The Role Of Proton Homeostasis Driven By Glucose Metabolism In The Development Of Tumor

Objective:Glucose is an important source of energy for maintaining normal physiological activity and cell proliferation.Oxidative phosphorylation,glycolysis,and aerobic glycolysis are the three main forms of glucose metabolism,and tumor cells use aerobic glycolysis as the main metabolic system.This is also known as the "Warburg effect." Why tumor cells choose inefficient aerobic glycolysis metabolism is still controversial.This study aims to elucidate the existence of the Warburg effect and its influence on tumor cell survival and tumor growth from a biochemical perspective.Methods: 1.Use a tool that can detect apoptosis in real time(GC3AI)and PI to detect cell death in the absence of glucose.2.Use RNA interference technology to down-express Caspase in MCF-7 cells or use caspase inhibitors to observe its effect on TNF-? induced cell death.3.MCF-7 cells over-expressed BCL-XL/BCL-2 to protect mitochondria and observed its effect on TNF-a induced cell death.4.Treat cells with mitochondrial respiratory chain inhibitors and observe their effect on TNF-? induced cell death.5.Separately add different amino acid and citric acid cycle intermediate metabolites in the nutrient-deficient medium to observe the effects of different substances metabolism on TNF-a induced cell death.6.Change the pH inside and outside the cell to observe the effect of glucose-induced cell necrosis.7.Treat the cells with Bafilomycin A or CQ and observe the effect on glucose-induced cell necrosis.8.Use CRISPR/Cas9 technology to knock out the LDH and PKM genes in HeLa or 4T1 cells and observe their effects on lactic acid production,cell proliferation,glucose metabolism,and glutamine metabolism.9.The mice were inoculated subcutaneously with 4T1/Cas9,4T1/PKM-KO,4T1/PKM-KO-hPKM1 and 4T1/PKM-KO-hPKM2 cells,and the tumor growth and tumor growth rate were observed.The expression of PKM was detected by immunohistochemistry.Results: 1.TNF-? promotes glucose deprivation-induced necrosis in MCF-7 cells.2.Mitochondrial impairment promotes glucose deprivation-induced necrosis.3.Mitochondrial ATP generation is required for TNF-? promoted necrosis in the absence of glucose.4.Mitochondrial ATP generation-associated catabolism has a decisive role in TNF-? promoted necrosis.5.Mitochondrial ATP generation-driven proton homeostasis plays a critical role in determing TNF-? promoted necrosis.6.Mitochondrial ATP generation-driven severely lysosomal alkalinization is required for TNF-?-promoted necrosis under the condition of glucose deficiency.7.Glucose is the major source to maintain proton homeostasis.8.Lactate excretion can be mediated by both PKM1 and PKM2.9.The reverse Warburg effect is mainly mediated by PKM2 in stroma.10.PKM2 suppresses the entry of glucose into acetyl-Coenzyme A.11.PKM2 promotes the entry of glutamine into acetyl-Coenzyme A.Conclusion:We show that glucose deprivation can directly elicit necrosis,which is promoted by mitochondrial impairment,depending on mitochondrial adenosine triphosphate(ATP)generation instead of ATP depletion.We demonstrate that glucose metabolism is the major source to produce protons.Glucose deficiency leads to lack of proton provision while mitochondrial electron transfer chain continues consuming protons to generate energy,which provokes a compensatory lysosomal proton efflux and resultant increased lysosomal pH.This lysosomal alkalinization can trigger apoptosis or necrosis depending on the extent of alkalinization.Taken together,our results build up a metabolic connection between glycolysis,mitochondrion,and lysosome,and reveal an essential role of glucose metabolism in maintaining proton homeostasis to support cell survival.Here we formulate a theoretical model of cellular metabolic states based on proton homeostasis.Our model indicates that an essential role of the Warburg Effect is to maintain proton homeostasis in proliferating cells whose active anabolism scavenges a large quantity of protons.The model also predicts the reverse Warburg effect in the tumor-associated-stromal cells,which is consistent with previous observations and supported by our results.We found that both PKM1 and PKM2 can enable aerobic glycolysis but PKM2 converts glucose to lactate much more efficiently than PKM1.As a result,PKM2 reduces glucose held for intracellular utilization,especially for production of citrate,and thus increases the ?-ketoglutarate/citrate ratio to promote generation of glutamine-derived acetyl-coenzyme A through the reductive pathway.Taken together,our data demonstrate that PKM2 plays a critical role in maintaining proton homeostasis and promoting reductive glutamine metabolism,and provide a better understanding of the physiological role of PKM2 in cancer cells.