Global metabolic effect of manipulating pyruvate dehydrogenase complex activity in mammalian cells

Abstract

Aerobic glycolysis is an inefficient metabolic phenotype displayed by many rapidly proliferating cells during growth. It is characterized by high glycolytic activity and only partial oxidation of glucose resulting in the production of high amounts of lactate. This phenotype was originally reported by Otto Warburg in 1927 as a hallmark of cancer andwhile it is now known to occur in other fast growing cells as well -it remains an interesting target for cancer therapy. Aerobic glycolysis also has major implications for biopharmaceutical production, since lactate accumulation can be growth inhibiting, limiting the cell density that can be achieved in culture.Due to its association with various diseases and being an unfavorable metabolic phenotype in industrial applications, reducing the Warburg effect and analyzing accompanying effects on the cell as a whole are of great interest. Whereas in cancer therapy the objective is to kill cells relying on aerobic glycolysis, the aim in industrial applications is to reduce aerobic glycolysis without inducing cell death or inhibiting cell growth. Pyruvate dehydrogenase complex (PDC) is a mitochondrial gatekeeping enzyme determining how much pyruvate is converted to acetyl-CoA and subsequently enters the TCA cycle. PDC activity is regulated by reversible phosphorylation catalyzed by pyruvate dehydrogenase kinase (PDK) (phosphorylation → inactivation) and pyruvate dehydrogenase phosphatase (dephosphorylation → activation). PDC activity can be increased by inhibiting PDK using dichloroacetate (DCA) a known PDK inhibitor, hereby reducing aerobic glycolysis.The objective in this thesis was twofold; i) analyzing metabolic as well as growth inhibitory effects of DCA in human embryonic kidney 293 (HEK293) cells, and ii) investigating the effects of a non growth inhibiting DCA concentration using Chinese hamster ovary (CHO) cells in industrial relevant bioprocesses. In both studies aerobic glycolysis decreased with increasing DCA concentration characterized by reduced glucose consumption and lactate production. At lower DCA concentrations cell growth was unaffected. Furthermore, no increase in oxidative metabolism was detected at low DCA concentration indicating that the cells adopt a more energy efficient metabolism without directing more pyruvate into the TCA cycle. However, it appears that the cytoplasmic pyruvate fraction is reduced as not only less lactate but also less alanine is produced. The metabolic changes observed were mostly attributable to post-translational regulation since transcriptomics and proteomics analyses revealed only minor changes to metabolic enzymes. However, in the absence of iv increased TCA cycle activity, allosteric regulation of glycolytic enzymes did not readily explain reduced glycolysis.Cell growth in HEK293 cells was reduced only at higher DCA concentration when increased cellular stress and TCA cycle activity were detected. Since DCA was found to depolarize mitochondria the increased TCA cycle activity may not result in higher ATP productio...