| Gansu zokor(Eospalax cansus)belongs to Rodentia,Spalaccidea,Myospalactinae,Eospalax and lives in underground tunnels with low oxygen and high carbon dioxide for life.It is a typical underground rat.In order to adapt to the living environment,Gansu zokor has evolved some abilities to adapt to hypoxia.Energy metabolism regulation is an important strategy for hypoxia adaptation.At present,the physiological and biochemical mechanism of Gansu zokor's hypoxia adaptation has been studied,but the energy metabolism mechanism of Gansu zokor under hypoxia has been seldom studied.Therefore,it is of great significance to explore glycolysis in hypoxia tolerance of Gansu zokor.In this study,key regulatory genes in glycolysis pathway of Gansu zokor were studied by biochemical and molecular biological methods,and the glycolysis metabolic mechanism of Gansu zokor under hypoxia was preliminarily explored.Gansu Zokor and SD rats were divided into three groups,and treated with hypoxia at 21%,10.5%and 6.5%oxygen concentration.The contents of glucose and lactic acid in serum,brain and liver were determined by spectrophotometry.The levels of gene expression and protein expression were detected by RT-PCR and Western Blot.The activities of HK,PFK and ALO were detected by ELISA kit.The SD rats were used as control mice to compare with the Gansu zokor.The glycolytic reaction of Gansu zokor under hypoxic conditions was preliminarily studied,aiming to lay a foundation for studying hypoxia adaptation mechanism in underground mice.The main experimental results are as follows:1.Glucose content in different tissues of Gansu Zokor and SD rat:The serum glucose content of Gansu zokor increased with the increase of hypoxia,but there was no significant difference in glucose content between SD rats in three oxygen concentrations.The blood glucose content of Gansu zokor was lower than SD rats under three oxygen concentrations.The glucose content in the brain of Gansu zokor and SD rats was significantly lower than that of 21%and 10.5%,but the Gansu zokor was significantly higher than SD rats.The glucose concentration in the liver of Gansu zokor was 10.5%.The oxygen concentration was significantly higher than the 21%oxygen concentration,which was significantly higher than the 6.5%oxygen concentration.The 21%oxygen concentration in SD rats was significantly higher than 10.5%and 6.5%oxygen concentration.At 21%and 6.5%oxygen concentration,the glucose content of SD rats was significantly higher than that of Gansu zokor,while at 10.5%oxygen concentration,Gansu zokor was significantly higher than SD rats.2.The content of lactate in different tissues of Gansu Zokor and SD rat:The content of lactic acid in Gansu zokor serum was significantly lower than that in 21%and 6.5%oxygen concentration at 10.5%oxygen concentration,while that in SD rats was significantly higher than that in 21%and 6.5%oxygen concentration at 10.5%oxygen concentration.SD rats were significantly higher than Gansu Zokors at 10.5%oxygen concentration.The oxygen concentration of Gansu zokor was significantly higher than that of 10.5%at 6.5%oxygen concentration,and that of SD rats was significantly higher than that of 21%at 10.5%and 6.5%oxygen concentration.At 10.5%oxygen concentration,the lactate content of SD rats was significantly higher than that of Gansu zokor.There was no significant difference between Gansu Zokor and SD rat in liver at 21%,10.5%and 6.5%oxygen concentration,and the lactic acid content of Gansu zokor was significantly higher than that of SD rat at 21%and 6.5%oxygen concentration.3.Expressions of GLUT1,GLUT2,HK1,PFK and ALDOA in different tissues of Gansu Zokor and SD rat:Brain expression:The GLUT1 mRAN of Gansu zokor was significantly down-regulated with respect to 21%oxygen concentration at 10.5%and 6.5%oxygen concentration;the expression level of SD rats at 6.5%oxygen concentration was significantly higher than 21%and 10.5%oxygen concentration.The expression levels of Gansu zokor were significantly higher than those of SD rats at the three oxygen concentrations.There was no significant difference in protein between SD rats and Gansu zokor at different oxygen concentrations.The expression level of HK1 mRNA at 6.5%oxygen concentration in Gansu zokor was significantly higher than that in 21%oxygen concentration.There was no significant difference in protein concentration under three oxygen concentrations.The expression of HK1 mRNA at 21%oxygen concentration in SD rats was significantly higher than 10.5%.At a concentration of 6.5%oxygen,the protein was significantly higher than 21%at 10.5%and 6.5%oxygen concentration.There was no significant difference in the expression of PFK mRNA and protein in Gansu zokor at different oxygen concentrations.The expression of PFK mRNA and protein in 6.5%oxygen concentration in SD rats was significantly higher than that in 21%oxygen concentration.The mRNA and protein expression levels of Gansu zokor were higher than those of SD rats at different oxygen concentrations.The expression