Fructose-driven Glycolysis In The Heart, Liver And Brain Of Gansu Zokor Under Hypoxic Conditions

Eospalax cansus(E.cansus)is a typical subterranean rodent which belong to the genus Myospalax family of Rodentia,Spalacidae,Myospalacinae,Eospalax.It has adapted to the underground environment with typical hypoxia and high carbon dioxide.In order to adapt to the subterranean hypoxia environment,it has produced a series of adaptation mechanisms during evolution and is an ideal model for studying hypoxia adaptation.One of the challenges faced by the organism under hypoxia is the supply of energy,and E.cansus is constantly conducting high-energy activities such as digging caves and finding food under hypoxia.Therefore,energy supply under low oxygen is particularly important.At present,the mechanism of hypoxia adaptation of E.cansus is studied in physiological and biochemical aspects,but there are few studies on the energy metabolism mechanism of E.cansus under hypoxia.Fructose as a monosaccharide exists in many foods.The fructose-driven glycolytic metabolic pathway does not require the main rate-limiting step of catabolism of glycolysis by phosphofructokinase in glucolysis and therefore enters glycolysis faster than glucose,allowing it to enter glycolysis faster than glucose,allowing it to quickly and efficiently provide energy for organisms.However,long-term ingestion of high fructose can cause cancer,metabolic syndrome and heart failure.Recent studies have found that subterranean rodent,the naked mole rat,can use fructose as an energy substance under almost anaerobic conditions,and high expression of fructose-driven glycolysis-related genes has appeared in many organs such as heart,brain and liver.E.cansus and naked mole rats are all subterranean mouse.How about the state of fructose-driven glycolysis providing energy to the body under normal living conditions of hypoxia?Whether the increase of fructose content will cause the body to appear metabolic syndrome caused by fructose metabolism?Therefore,it is necessary to study the characteristics and molecular mechanism of fructose-driven glycolysis in E.cansus to adapt to hypoxia.Based on the transcriptome data of hearts,livers and brains of E.cansus which are treated with 21%,10.5%and 6.5%oxygen concentration,we measured the fructose levels in serum,heart,liver and brain at different oxygen concentrations.Real-time technique was used to study the mRNA expression of related genes glucose transporter(GLUT5),hexoketokinase(KHK)and aldolase C(ALDOC)in fructose-driven glycolytsis pathways in heart,liver and brain of E.cansus.Western blot technique was used to study the expression of related proteins in the fructose-driven glycolysis pathway in the liver tissue where fructose-driven glycolysis pathway was obvious.Fructose-driven glycolysis of E.cansus under hypoxia was studied from the level of genes and proteins.Finally,the CDS area sequences of the related genes glucose GLUT5,KHK and ALDOC in the fructose-driven glycolytic pathway were obtained by PCR technology.The main results and conclusions are as follows:1.The serum concentration of fructose in E.cansus serum was significantly higher than that of SD rats at 21%oxygen concentration.There was no significant difference between SD rats and E.cansus at oxygen concentration of 10.5%.The fructose content of E.cansus was significantly higher than that of SD rats at 6.5%oxygen concentration.There was a significant difference in the fructose content in 6.5%oxygen concentration in E.cansus serum compared with that in 21%and 10.5%oxygen concentration.There was a clear upward trend,that is,6.5%oxygen concentration.There was no significant difference between 21%and 10.5%oxygen concentration,indicating that E.cansus has an increased ability to use fructose under an oxygen concentration of 6.5%oxygen.There was no significant difference in fructose content of SD rats at 6.5%oxygen concentration compared with 21%and 10.5%oxygen concentration,and there was a downward trend.This indicated that the ability of SD rats to use fructose was weakened under an acute hypoxic environment with an oxygen concentration of 6.5%.2.The expression of GLUT5,KHK and ALDOC mRNA in the glycolytic pathway driven by fructose in the heart tissue of E.cansus showed an upward trend with decreasing oxygen concentration,indicating that the transport capacity and glycolytic capacity of fructose increased.However,there was no significant difference between 21%,10.5%and 6.5%oxygen concentrations,which remained at a stable level.The fructose content was significantly lower than that of SD rats at 10.5%and 6.5%oxygen concentrations,and there was no significant difference with SD rats at 21%oxygen concentration.At 6.5%and 10.5%oxygen concentration,the fructose content in the heart of SD rats was significantly higher than that at 21%oxygen concentration,but the expression of GLUT5,KHK and ALDOC mRNAs in the fructose-driven glycolytic pathway was not consistent.In the change,GLUT5 was significantly higher than 21%and 6.5%at 10.5%oxygen