8,ordinary feed)and high-fat diet group(High fat diet-induced obesity,DIO,n = 40,feed containing 60% fat,D12492,Research Diets),mice had ad libitum access to food and water.After 8 weeks of high-fat diet intervention,mice were evaluated for obesity modeling.After the successful establishment of the obesity model,40 obese mice were randomly divided into 4 groups: Control group(DIO,n=10),magnesium supplement group(Mg,n=10),Hypoxia Training group(HT,n=10)and Hypoxia Training plus magnesium supplement group(HT+Mg,n=10).Mice in each group were fed with highfat diet,and four-week hypoxic training and magnesium supplement intervention experiments begin at the ninth week: 1)Group Mg: Add Mg Cl2 in drinking water at a dose of 100mg/kg/day.2)Group HT: Hypoxia training intervention method is the same as that in research 1.3)Group HT+Mg: Hypoxia training intervention combined with magnesium supplement intervention.No intervention in the DIO group,and the measurement indicators are shown in the technology roadmap 2.Statistical analysis All data are represented by mean ± standard error(Mean ± SEM).Statistical analysis was performed in SPSS19.0 software.Independent sample t-test was used between the two groups and the four groups were compared by Kruskal-Wallis analysis of variance and the Mann-Whitney test(all the results show the p value with Bonferroni correction).p<0.05 means statistically significant difference,p<0.01 means statistically intense significant difference.Results(1)Changes in metabolism-related phenotypic parameters of obese mice after hypoxia training intervention: compared with DIO group,the body weight(p<0.05,p<0.01)of T and HTgroup,visceral fat weight(p<0.05,p<0.01,p<0.01)of T,H and HT group,and serum TG content(p<0.05,p=0.063)of H and HT group mice were significantly reduced,and the serum FFA content(p<0.05,p<0.01)of H and HT group was significantly increased;(2)Changes in liver-related phenotypic parameters of obese mice after hypoxia training intervention: compared with DIO group,the liver weight(p<0.01)of HT group,liver weight/body weight ratio(p<0.05,p<0.01)and serum ALT content(p<0.01,p<0.01)of H and HT group,hepatic FFA content(p<0.01,p<0.01,p<0.01)of T,H and HT group was significantly reduced,and hepatic lipid accumulation was also significantly reduced,with the most significant effect in the HT group;(3)After hypoxia training intervention,compared with mice in the DIO group,the content of liver glycogen(p<0.05),branched-chain amino acids like leucine(p <0.01)and valine(p <0.01)of mice in HT group is significantly increased,liver glucose metabolism are enhanced;(4)After hypoxia training intervention,compared with mice in the DIO group,the content of PUFA(p<0.01),n6-PUFA(p<0.01),C20:3n6(p<0.05),C20:4n6(p<0.05),C20:5n3(p<0.01),C22:6n3(p<0.01),n6/n3(p<0.05)in liver of mice in HT group is significantly reduced,the expression level of fatty acid synthase Fads1(p=0.057)and Fads2(p=0.055)has a distinct downward trend,the expression level of inflammatory factors IL-1β(p<0.05),TGF-β(p<0.01),and TNF-α(p<0.01)is significantly decreased;(5)Changes of metabolism-related phenotype in obese mice after hypoxia training intervention combined with magnesium supplementation intervention: compared with mice in the DIO group,the body weight(p<0.001)and body fat weight(p<0.001)of mice in HT group and HT+Mg group are significantly reduced,the exhaustion time(p <0.001)and running distance(p<0.001)in the exhaustion experiment are significantly increased;There is no significant change in food intake and water intake in the 24 h period during intervention;(6)Changes of glucose-related metabolism in obese mice after hypoxic training intervention combined with magnesium supplementation intervention: Compared with mice in group DIO,the fasting blood glucose level(p<0.001),postprandial blood glucose level(p<0.001)and the area under the curve in insulin resistance test(p <0.05)of mice in HT group and HT+Mg group are significantly decreased;(7)Changes of liver-related phenotypes in obese mice after hypoxia training intervention combined with magnesium supplementation intervention: Compared with mice in group DIO,liver weight and liver