level of ALDOA mRNA in Gansu zokor was significantly higher than that in 21%oxygen concentration at 10.5%and 6.5%;the expression level in 6.5%oxygen concentration in SD rats was significantly higher than 10.5%oxygen concentration.The expression levels of SD rats in the three oxygen concentrations were significantly higher than those in Gansu zokor.There was no significant change in the protein expression of Gansu zokor and SD rats at three different oxygen concentrations,but it increased with the decrease of oxygen concentration.Expression in the liver:The expression of 10.5%oxygen concentration of GLUT2 mRNA in Gansu zokor and SD rats was significantly higher than 21%and 6.5%oxygen concentration.The expression levels of mRNA and protein in Gansu zokor were higher than those in SD rats at three oxygen concentrations.The expression of HK1 mRNA and protein at 21%oxygen concentration in Gansu zokor was significantly higher than 10.5%and 6.5%oxygen concentration;the expression of HK1 mRNA and protein at 6.5%oxygen concentration in SD rats was significantly higher than 10.5%oxygen concentration.Under the three oxygen concentrations,the expression of HK1 protein in Gansu zokor was higher than that in SD rats.The expression levels of PFK mRNA and protein in Gansu zokor were significantly higher than that in 21%oxygen concentration.The expression of 6.5%oxygen concentration in SD rats was significantly higher than 21%and 10.5%oxygen concentration.There were no significant differences at the three oxygen concentrations.The expression of PFK protein in Gansu zokor was higher than that in SD rats at different oxygen concentrations.There was no significant difference in the expression of ALDOA mRNA in Gansu zokor at three different oxygen concentrations.The expression of 6.5%oxygen in protein was significantly higher than 21%and 10.5%.The expression of 6.5%oxygen in SD rats was significantly higher than 21%.The 10.5%oxygen concentration,protein 21%oxygen concentration was significantly higher than the 10.5%and 6.5%oxygen concentrations.At three different oxygen concentrations,the expression of ALDOA mRNA and protein in SD rats was significantly higher than that in Gansu zokor.4.The results of HK,PFK and ALD enzyme activities in different tissues Gansu zokor and SD rats:Enzyme activity in brain:The activities of HK and ALD in Gansu zokor were 10.5%oxygen concentration significantly higher than 21%oxygen concentration,significantly higher than 6.5%oxygen concentration,while the activities of PFK were 21%and 6.5%oxygen concentration significantly higher than 10.5%oxygen concentration.The activities of HK,PFK and ALD in SD rat were 10.5%oxygen concentration significantly higher than 21%oxygen concentration,significantly higher than 6.5%oxygen concentration.The activities of PFK and ALD in SD rats at 10.5%oxygen concentration were significantly higher than those in Gansu zokors.Liver enzyme activity:The activity of HK in Gansu zokor was significantly higher than 10.5%and 6.5%,the activity of PFK was significantly higher than 21%and 6.5%,the activity of ALD was significantly higher than 6.5%and 6.5%,and the activity of HK and ALD was significantly higher than that of 6.5%and 21%.The activity of HK and ALD in SD rats was significantly higher than that of 21%,and significantly higher than that of 6.5%,while the activity of PFK was 2%.Oxygen concentrations of 1%and 10.5%were significantly higher than those of 6.5%.The activities of HK,PFK and ALD in SD rats at 10.5%oxygen concentration were significantly higher than those in Gansu zokors.The above results show that the blood sugar content of Gansu zokor is always lower than that of SD rats,and the activity of glycolytic enzymes,especially the rate-limiting enzymes PFK,is lower than that of SD rats.This indicates that Gansu zokor has a lower level of glycolysis than SD rats.Hypoxia can stimulate the increase of glucose content in serum of Gansu zokor,transport glycolytic metabolic substrates for tissues,and increase the glycolysis to provide energy for tissues.The expression of glucolysis-related genes and proteins in liver and brain of Gansu zokor and SD rats increased in varying degrees under hypoxia,suggesting that hypoxia stimulated the glycolysis ability of Gansu Zokor and SD,especially Gansu zokor increased the levels of glucolytic enzymes and proteins and enzymes under chronic hypoxia,and increased the level of Glucolysis to meet the energy demand,but acute hypoxia stimulated the glycolysis ability of Gansu Zokor and SD.The transcription,translation and enzymatic activity of glycolytic enzymes decreased,suggesting that energy requirements might be met by fructose hydrolysis or other metabolic methods.At the same time,Gansu zokor did not accumulate lactic acid when glycolysis was enhanced,which indicated that there might be an internal mechanism to inhibit the accumulation of lactic acid in Gansu zokor,which could remove lactic acid in time and avoid acidosis. |
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