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Regulation of pyruvate dehydrogenase complex related to lactate switch in CHO cells.

ABSTRACT: The metabolism of Chinese hamster ovary (CHO) cell lines is typically characterized by high rates of aerobic glycolysis with increased lactate formation, known as the "Warburg" effect. Although this metabolic state can switch to lactate consumption, the involved regulations of the central metabolism have only been partially studied so far. An important reaction transferring the lactate precursor, pyruvate, into the tricarboxylic acid cycle is the decarboxylation reaction catalyzed by the pyruvate dehydrogenase enzyme complex (PDC). Among other mechanisms, PDC is mainly regulated by phosphorylation-dephosphorylation at the three sites Ser232, Ser293, and Ser300. In this work, the PDC phosphorylation in antibody-producing CHO DP-12 cell culture is investigated during the lactate switch. Batch cultivations were carried out with frequent sampling (every 6 h) during the transition from lactate formation to lactate uptake, and the PDC phosphorylation levels were quantified using a novel indirect flow cytometry protocol. Contrary to the expected activation of PDC (i.e., reduced PDC phosphorylation) during lactate consumption, Ser293 and Ser300 phosphorylation levels were 33% higher compared to the phase of glucose excess. At the same time, the relative phosphorylation level of Ser232 increased steadily throughout the cultivation (66% increase overall). The intracellular pyruvate was found to accumulate only during the period of high lactate production, while acetyl-CoA showed nearly no accumulation. These results indicate a deactivation of PDC and reduced oxidative metabolism during lactate switch even though the cells undergo a metabolic transition to lactate-based cell growth and metabolism. Overall, this study provides a unique view on the regulation of PDC during the lactate switch, which contributes to an improved understanding of PDC and its interaction with the bioprocess.

SUBMITTER: Moller J

PROVIDER: S-EPMC7923601 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:Background & aimsAcute liver failure is a rapidly progressive deterioration of hepatic function resulting in high mortality and morbidity. Metabolic enzymes can translocate to the nucleus to regulate histone acetylation and gene expression.MethodsLevels and activities of pyruvate dehydrogenase complex (PDHC) and lactate dehydrogenase (LDH) were evaluated in nuclear fractions of livers of mice exposed to various hepatotoxins including CD95-antibody, α-amanitin, and acetaminophen. Whole-genome gene expression profiling by RNA-seq was performed in livers of mice with acute liver failure and analyzed by gene ontology enrichment analysis. Cell viability was evaluated in cell lines knocked-down for PDHA1 or LDH-A and in cells incubated with the LDH inhibitor galloflavin after treatment with CD95-antibody. We evaluated whether the histone acetyltransferase inhibitor garcinol or galloflavin could reduce liver damage in mice with acute liver failure.ResultsLevels and activities of PDHC and LDH were increased in nuclear fractions of livers of mice with acute liver failure. The increase of nuclear PDHC and LDH was associated with increased concentrations of acetyl-CoA and lactate in nuclear fractions, and histone H3 hyper-acetylation. Gene expression in livers of mice with acute liver failure suggested that increased histone H3 acetylation induces the expression of genes related to damage response. Reduced histone acetylation by the histone acetyltransferase inhibitor garcinol decreased liver damage and improved survival in mice with acute liver failure. Knock-down of PDHC or LDH improved viability in cells exposed to a pro-apoptotic stimulus. Treatment with the LDH inhibitor galloflavin that was also found to inhibit PDHC, reduced hepatic necrosis, apoptosis, and expression of pro-inflammatory cytokines in mice with acute liver failure. Mice treated with galloflavin also showed a dose-response increase in survival.ConclusionPDHC and LDH translocate to the nucleus, leading to increased nuclear concentrations of acetyl-CoA and lactate. This results in histone H3 hyper-acetylation and expression of damage response genes. Inhibition of PDHC and LDH reduces liver damage and improves survival in mice with acute liver failure. Thus, PDHC and LDH are targets for therapy of acute liver failure.Lay summaryAcute liver failure is a rapidly progressive deterioration of liver function resulting in high mortality. In experimental mouse models of acute liver failure, we found that two metabolic enzymes, namely pyruvate dehydrogenase complex and lactic dehydrogenase, translocate to the nucleus resulting in detrimental gene expression. Treatment with an inhibitor of these two enzymes was found to reduce liver damage and to improve survival.

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Regulation of pyruvate dehydrogenase complex related to lactate switch in CHO cells.

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