weight/body weight ratio of mice is significantly reduced in group Mg(p<0.01,p<0.01),group HT(p<0.001,p<0.05),and group HT+Mg(p<0.001,p<0.05),the size and number of liver lipid droplet are also significantly decreased in these group;(8)Changes of adipose tissue-related phenotypes in obese mice after hypoxia training intervention combined with magnesium supplementation intervention: Compared with mice in group DIO,the weight of subcutaneous fat(p<0.001)and epididymal fat(p<0.001)of mice in HT group and HT+Mg group are significantly reduced,the size and number of lipid droplet of subcutaneous fat and epididymal fat of mice in HT group and HT+Mg group are also significantly decreased,and the transcriptional level of thermogenic genes like UCP1,UCP2,and PGC-1α is increased in brown adipose tissue;(9)Based on the KEGG pathway enrichment analysis of transcriptome results,intervention of hypoxia training and/or magnesium supplementation may regulate liver lipid metabolism partly through the PPAR signaling pathway;(10)Changes of liver lipid-related metabolism in obese mice after magnesium supplementation intervention: Compared with mice in group DIO,the TG concentration(p<0.05)and AST / ALT concentration ratio(p<0.01)in plasma and TG concentration in liver(p<0.01)are significantly decreased in mice of group Mg;transcriptome analysis showed that liver fat biosynthesis,lipolysis,fatty acid oxidation,and lipid transportability are significantly increased in obese mice after magnesium supplementation intervention;transcriptional level of liver lipolysis related genes like PPARα(p<0.01),ATGL(p<0.01),HSL(p<0.001)and fatty acid oxidation related gene CPT-1α(p<0.001)and liposynthesis related gene SREBP-1c(p<0.05),LXRa(p<0.001)are also significantly increased in mice after magnesium supplementation intervention;(11)Changes of lipid metabolism in primary hepatocytes after magnesium supplementation treatment: In the experiment of Mg SO4 supplementation treatment,the transcriptional level of PPARα(p<0.01)and SREBP-1c(p<0.01)of primary hepatocytes in the 2.0m M treatment are significantly increased compared with the 0.8m M group.The steatosis was induced by PA treatment in primary hepatocyte culture,and then the cells were treated with Mg SO4 supplementation,compared with the 0.8 m M group,the expression level of gene PPARα(p<0.01)are significantly increased in the 2.0 m M group.(12)According to the KEGG enrichment analysis,the Hippo,TGF-β,and p53 signal pathways are significantly down-regulated and the AMPK signal pathway is significantly up-regulated in the liver of mice after magnesium supplementation;Main conclusions(1)Hypoxia training can significantly reduce body weight,fat mass and liver weight,improve hepatocyte injury and liver lipid accumulation;significantly improve hepatic lipid metabolism in obese mice.The mechanism is partly due to the inhibition of fads1/2 expression which induced reduction of n6-PUFAs synthesis and improvement of fatty liver inflammatory response;(2)Hypoxia training combined with magnesium supplement intervention can significantly reduce body weight and body fat content,improve the exercise performance,glucose tolerance and insulin resistance,increase the insulin sensitivity;in addition,hypoxia training combined with magnesium supplement intervention has the same effect on metabolism improvement in obese mice as hypoxia training intervention,hypoxic training combined with low-dose magnesium supplement had no superposition effect;(3)Magnesium supplement,hypoxia training and hypoxia training combined with magnesium supplement can significantly reduce liver weight and lipid accumulation in obese mice,which may partly be regulated through the PPAR signaling pathway;(4)Magnesium supplement significantly improve hepatic lipid accumulation,liver cell injury and hepatic lipid metabolism without reducing body wight and body fat mass,the regulation mechanism of magnesium supplement which may be interact with multiple signaling pathways: the significant down-regulation of the Hippo,TGF-β,p53 signaling pathway,and up-regulation of the PPAR,AMPK signaling pathway. |
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