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Global Metabolic Effect of Manipulating Pyruvate Dehydrogenase Complex Activity in Mammalian Cells

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Global Metabolic Effect of Manipulating Pyruvate Dehydrogenase Complex Activity in Mammalian Cells Global metabolic effect of manipulating pyruvate dehydrogenase complex activity in mammalian cells Maria Buchsteiner (Dipl.-Ing., M. Sc.) A thesis submitted for the degree of Doctor of Philosophy at The University of Queensland in 2014 Australian Institute of Bioengineering and Nanotechnology ii 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 and – while 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 iii 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 production and in fact the ATP yield may be insufficient leading to a “metabolic crisis” and reduced cell growth. Additionally, in the HEK293 study asparagine synthetase (ASNS) activity, amino acid transporter gene expression and mitochondrial one-carbon metabolism were increased in DCA cultures. Increased ASNS activity and one-carbon metabolism have been linked to various human diseases including cancer. However, further investigations are necessary to explain the upregulation of these reactions during increased PDC activity. With regard to industrial applications, reduced aerobic glycolysis induced by DCA led to longer cultivation periods and higher final antibody titers in CHO cell cultures due to lower lactate accumulation and slower increase in osmolality. Cell specific productivity and antibody quality in terms of charge variants, aggregation and glycan pattern were unaffected by DCA. In summary, we investigated the effects of increased PDC activity induced by DCA with regard to metabolic effects and growth inhibition. At lower DCA concentration the cells appear to have a more energy efficient metabolism and reduced aerobic glycolysis proved to be beneficial for culture performance and antibody production. However, the exact mechanisms leading to growth inhibition caused by DCA remain to be further investigated. iv Declaration by author This thesis is composed of my original work, and contains no material previously published or written by another person except where due reference has been made in the text. I have clearly stated the contribution by others to jointly-authored works that I have included in my thesis. I have clearly stated the contribution of others to my thesis as a whole, including statistical assistance, survey design, data analysis, significant technical procedures, professional editorial advice, and any other original research work used or reported in my thesis. The content of my thesis is the result of work I have carried out since the commencement of my research higher degree candidature and does not include a substantial part of work that has been submitted to qualify for the award of any other degree or diploma in any university or other tertiary institution. I have clearly stated which parts of my thesis, if any, have been submitted to qualify for another award. I acknowledge that an electronic copy of my thesis must be lodged with the University Library and, subject to the General Award Rules of The University of Queensland, immediately made available for research and study in accordance with the Copyright Act 1968. I acknowledge that copyright of all material contained in my thesis resides with the copyright holder(s) of that material. Where appropriate I have obtained copyright permission from the copyright holder to reproduce material in this thesis. Maria Buchsteiner v Publications during candidature Oral presentations Buchsteiner M, Quek LE, Gray P, Nielsen LK (2013) Improving Culture Performance and Antibody Production in CHO Cell Culture Processes by Reducing the Warburg Effect. Presented at BioProcessing Network Annual Conference 2013. October 22nd – 24th. Gold Coast, Australia. Buchsteiner M, Quek LE, Gray P, Nielsen LK (2012) Characterization of Increased Pyruvate Dehydrogenase Complex Activity Caused by Dichloroacetate in HEK 293 Cells. Presented at School of Chemistry and Molecular Biosciences 8th Annual Research Students Symposium 2012. November 22nd. Brisbane, Australia Poster presentations Buchsteiner M, Quek LE, Gray P, Nielsen LK (2014) Improving culture performance and antibody production in CHO cell culture processes by reducing the Warburg effect. Presented at Cell Culture Engineering XIV, May 4th – 9th. Quebec, Canada Buchsteiner M, Quek LE, Gray P, Nielsen LK (2012) Systems Level Characterisation of HEK293 Cells with Dichloroacetate Enhanced Pyruvate Dehydrogenase Activity. Presented at 23 rd meeting of the European Society for Animal Cell Technology. June 23rd – 26th. Lille, France Publications included in this thesis No publications included vi Contributions by others to the thesis Chapter 3: Dynamic Metabolic Flux Analysis using B-splines to study the Effects of Temperature Shift on CHO Cell Metabolism Contributor Statement of contribution Maria Buchsteiner (Candidate) Designed experiments (30%) Performed experiments (100%) Analysed data (30%) Wrote chapter (30%) Edited chapter (10%) Verónica Martínez Designed experiments (30%) Developed software (90%) Analysed data (70%) Wrote chapter (70%) Edited chapter (19%) Peter Gray Edited chapter (1%) Lake-Ee Quek Designed experiments (10%) Developed software (10%) Edited chapter (40%) Lars Keld Nielsen Designed experiments (30%) Edited chapter (30%) Chapter 4: Improving Culture Performance and Antibody Production in CHO Cell Culture Processes by Reducing the Warburg Effect Contributor Statement of contribution Maria Buchsteiner (Candidate) Designed experiments (80%) Performed experiments (97%) Analysed data (80%) Wrote chapter (100%) Edited chapter (20%) Lake-Ee Quek Analysed data (10%) Edited chapter (9%) Verónica Martínez Analysed data (10%) Camila Orellana & Esteban Marcellin Performed experiments (3%) Peter Gray Edited chapter (1%) Lars Keld Nielsen Designed experiments (20%) Edited chapter (70%) vii Chapter 5: Characterization of Increased Pyruvate Dehydrogenase Complex Activity Induced by Dichloroacetate in HEK293 Cells at a Cellular System Level Contributor Statement of contribution Maria Buchsteiner (Candidate) Designed experiments (60%) Performed experiments (100%) Analysed data (90%) Wrote chapter (100%) Edited chapter (30%) Lake-Ee Quek Analysed data (10%) Peter Gray Edited chapter (1%) Lars Keld Nielsen Designed experiments (40%) Edited chapter (69%) Appendix C: Metabolic Flux Analysis method Contributor Statement of contribution Lake-Ee Quek Developed software (100%) Wrote appendix (100%) Appendix H: Generating a stable HEK293 cell line overexpressing resistin-like molecule β using a Tet-On 3G inducible expression system Contributor Statement of contribution Maria Buchsteiner (Candidate) Designed experiments (30%) Performed experiments (80%) Analysed data (100%) Wrote appendix (100%) Edited appendix (70%) Stacey
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