| With an average altitude of 4,500 m,the qinghai-tibet plateau is the highest plateau in the world.Due to its extreme environment of low oxygen,low pressure,high cold and strong ultraviolet,it has a profound impact on the distribution,heredity,differentiation,population structure,survival status and evolution pattern of the organisms living in it.Hypoxia is the most important factor restricting the survival of animals on the qinghai-tibet plateau.As a native mammal unique to the plateau,the plateau pika has shown a high tolerance to the hypoxic environment at the plateau and is considered to be a good animal model for the study of plateau hypoxic adaptation.The PKLR gene encodes two types of Pyruvate Kinase(PK),including liver type(PKL)and erythrocyte type(PKR).Pyruvate kinase is one of the three key enzymes in the glycolysis pathway that catalyze the final reaction of glycolysis,the conversion of phosphoenolpyruvate(PEP)and adenosine diphosphate(ADP)into pyruvate and the production of adenosine triphosphate(ATP).Previous studies have found that the convergence mutation from histidine(H)to glutamine(Q)occurred in many plateau mammal species such as pika at the 303 rd amino acid site of pyruvate kinase gene and it was found that PKLR gene was positively selected in many plateau native mammals which suggesting that PKLR may play an important role in the adaptation of plateau mammals to the environment.But the biological function and physiological and biochemical phenotype of PKLR in hypoxic adaptation are not clear.In this study,PKLR-M1(303Q-glutamine)mutant in the PKLR coding region was constructed in mouse model first time.Hypoxic induction experiments under10% and 8% oxygen conditions were conducted on homozygous HO(Q/Q)and wild-type WT(-/-)mice knocked in by PKLR-M1.To observe the changes in the physiological phenotypes of degree of right ventricular hypertrophy,peripheral blood oxygen saturation,spleen volume,red blood cell phenotype,fasting blood glucose,serum erythropoietin,thyroid hormone T4,lung staining section,spleen stained section and the changes in biochemical phenotype of pyruvate kinase activity of liver,the content of key enzymes and substrates of anaerobic glycolysis,aerobic oxidation and gluconeogenesis and glycogen synthesis in liver glucose metabolism in mice under long-term chronic hypoxia.In addition,in order to reveal the molecular mechanism of the physiological and biochemical changes observed in mice,transcriptome analysis was performed on eight types of tissues(left ventricle and diaphragm,right ventricle,liver,spleen,lung,kidney,brain and adrenal gland)in mice with two genotypes.The results showed that :(1)hypoxic mice of both genotypes had the same degree of weight loss,right ventricular hypertrophy and pulmonary vascular wall thickening.From normoxia to hypoxia,the hemoglobin concentration and average erythrocyte hemoglobin content in HO mice were higher than WT(P=0.0135,P=0.0281,P>0.05)(P< 0.0001,P=0.0253,P=0.0001).At 8% hypoxia,the blood oxygen saturation of HO mice was higher than WT(P< 0.0001),Similar differences between the two genotypes of mice were observed,though not significant,under other oxygen conditions.This indicates that the oxygen carrying capacity of red blood cells of HO mice is enhanced,which makes the body get more oxygen.With lower oxygen levels,the spleens of the two genotypes of mice grew larger,with a proliferation of B lymphocytes in the spleen,Mouse spleen enlargement under hypoxia may be caused by increased erythropoietin and erythrocyte count,increased non-specific immune response of the spleen and B lymphocyte proliferation,and increased spleen volume is not caused by increased thyroid hormone T4.(2)The PK activity of HO mice under normoxic and 8% hypoxic conditions was significantly lower than WT(P=0.0007,P=0.0033).PK activity of HO and WT mice under hypoxia was significantly higher than that of normoxia(P=0.0166,P=0.0052).Under hypoxic conditions,the liver of HO mice showed lower levels of anaerobic glycolysis and aerobic oxidation than that of WT mice.Anaerobic glycolysis and aerobic oxidation pathways were enhanced in both genotypes of mice under hypoxia.We speculated that HO mice were likely to inhibit their glycolysis to some extent through low pyruvate kinase activity.However,in the presence of a high concentration of gluconeogenic matrix,compared with WT,HO mice could rapidly improve liver gluconeogenic level to convert lactate,pyruvate and other non-glycolic substances into glucose and glycogen and regulate blood glucose balance.(3)In transcriptome analysis,differential expression analysis showed that the number of differentially expressed genes in the spleen and liver of HO and WT mice was significantly higher than that of other tissues.The differential high-expression genes in the liver of HO mice were mainly enriched in glucose metabolism and lipid metabolism related pathways,indicating that the gene PKLR-M1 plays an important role in liver metabolism.In addition,differential high-expression genes in HO liver were also enriched in insulin-related pathways,suggesting that lipid metabolism and insulin signaling pathways were closely related to glucose metabolism in HO mice.The differentially expressed genes in the spleen are mainly concentrated in the pathways related to cell division and DNA repair,which are related to the increased non-specific immune response and b-lymphocyte proliferation in the spleen under hypoxia.This study reveals the biological function and role of PKLR in the adaptive evolution of mammals in the plateau.We found that there were significant differences in erythrocyte phenotypes between HO mice and WT under hypoxia,which caused differences in erythrocyte oxygenation capacity and spleen enlargement.Low pyruvate kinase activity in the liver of HO mice may also be an adaptation to a hypoxic environment.Low pyruvate kinase activity in HO mice may inhibit glycolysis to some extent,but in the presence of a high concentration of gluconeogenic matrix,HO mice can rapidly improve liver gluconeogenic level compared with WT.Transcriptome analysis revealed that the gene pklr-m1 plays an important role in the regulation of liver metabolism,suggesting that lipid metabolism and insulin signaling pathways are closely related to the glucose metabolism of HO mice,so as to help us fully understand the complex mechanism of glucose metabolism regulation in organisms. |
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