The Role Of SERPINA3K In Different Obesity Phenotypes Induced By A High-fat Diet And The Effect Of Aerobic Exercise On It

Objective:The incidence of obesity has increased dramatically worldwide.There are two different phenotypes called obesity susceptibility and obesity resistance during constructing the high-fat diet-induced obesity model,however,the specific mechanism is still unknown.Energy imbalance contributes to obesity.The regulation of energy balance involves multiple organs and tissues such as the liver,muscle,and adipose tissue,which are regulated by the central nervous system in the hypothalamus.High-fat diet would induce oxidative stress in the brain,liver,and adipose tissue,which contributes to cell damage and dysfunction,ultimately disrupting various cellular physiological processes such as energy metabolism and mitochondrial function,and promoting the development of obesity.Exercise could prevent and treat obesity effectively.Previous studies have demonstrated that aerobic exercise could suppress excessive oxidative stress,improve antioxidant capacity,and improve glucose and lipid metabolism.Based on screening the hypothalamic differential protein serine protease inhibitor A3K（SERPINA3K）in high-fat diet-induced obesity susceptibility and resistance mice,this study verified SERPINA3K and the role of its downstream signal pathway factors in high-fat diet-induced obesity susceptibility and obesity resistance phenotypic differences in vivo and in vitro,and investigated the effect of aerobic exercise on its expression.Method:1.This study constructed the mouse model of obesity susceptibility and obesity resistance induced by high-fat diet:160 6-week-old SPF C57BL/6 male mice were adapted for one week and then subjected to HFD intervention for 16 weeks.Mouse ranked in the top and bottom thirds for weight gain during this feeding period were designated as DIO and DR,respectively.The DR group and DIO group mice continued to receive high-fat feeding for 12 weeks.After the high-fat intervention,the body weight,body composition,energy metabolism and other indicators of the two groups of mice were measured.2.Observation of serum and liver metabolic indicators in mice:The levels of serum TG,TC,HDL-C,LDL-C in mice were detected by colorimetry.The histological structure and lipid aggregation of liver,epididymal fat,brown adipose tissue,and the content of TG and TC in liver were observed by H&E and oil red O staining.3.Proteomics TMT sequencing identifies differentially expressed proteins:Proteomics TMT sequencing was performed on the hypothalamus of DR and DIO mice,and the differential protein SERPINA3K in the hypothalamus of the two groups of mice was analyzed and screened.Western Blot was used to detect the expression of SERPINA3K,downstream antioxidant factors and ROS producing key proteins（NOX4,NRF2,p NRF2,SOD2,NQO1,HO-1）in the hypothalamus,liver,epididymal fat,and gastrocnemius muscle tissues of the two groups of mice.4.Detection of antioxidant levels and oxidative stress levels in mice:Colorimetry and DHE staining were used to detect the total SOD activity,GSH and MDA content,and ROS production in hypothalamus,liver,epididymal fat,and gastrocnemius muscle tissues in response to oxidative stress.5.In vitro validation of the effect of SERPINA3K on the reduction of antioxidant levels induced by PA:Two parts experiments were designed.Experiment 1 intervened with different concentrations of SERPINA3K recombinant protein in hypothalamic cells（m Hypo EN43/5）,and Experiment 2 treated with PA and PA+SERPINA3K（320n M,640 n M）.After 48 h intervention,Western Blot was used to detect the expression level of downstream antioxidant factors of SERPINA3K in cells,further verifying the effect of SERPINA3K on antioxidant factors.6.The second part constructs an aerobic exercise mouse model obesity.Obese mice were subjected to 12 weeks of moderate intensity（75%VO₂max）treadmill exercise,5 days a week,for 1 h per day.After exercise,Western Blot was used to detect the expression levels of SERPINA3K and downstream antioxidant factors in the hypothalamus of mice,as well as key proteins that generate ROS（NOX4,NRF2,p NRF2,SOD2,NQO1,HO-1）,to explore the effects of aerobic exercise on SERPINA3K and downstream antioxidant factors.Results:1.Phenotypic differences in obesity susceptibility and obesity resistance mice induced by high-fat diet:（1）The DIO group mice were larger in size,with significantly higher body weight,body fat mass,body fat percentage,lean body weight,and serum TC than the DR group（P<0.05）.There was no significant difference in serum TG,HDL-C,and LDL-C levels between the two groups（P>0.05）;（2）The DIO group mice had larger adipocytes,significant lipid accumulation in the liver,and higher levels of TG and TC in the liver（P<0.05）;（3）The food intake,energy intake,energy consumption,oxygen consumption and total activity of DIO mice were significantly lower than those of DR mice（P<0.05）,but there was no significant difference in respiratory quotient between the two phenotypes of mice（P>0.05）.2.Differential expression of SERPINA3K protein in the hypothalamus of DIO and DR mice was identified by TMT labeling proteomics analysis.Sequencing revealed that compared with DIO,the expression of SERPINA3K protein in the hypothalamus of DR mice was higher.Western Blot further confirmed that the expression level of SERPINA3K protein in hypothalamus,liver,epididymal fat and gastrocnemius muscle of DIO mice was significantly lower than that of DR mice（P<0.05）.3.Antioxidant levels in the hypothalamus and other metabolic organs of DIO and DR mice:The expression levels of antioxidant factors NRF2,p NRF2,SOD2,NQO1,HO-1 in the hypothalamus,liver,epididymal fat,gastrocnemius muscle of DR mice,as well as the total SOD activity and GSH content of antioxidant enzymes were significantly higher than those of DIO mice（P<0.05）.4.Oxidative stress in the hypothalamus and other metabolic organs of DIO and DR mice:The expression level of NOX4,the key protein for generating ROS in the liver of DIO mice,was significantly lower than that of DR mice（P<0.05）,while the expression of NOX4 protein in epididymal fat was significantly higher than that of DR mice（P<0.05）,but there was no significant difference in the expression level of NOX4protein in the hypothalamus and gastrocnemius muscle tissues between the two groups（P>0.05）;In addition,the MDA content and ROS production of lipid peroxidation products in liver,epididymal fat and gastrocnemius muscle of DR mice were significantly lower than those of DIO mice（P<0.05）,but there was no significant difference between the two groups in the MDA content of hypothalamus tissue（P>0.05）5.In vitro experiments have confirmed that SERPINA3K antagonizes the decrease in antioxidant levels in hypothalamic cells induced by PA,promoting the expression of antioxidant factors.Different concentrations of SERPINA3K,as well as PA+SERPINA3K（320 n M,640 n M）treatment of hypothalamic cells,can upregulate the expression of downstream antioxidant factors NRF2,p NRF2,SOD2,NQO1,and HO-1（P<0.05）,but has no significant effect on the key protein NOX4 that generates ROS（P>0.05）.6.The effect of aerobic exercise on the SERPINA3K antioxidant signaling pathway:Aerobic exercise significantly upregulated the expression of SERPINA3K and downstream antioxidant factors NRF2,p NRF2,SOD2,NQO1,and HO-1 in the hypothalamus of DIO mice（P<0.05）,but had no significant effect on the key protein NOX4 that generates ROS（P>0.05）.Conclusion:1.The occurrence of differences in obesity susceptibility and obesity resistance phenotypes probably related to the expression of SERPINA3K and its downstream antioxidant factors.2.Aerobic exercise could upregulate the expression of SERPINA3K and its downstream antioxidant factors in obesity mice.