A thesis submitted in fulfilment of the requirements for the degree of Doctor of Philosophy Metabolic tracing of NAD+ precursors using strategically labelled isotopes of NMN by Lynn-Jee Kim Supervisors Dr. Lindsay E. Wu (Primary) Prof. David A. Sinclair (Joint-Primary) Dr. Lake-Ee Quek (Co-supervisor) School of Medical Sciences Faculty of Medicine Thesis/Dissertation Sheet Surname/Family Name: Kim Given Name/s: Lynn-Jee Abbreviation for degree as give in PhD the University calendar: Faculty: Faculty of Medicine School: School of Medical Sciences + Thesis Title: Metabolic tracing of NAD precursors using strategically labelled isotopes of NMN Abstract 350 words maximum: (PLEASE TYPE) Nicotinamide adenine dinucleotide (NAD+) is an important cofactor and substrate for hundreds of cellular processes involved in redox homeostasis, DNA damage repair and the stress response. NAD+ declines with biological ageing and in age-related diseases such as diabetes and strategies to restore intracellular NAD+ levels are emerging as a promising strategy to protect against metabolic dysfunction, treat age-related conditions and promote healthspan and longevity. One of the most effective ways to increase NAD+ is through pharmacological supplementation with NAD+ precursors such as nicotinamide mononucleotide (NMN) which can be orally delivered. Long term administration of NMN in mice mitigates age-related physiological decline and alleviates the pathophysiologies associated with a high fat diet- and age-induced diabetes. Despite such efforts, there are certain aspects of NMN metabolism that are poorly understood. In this thesis, the mechanisms involved in the utilisation and transport of orally administered NMN were investigated using strategically labelled isotopes of NMN and mass spectrometry. A mass spectrometry method was developed to trace the incorporation of labelled NMN moieties in NAD+ metabolites following supplementation with labelled NMN compounds. This was validated in biologically relevant models such as mammalian cell lines (Chapter 3) and bacteria (Chapter 4), the latter serving as a proof-of-concept model to investigate NMN metabolism through bacterial routes before investigating its metabolic fate in vivo (Chapter 5). Following oral administration with labelled NMN compounds in mice, labelled NAD+ metabolites were detected in abundance in the peripheral tissues of mice treated with antibiotics but were largely absent in control mice. This suggests the majority of orally administered NMN is consumed by gut bacteria, limiting its availability to host peripheral tissues and insinuates host-microbe competition for NAD+ precursors. Interestingly, an abundance of nicotinamide riboside (NR) was observed both in vitro and in vivo following supplementation with NMN supporting the indirect NMN transport mechanism whereby it is dephosphorylated to NR prior to entering the cell. Overall, these findings have therapeutic implications in the dosing and route of administration of NMN as an NAD+-boosting strategy to treat conditions related to metabolic dysfunction and age-related diseases and further to promote healthy ageing and longevity. Declaration relating to disposition of project thesis/dissertation I hereby grant to the University of New South Wales or its agents the right to archive and to make available my thesis or dissertation in whole or in part in the University libraries in all forms of media, now or here after known, subject to the provisions of the Copyright Act 1968. I retain all property rights, such as patent rights. I also retain the right to use in future works (such as articles or books) all or part of this thesis or dissertation. I also authorise University Microfilms to use the 350 word abstract of my thesis in Dissertation Abstracts International (this is applicable to doctoral theses only). …………………………………… …………………………… ………...… Signature Witness Signature Date The University recognises that there may be exceptional circumstances requiring restrictions on copying or conditions on use. Requests for restriction for a period of up to 2 years must be made in writing. Requests for a longer period of restriction may be considered in exceptional circumstances and require the approval of the Dean of Graduate Research. FOR OFFICE USE ONLY Date of completion of requirements for Award: ii iii iv INCLUSION OF PUBLICATIONS STATEMENT UNSW is supportive of candidates publishing their research results during their candidature as detailed in the UNSW Thesis Examination Procedure. Publications can be used in their thesis in lieu of a Chapter if: • The candidate contributed greater than 50% of the content in the publication and is the “primary author”, ie. the candidate was responsible primarily for the planning, execution and