Activation Of The PyLKB1-PyAMPKα Signaling Pathway And Its Regulation On Glycogen Metabolism Under High Temperature Stress In The Patinopecten Yessoensis

Patinopecten yessoensis is a cold-water shellfish characteristic in northern China,in recent years,due to global warming,floating raft farming scallops often break out in summer mass death,seriously affecting the green and healthy development of scallop aquaculture industry.Studies have shown that shellfish can maintain homeostasis in the organism by regulating their own energy metabolism levels under high temperature stress.However,there are no reports on the regulation mechanism of shellfish energy metabolism under high temperature stress.AMP-activated protein kinase(AMPK)is a serine/threonine protein kinase that is a core molecule that senses the level of energy metabolism within cells.Changes in intracellular energy levels lead to phosphorylation of the Thr172 site of AMPK,which in turn regulates downstream molecules to promote cellular productivity pathways and inhibit energy consumption pathways.In this paper,PyAMPKα and PyLKB1(liver kinase B1,LKB1,a homologous analogue of LKB1)was identified from scallops,its expression characteristics under high temperature stress were detected,and its regulatory effect on metabolic processes such as glycogenolysis,glycolysis and glucose transport were explored.The main results are as follows:1.The full length of PyAMPKα c DNA is 1599 bp,encoding 533 aa with a molecular weight of 60.4 k Da.It contains a serine/threonine kinase domain(S＿TKc)and an adenylate Sensor domain.The full length of PyLKB1 c DNA is 1251 bp long,encodes 417 amino acids,has a molecular weight of 47.8 k Da,and contains only one S＿TKc domain.One phosphorylation site Thr170 was predicted in the kinase domain of PyAMPKα,33 phosphorylation sites and three SUMOylation sites and two nuclear localization sequences(NLS)were predicted on the PyLKB1 sequence.Multiple sequence alignment results showed that the amino acid sequences of PyAMPKα and PyLKB1 were highly conserved compared with homologous proteins of other species.The results of the evolutionary tree showed that PyAMPKα and Azumapecten farreri AMPKα were clustered into one branch,and then a large branch with other shellfish AMPKα.PyLKB1 is clustered with Mytilus edulis LKB1 and then with other shellfish AMPKα into one large branch.2.PyAMPKα and PyLKB1 m RNA were expressed in all tissues of scallops,and the m RNA expression were highest in hepatopancreas and adductor muscle,respectively.PyAMPKα protein was mainly localized in the cytoplasm of scallop haemocytes PyLKB1 protein was distributed in both the cytoplasm and nucleus of haemocytes.3.Under high temperature stress(25 °C)treatment,the expression of m RNA of PyAMPKα and PyLKB1 in adductor muscle of scallop showed a pattern of ‘rise-fallrestore’.After 3 h of high temperature stress,the m RNA expression of PyAMPKα and PyLKB1 increased to the highest level,which was 3.09-fold(p < 0.05)and 17.53-fold(p < 0.001)in the blank group(15 °C),respectively.The phosphorylation level of PyAMPKα under high temperature stress(25 °C)treatment was significantly increased and was 12.9-fold(p < 0.05)compared with the blank group.After 3 h treatment with AMPK activator(Acadesine,AICAR),the m RNA expression and phosphorylation level of PyAMPKα increased significantly(p < 0.05).Protein-protein interaction networks(PPI)database was used for predictive analysis,and it was found that there was an interaction between PyAMPKα and PyLKB1 in the high temperature stress(25 °C)group and AICAR group,the expression of glycogen synthase kinase-3β(PyGSK-3β)m RNA was significantly reduced(p < 0.05).The expression of m RNA in glycogen phosphorylase(PyGPa)was significantly increased(p < 0.05).The glycogen content was significantly reduced;The expression of m RNA in Glucose transporter 1(PyGLUT1)was significantly increased(p < 0.05).The m RNA and enzyme activities of phosphofructokinase-1(PyPFK)and pyruvate kinase(PyPK)were significantly increased during glycolysis(p < 0.05).4.RNAi technology was used to interfere with the m RNA expression of PyLKB1,and it was found that after high temperature stress(25 °C)for 3 h,the expression of m RNA and protein of PyLKB1 decreased,the expression of m RNA and protein of PyAMPKα did not change significantly,and the m RNA of PyGLUT1 decreased significantly,which was 0.68-fold that of the control group(p < 0.05).The phosphorylation level of PyAMPKα decreased,and the protein content of PyGLUT1 decreased but did not change significantly.In summary,PyAMPKα and PyLKB1 are evolutionarily highly conserved members of the serine/threonine protein kinase family.Under high temperature stress(25 °C),PyLKB1 in adductor muscle activates PyAMPKα,thereby promoting glycogen decomposition and glycolytic pathways,promoting cellular glucose transport,and then promoting scallops to produce more energy in response to high temperature stress.The results of this study lay a foundation for in-depth study of the activation of AMPK signaling in scallops under high temperature stress and its regulatory effect on glycogen metabolism,and also provide ideas for further study of the regulatory mechanism of shellfish energy metabolism under high temperature stress.