concentration,and there was no significant difference between 21%and 6.5%oxygen concentration.There was no significant difference in KHK at the three concentrations.ALDOC expression at 10.5%and 6.5%oxygen concentration was significantly higher than 21%oxygen concentration.Overall,the expression was higher at 10.5%oxygen concentration,indicating that SD rats have a stronger ability to use fructose in a chronic hypoxic environment.3.The content of fructose in the liver tissue of E.cansus was increased with the decrease of oxygen concentration,and compared with 21%,there was a significant difference in oxygen concentration of 10.5%and 6.5%.The expression of GLUT5,KHK and ALDOC mRNA and protein in the liver-driven glycolytsis pathway increased with the decrease of oxygen concentration.In particular,there was a significant difference in the expression of related proteins at oxygen concentrations of 10.5%and 6.5%compared to 21%oxygen concentration.It was demonstrated that the liver tissue of E.cansus can use fructose to carry out efficient fructose-driven glycolysis under low-oxygen conditions,thus providing energy for the organization.The fructose content of SD rats decreased with the decrease of oxygen concentration.Fructose content at 6.5%oxygen concentration was significantly less than 21%oxygen concentration.The fructose content at 10.5%oxygen concentration was not significantly different with 10.5%and 21%oxygen concentration.However,with the decrease of oxygen concentration,the expression changes of related genes in the glycolytic pathway driven by fructose were inconsistent.The expression of GLUT5 at 10.5%oxygen concentration was significantly higher than that of 21%and 6.5%oxygen concentration,and there was no significant difference between 21%and 6.5%oxygen concentration.There was no significant difference in the expression of KHK and ALDOC between the three concentrations.The expression of related proteins in the fructose-driven glycolytic pathway increases as the oxygen concentration decreases.It is suggested that the liver tissue of SD rats may also use fructose under hypoxia,but its ability is weak.The fructose content of E.cansus was significantly higher than that of SD rats at 10.5%and 6.5%oxygen concentrations,and there was no significant difference with SD rats at 21%oxygen concentration.The expression of GLUT5 and KHK mRNA in the glycolytic pathway driven by fructose in E.cansus was significantly higher than that in SD rats at 6.5%oxygen concentration,and there was no significant difference at 21%and 10.5%oxygen concentration.The expression of ALDOC was significantly higher at 21%and 10.5%oxygen concentration than SD rats,and was extremely significantly higher at 6.5%oxygen concentration than SD rats.The expression of GLUT5 and KHK,which was the related proteins in the glycolytic pathway driven by fructose from E.cansus were significantly higher than SD rats at three concentrations.4.The expression of GLUT5,KHK and ALDOC mRNAs in the glycolytsis pathway driven by fructose in the brain tissue of E.cansus increased with decreasing oxygen concentration,but fructose content appeared with the decrease of oxygen concentration.This may be due to the brain is an important organ that regulates the function of the body.In a hypoxic environment,the brain can still get sufficient oxygen for aerobic respiration.The fructose content in SD rat brain had no significant difference at 21%,10.5%and 6.5%oxygen concentration,although the 6.5%oxygen concentration fructose content increased compared to the 10.5%oxygen concentration,but the amplitude was not large.And the expression of the related genes GLUT5 and ALDOC mRNA in the fructose-driven glycolysis pathway is very low,indicating that the ability of SD rats to use fructose under hypoxia is limited.There was no significant difference in fructose content between E.cansus and SD rats at each oxygen concentration.5.The genes with gene IDs c89203.graph_c0,c77226.graph_c0 and c25582.graph_c0 are annotated as GLUT5,KHK and ALDOC genes.Using the ORF Finder on NCBI to predict the CDS of GLUT5,KHK and ALDOC gene sequences,it was concluded that the GLUT5 gene CDS of E.cansus coded 499 amino acids,KHK gene CDS of E.cansus coded 213 amino acids,ALDOC gene CDS of E.cansus coded 364 amino acids.The full-length CDSs of GLUT5,KHK and ALDOC genes were amplified by PCR,and the three genes were synthesized and subsequently aligned with the corresponding sequences obtained from transcriptome sequencing.The homology was as high as 99%.Sequences and amino acid comparisons were performed at NCBI and the results showed high homology with Spalax,up to 98%.Therefore,genes with gene IDs of c89203.graph_c0,c77226.graph_c0 and c25582.graph_c0 were identified as GLUT5,KHK and ALDOC genes of E.cansus.From the above results,we can conclude that the heart,liver and brain tissue of E.cansus can use fructose as a substrate under low oxygen for fructose-driven glycolysis to supply energy to the body and the glycolysis of fructose in the liver is the strongest.