preparation of the work for publication • The candidate has approval to include the publication in their thesis in lieu of a Chapter from their supervisor and Postgraduate Coordinator. • The publication is not subject to any obligations or contractual agreements with a third party that would constrain its inclusion in the thesis Please indicate whether this thesis contains published material or not: This thesis contains no publications, either published or submitted for ☐ publication (if this box is checked, you may delete all the material on page 2) Some of the work described in this thesis has been published and it has ☒ been documented in the relevant Chapters with acknowledgement (if this box is checked, you may delete all the material on page 2) This thesis has publications (either published or submitted for publication) ☐ incorporated into it in lieu of a chapter and the details are presented below CANDIDATE’S DECLARATION I declare that: • I have complied with the UNSW Thesis Examination Procedure • where I have used a publication in lieu of a Chapter, the listed publication(s) below meet(s) the requirements to be included in the thesis. Candidate’s Name Signature Date (dd/mm/yy) Lynn-Jee Kim v Acknowledgements First and foremost, I would like to sincerely thank my supervisors, Lindsay, David and Lake- Ee for their support and for providing me with all the necessary means and opportunities during my PhD. I thank each of you for all the intellectual insight, genuine advice and guidance I have received over the years. Thank you for all the invaluable memories, both within and outside the lab and for always making the extra effort to make the research experience in our lab unique and inspiring. A special thank you to all the members, past and present, of the Laboratory for Ageing Research. Thank you for being the most amazing team to work with, as well as learn alongside and from. Thank you especially to Abhi, Catherine, Jon, Ashley, Jin, and Tim for their assistance in experiments and for helping me solve experimental and technical dilemmas. This gratitude is also extended to faculty members and fellow peers of 3East, Allison and BRC technical staff for all their guidance and support. Support as part of the Australian Post-Graduate award is kindly acknowledged and I sincerely thank the post-graduate research support scheme for funding domestic and international conference travels. Finally, to all my friends and family, I thank you all for the undying love and encouragement I have received throughout my PhD. Here’s to growing older and healthier together. vi Publications and Presentations Research Publications NAD+ Repletion Rescues Female Fertility During Reproductive Ageing. Cell Reports, Feb 2020 Michael J. Bertoldo, Dave R. Listijono, Wing-Hong Jonathan Ho, Angelique H. Riepsamen, Xing L. Jin, Kaisa Selesniemi, Dale M. Goss, Saabah Mahbub, Jared M. Campbell, Abbas Habibalahi, Wei-Guo Nicholas Loh, Neil A. Youngson, Jayanthi Maniam, Ashley S.A. Wong, Dulama Richani, Catherine Li, Yiqing Zhao, Maria Marinova, Lynn-Jee Kim, Laurin Lau, Rachael M Wu, A. Stefanie Mikolaizak, Toshiyuki Araki, David G. Le Couteur, Nigel Turner, Margaret J. Morris, Kirsty A. Walters, Ewa Goldys, Christopher O’Neill, Robert B. Gilchrist, David A. Sinclair, Hayden A. Homer, Lindsay E. Wu + Impairment of An Endothelial NAD -H2S Signalling Network Is A Reversible Cause Of Vascular Aging. Cell, Mar 2018. Abhirup Das, George X. Huang, Michael S. Bonkowski, Alban Longchamp, Catherine Li, Michael B. Schultz, Lynn-Jee Kim, Brenna Osborne, Sanket Joshi, Yuancheng Lu, Jose Humberto Treviño-Villarreal, Myung-Jin Kang, Tzong-tyng Hung, Brendan Lee, Eric O. Williams, Masaki Igarashi, James R. Mitchell, Lindsay E. Wu, Nigel Turner, Zolt Arany, Leonard Guarente, David A. Sinclair Nicotinamide mononucleotide (NMN) deamidation and indirect regulation of the NAD metabolome Submitted to Nature Communications on 02/10/2020 Lynn-Jee Kim, Timothy J. Chalmers, Greg C. Smith, Abhirup Das, Eric Wing Keung Poon, Jun Wang, Simon P. Tucker, David A. Sinclair, Lake-Ee Quek, Lindsay E. Wu Fertility preservation during chemotherapy treatment without compromising oncological efficacy by NAD+ repletion. In preparation for resubmission Wing-Hong Jonathan Ho, Dave R. Listijono, Michael J. Bertoldo, Angelique H. Riepsamen, Kaisa Selesniemi, Yiqing Zhao, Wei-Guo Nicholas Loh, Neil A. Youngson, Safaa Cabot, vii Ashley S.A. Wong, Dulama Richani, Catherine Li, Lynn-Jee Kim, Laurin Lau, Rachael M Wu, Pawel Kordowitzki, Toshiyuki Araki, A. Stefanie Mikolaizak, Sonia Bustamante, Abhirup Das, Jayanthi Maniam, David G. Le Couteur, Nigel Turner, Lake-Ee Quek, Margaret J. Morris, Kirsty A. Walters, Robert B. Gilchrist, David A. Sinclair, Hayden A. Homer, Lindsay E. Wu Oral Presentations