The Study On The Mechanism Of AMPK Signaling Pathway Regulating Bovine Muscle Tenderness In Postmortem

Beef is one of the most popular foods in the world,and its tenderness is of great importance,which determines customers’ desire for repurchasing and the premium they would like to pay.The global meat industry highly recognize that the postmortem maturation process plays a vital role in improving the tenderness of beef.During the process,endogenous protease,including Calpain and Caspase promotes muscle tenderization and conversion of muscle to meat when hydrolyzing myofibrillar protein of muscle skeleton.When the muscle is confronted with oxygen-deprived response,AMPK is the first protein kinase to be activated.The activated AMPK controls the downstream complex biochemical reaction and meat quality through signal transduction.AMPK’s controlling ways and mechanisms on the tenderness of cattle muscle happened in the postmortem maturation process is still under exploration.Taking hindquarter beef(Semimembranosus)as the research object,the thesis discussed the effects of AMPK on tenderness of cattle muscle and related proteome,specified the regulation mechanism of AMPK on glycolysis,analyzed the effects of AMPK mediated glycolytic metabolism on Calpain-1 activation and activity,revealed the potential controlling method of AMPK in Caspase-3 activation,clarified the relationship between autophagy and apoptosis induced by mitochondrial dysfunction,systematically introduced the mechanism of AMPK on tenderness of beef,which lays a foundation for revealing the controlling method of hypoxia response in tenderness of muscle during postmortem maturation and determining the biomarker sites controlling the tenderness of beef.The primary research results are as follows:1.Studied the effects of AMPK on postmortem tenderness of cattle muscle and related proteome;the phosphorylation of cattle muscle’s AMPKα2 reached to a maximum level at postmortem 6 h.Activated AMPK controlled the tenderness of cattle muscle during maturation,which reflected in the activation of AMPK was not beneficial to tenderness of beef before postmortem 12 h(P<0.05),while promoted tenderness of beef after 12 h,and increased tenderness of beef by 15.75%(P<0.05)at168 h.After analyzing proteomics,the metabolic pathway that AMPK may be involved was including glucose metabolism,energy metabolism,cytoskeleton protein controlling,mitochondrial pathway,oxidative stress during postmortem maturation of beef,which are connected with tenderization of muscle.2.Studied the effects of AMPK on beef glycolysis and Calpain-1 activity;through the function of phosphorylation,AMPK increased the activity of glycogen phosphorylase(6 h),hexokinase and lactate dehydrogenase(6～12 h)(P<0.05),promoted the translocation of GLUT4 to cytomembrane.In earlier stage of postmortem,AMPK promoted the decline of p H of cattle muscle and degeneration of sarcoplasmic protein(P<0.05),hampered the Calpain-1 autolysis(P<0.05),limited the Calpain-1 catalytic activity(P<0.05)caused by muscle acidification.Therefore,through increasing the activities of glycogen phosphorylase,hexokinase and lactate dehydrogenase and inducing the translocation of GLUT4 protein to cell membrane,the activated AMPK promoted glycolysis and muscle acidification,resulting in increased denaturation of sarcoplasmic protein and decreased autolysis and activity of Calpain-1 and decreased tenderness of muscle before postmortem 12 h.3.Studied the effects of mitochondria-dependent apoptosis mediated by AMPK on tenderness of muscle;within postmortem 12～24 h,AMPK promoted the accumulation of ROS of cattle muscle(P<0.05),increased the ratio of Bax/Bcl-2 on mitochondrial membrane within postmortem 6～12 h(P<0.05),induced mitochondrial dysfunction of cattle muscle,promoted the release of Cyt-c from mitochondria,the oxidation of Cyt-c in cytoplasm and Caspase-3 activation(P<0.05).Therefore,through increasing the accumulation of muscle cells ROS and inducing the translocation of cytoplasmic Bax to mitochondrial membrane,activated AMPK enhanced mitochondrial dysfunction,promoted the release and oxidation of mitochondrial Cyt-c,and then induced the apoptosis of Caspase-3 and cells during the postmortem maturation of cattle muscle.Though the approach of mitochondria-dependent apoptosis,AMPK improved the tenderness of cattle muscle by 10.49% after postmortem 168 h.4.Studied the effects of autophagy to apoptosis induced by AMPK during postmortem maturation;the activated AMPK decreased the compound content of m TOR1 and increased the content of ULK1(P<0.05),and promoted the occurrence of autophagy;while,the activated AMPK had limited effects on content of Beclin-1.The occurrence of autophagy promoted lysosomal membrane instability(P<0.05)increased cathepsin D activity,mitochondrial membrane dysfunction,Caspase-3activity as well as tenderization of cattle muscle(P<0.05).Therefore,through decreasing the compound content of m TOR1,activated AMPK increased the level of ULK1 and promoted autophagy;autophagy protease D induced by AMPK promoted apoptosis and tenderness of muscle.Based on the above mentioned studied,it can be concluded that the process of cell death was from autophagy to apoptosis to necrosis.5.Studied the effects of endoplasmic reticulum stress mediated by AMPK on Caspase-3 activation and tenderness of cattle muscle;the activated AMPK increased the phosphorylation level of IRE1(P<0.05)and induced endoplasmic reticulum stress during postmortem maturation;furthermore,through activating downstream Caspase-12 and-9,the activated AMPK promoted Caspase-3 activation(P<0.05)and further increased the degradation of myofibrillar protein and the degree of tenderness of muscle(P<0.05).The tenderness of muscle was increased by 6.29% at 168 h under the condition that(P<0.05)the apoptosis way was inhibited by mitochondrial dysfunction.Therefore,it can be concluded that AMPK promoted Caspase-3activation and tenderness of cattle muscle through endoplasmic reticulum stress mediated by IRE1 channel.In conclusion,activated AMPK promoted the decrease of p H of muscle through accelerating glycolytic metabolism,resulting in autolysis and activity decline of Calpain-1,which hampered the tenderization of muscle during the postmortem earlier stage of cattle muscle(0～12 h).While AMPK promoted Caspase-3 activation and tenderness of muscle through activating autophagy,apoptosis induced by mitochondrial dysfunction and endoplasmic reticulum stress.Consequently,as an effective controlling target,AMPK can significantly improve the tenderness of beef.