Effect Of AMPK On Energy Metabolism Of Yak (Bos Grunniens) During Postmortem Aging Under Hypoxia Adaptation

Yaks are in hypoxia environment for a long time,which not only keeps normal growth and physiological activities.In order to balance the energy supply and demand under the condition of low oxygen,there is a special metabolic mechanism in Yak to adapt to the low oxygen environment.AMPK is one of the important"energy regulators".The activated AMPK can regulate the energy balance of body by closing the pathway of anabolism and opening the pathway of catabolism.At present,the research on AMPK is mainly focused on living animals.There is no report on the regulation of AMPK and its effect on muscle energy metabolism and meat quality.Therefore,it is necessary to study the AMPK regulatory pathway of yaks to adapt to hypoxia environment under different altitude.To reveal the mechanism of AMPK activity on energy metabolism and meat quality change during postmortem maturation.In this paper,three kinds of altitude beef were selected as the research object,and the longissimus dorsi was selected as the test sample.Through the phosphorylation of AMPK and the regulation of AMPK by AMPK/SIRT1 signal pathway,the role of phosphorylation post-translational modification in AMPK regulation was clarified,and the signal pathway in the energy metabolism process of postslaughter yak was confirmed.Immunocoprecipitation and RT-PCR(real-time fluorescence quantitative polymerase)were used Chain reaction and other methods to determine the regulation of AMPK on the glycolysis,and combined with the speed and degree of glycolysis to analyze the meat quality.The main results are as follows:1.To study the changes of AMPK activity,glycolysis metabolism and energy metabolism regulation of yak at different altitudes.The results showed that the AMPK activity of Gannan yak was significantly lower than that of Yushu yak in 12-168 h;the lactate content and free glucose content of Gannan yak were significantly lower than Yushu yak in 72-168 h(P<0.05).The content of ATP,ADP and AMP in Gannan yak was significantly higher than that in Yushu yak(P<0.05).In the process of maturity,the hardness of Gannan yak is significantly lower than Yushu yak.The chewiness of Yushu yak is significantly higher than Gannan yak,and the elasticity of Gannan yak is significantly higher than Yushu yak.To sum up,AMPK is activated by lactate myoglycogen,which significantly increases ATP muscle lactate concentration and muscle glycogen level,significantly increases AMP production,accelerates the glycolysis of lactate in muscle tissue,increases muscle lactate myoglycogen content,reduces muscle p H value,and directly affects yak nutrition and quality.2.To study the regulatory pathway of AMPK activity and its effect on energy metabolism after muscle slaughter.By adding the activator AICAR of ampkk and the inhibitor STO-609 of ampkk,the regulation mechanism of AMPKK on AMPK pathway was studied.AICAR significantly increased the activity of AMPK and the degree of glycolysis,increased the rate of energy metabolism and increased the expression of AMPK gene,which indicated that AICAR significantly increased the level of energy metabolism of muscle after slaughter by inducing the increase of AMPK activity.STO-609 could inhibit the activation of AMPK in the longissimus dorsi of yak.In addition,STO-609 treatment inhibited glycolysis and increased the p H value of postmortem muscle.According to the above studies,AMPK is an important regulator of glycolysis in postmortem muscles,which can be used as a useful target for meat quality control.The increased expression of PRKAA1 and PRKAA2 genes will increase the activity of AMPK.After the activation of AMPK,it will directly phosphorylate the glycolysis pathway,increase the glycolysis activity,promote the glycolysis process,produce a large amount of lactic acid,and then reduce the energy metabolism of postmortem animal muscles.3.To study the regulation of AMPK/SIRT1 signaling pathway on energy metabolism after slaughter.NAD~+and NADH,the deacetylation cofactors of SIRT1,were added to study the regulatory mechanism of AMPK/SIRT1 signaling pathway.NAD~+significantly increased the activity of AMPK and glycolysis,increased the rate of energy metabolism and increased the expression of AMPK protein,indicating that NAD~+significantly increased the level of energy metabolism of muscle after slaughter by inducing the increase of AMPK activity.NADH treatment can inhibit the activation of AMPK in the longissimus dorsi of postslaughter yak.In addition,NADH treatment inhibited glycolysis and increased the p H value of postmortem muscle.The results show that activation of SIRT1 can increase the activity of AMPK,and the activation of AMPK can directly phosphorylate the glycolysis pathway,increase the glycolysis activity,promote the glycolysis process,produce a large amount of lactic acid,and then lead to the decrease of energy metabolism of postmortem muscles.Therefore,the activity of AMPK increased under hypoxia adaptation,which accelerated glycolysis metabolism,more effectively regulated energy production,and played a certain role in improving muscle tenderness after slaughter.4.iTRAQ proteome was used to identify the differential proteins of the longissimus dorsi muscle samples of Gannan yak and Yushu yak at 0,12,72 h after slaughter.The total number of peptides identified in the six groups was 2880,and 2333 proteins were identified.There were 36 up-regulated proteins and 439 down-regulated proteins in 475 differential proteins in 0 h of yak longissimus dorsi muscle during cold storage,45 up-regulated proteins and 245 down-regulated proteins in 290 differential proteins in 12 h,380 up-regulated proteins and 56 down-regulated proteins in 436 differential proteins in 72 h,which were mainly concentrated in glycolysis,actin linkage,mitochondria,redox reaction,blood circulation,etc.Bioinformatics analysis of differential proteins in different altitudes during cold storage of longissimus dorsi showed that the metabolic pathways involved in differential proteins were mainly muscle contraction,glycolysis,heart development and HIF1 signaling pathways,which all affected energy metabolism and hypoxic adaptability of muscles after slaughter.5.The key proteins affecting energy metabolism and hypoxia adaptability of the longissimus dorsi muscle of yaks at different altitudes were screened.iTRAQ analysis screened out the proteins related to energy metabolism and hypoxic adaptability during 0,12and 72 hours of postmortem maturation of the longissimus dorsi muscle of yak.These proteins are mainly glycometabolism enzymes,muscle structural proteins,energy metabolism,mitochondria and hypoxic related proteins.Comprehensive analysis results 0 h,33(ALDOA?PDHA1?LDHA?PGM1et al.,),12 h 27(MDH1?PYGM?PHKB?SPAST et al.,)and 72 h24(PFKM?LDHA?PKM2?MDH1et al.,)proteins were differentially expressed in relation to energy metabolism and hypoxia adaptation.In summary,AMPK is activated to directly phosphorylate the glycolysis pathway,which increases the glycolysis activity,thereby promoting the glycolysis process and producing a large amount of lactic acid,which in turn leads to postmortem animal muscle Energy metabolism is reduced.As the altitude increases,the p-AMPK level continues to increase,which indicates that the phosphorylation level of AMPK increases significantly under high altitude.In a hypoxic environment where the intracellular ATP is depleted during the post-mortem maturation of beef at different altitudes,a specific systemic adaptive response is triggered.The iTRAQ proteome identified the differential proteins in Gannan yak and Yushu yak at 0 h,12 h and 72 h after slaughter.Bioinformatics analysis of differential proteins at different altitudes shows that the metabolic pathways involved in differential proteins are mainly muscle contraction,glycolysis,cardiac development and HIF1 signaling pathway;these metabolic pathways all affect muscle energy metabolism after slaughter.High-altitude beef has higher AMPK activity,enhanced glycolysis rate,increased muscle lactic acid content and lowered p H,which in turn affects meat quality.