Study On The Mechanism Of PDK1 Regulating Energy Metabolism In HepG2 Cells

The research on the mechanism of tumorigenesis is one of the hot topics in the life science.The change of metabolism is one of the main characteristics of tumor.Thus,studying the mechanism of energy metabolism of tumor cells is of great significance for tumorigenesis mechanism and treatment research.As a key enzyme linking glycolysis and oxidative phosphorylation,pyruvate dehydrogenase complex(PDH)activity has abnormal changes in varieties of tumor cells.Studies have shown that P32 is closely related to cell autophagy,oxidative phosphorylation and other processes.The interaction between P32 and PDH influence the mode of energy metabolism-glycolysis will be converted to oxidative phosphorylation.As a phosphorylation kinase of PDH and key node kinase for glycolysis and oxidative phosphorylation,pyruvate dehydrogenase kinase 1(PDK1)also involved in the regulation of the Warburg effect in tumor cells.Therefore,PDK1 plays an important role in energy metabolism of tumor cells,but the exact mechanism isn’t very clear.In order to explore the mechanism of PDKI effect in the energy metabolism of tumor cells,the liver cancer cell line HepG2 will be used as a model for the following experimental research in this project.Results are as follows:1.The effect of PDK1 on the energy metabolism of HepG2 cells:Dichloroacetic acid(DCA)was used to inhibit PDK1 activity in HepG2 cells.The results showed that cell viability and proliferation decreased of HepG2 cell.Lactic acid content decreased significantly.Glucose consumption,ATP content and the energy produced by HepG2 cells increased significantly.There were no significant changes in G6PDH activity and NADPH content.It was shown that the anaerobic lysis of HepG2 cells can be affected by PDK1 inhibition to some extent and the level of oxidative respiration was increased.2.The effect of PDK1 on the level and intracellular distribution of P32 and PDH in HepG2 cells:After PDK1 was inhibited in HepG2 cells,the results showed that the level of PDH and pPDHA1(Ser293)changed,indicating that the downstream PDH of PDK1 was activated and the metabolism was partially reversed to oxidative phosphorylation in HepG2 cells.The level of P32 was changed.The location of P32 and co-localization with PDH were both increased in mitochondria after PDK1 inhibition.Results above showed that PDH dephosphorylation is related to the interaction between P32 and PDH.The change of cellular localization of P32 has a certain relationship with PDK1 activity.3.The effect of PDK1 on the morphology of mitochondria in HepG2 cells:Using DCA to inhibit PDK1 in HepG2 cells,the expression of mitochondrial fusion protein mitofusin 2(Mfn2)increased significantly while the mitochondrial division protein dynamin-related protein 1(Drp1)decreased significantly,indicating that PDK1 not_only control the process of oxidative phosphorylation through regulating the activity of PDH,but also has an effect on the morphological changes of mitochondria.PDK1 plays multiple roles-in regulating the process of oxidative phosphorylation and also changes the level of oxidative phosphorylation in cells through affecting the balance of mitochondrial division and fusion.4.The effect of PDK1 on the apoptosis of HepG2 cells:When PDK1 activity was inhibited by DCA in HepG2 cells,the results showed that the level of apoptosis-related proteins,such as Cleaved Caspase9,Cleaved Caspase3 and Bax(Bcl-2 associated X protein)showed a trend of decreasing first and then increasing,indicating that apoptosis was increased after PDK1 inhibition in HepG2 cells.In conclusion,as a kinase of PDH,PDK1 can regulate the conversion of anaerobic lysis to aerobic respiration in cells.The level of oxidative respiration can increase when PDK activity was inhibited in cells.P32,PDK and PDH interact with each other and the interaction of P32 and PDH is related to PDH dephosphorylation.The change of localization of P32 is related to the activity of PDK 1 in cells.Based on the co-localization of P32,PDK1 and PDH,it is speculated that the three participate in the mitochondrial oxidative phosphorylation.PDK1 controls the oxidative phosphorylation process by regulating the activity of PDH and the process of mitochondrial division and fusion to change the mitochondrial morphology.PDK1 has multiple regulatory effects on the aerobic respiration process.The apoptosis of cells was increased after PDK1 inhibition,indicating that oxidative phosphorylation and mitochondrial morphology tightly related to endogenous apoptosis of mitochondria.Reversal the growth of tumor cells has a certain relationship with the balance of mitochondrial division and fusion.Results of this project provide an important foundation for revealing the mechanism of PDK1 regulating energy metabolism of tumor cells,meanwhile provide a new theoretical basis for tumor treatment based on targeted tumor metabolism.