Metabolic Tracing of NAD Precursors Using Strategically Labelled Isotopes of NMN

Home , Antony Galione, NADSYN1, Osamu Hayaishi, Tomas Lindahl, William John Young

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

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:

iii

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

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.

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

Metabolic Tracing of NAD+ Precursors Using Strategically Labelled Isotopes of NMN: Contribution of The Gut Microbiome to NAD+ Metabolism. Lynn-Jee Kim, Jonathan Ho, Jo Ann Yap, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu Neuroscience and Non-Communicable Diseases Department Seminar 2019, UNSW, Australia

Investigating Novel NAD+ Precursors and Their Therapeutic Potential Against Age- Associated Physiological Decline. (Poster selected for oral presentation) Lynn-Jee Kim, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu IUBMB Seoul 2018 Meeting, COEX Convention Centre 2018, Seoul, South Korea.

Investigating Novel NAD+ Precursors and Their Therapeutic Potential Against Age- Associated Physiological Decline. Lynn-Jee Kim, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu Neuroscience and Non-Communicable Diseases Department Seminar 2017, UNSW, Australia

Poster Presentations

Gut Microbiome Competes with Host For Orally Administered Nicotinamide Mononucleotide (NMN) By Reducing Its Availability And Altering Uptake Pathways. Lynn-Jee Kim, Jonathan Ho, Jo Ann Yap, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu Australian Biology of Ageing Conference 2019, University of Sydney, NSW, Australia. Awarded Best Poster Presentation.

Investigating Novel NAD+ Precursors and Their Therapeutic Potential Against Age- Associated Physiological Decline. Lynn-Jee Kim, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu

viii

IUBMB Seoul 2018 Conference, COEX Convention Centre, Seoul, South Korea. Awarded Top 10 Best Poster Presentations

Investigating Novel NAD+ Precursors and Their Therapeutic Potential Against Age- Associated Physiological Decline. Lynn-Jee Kim, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu Australian Biology of Ageing Conference 2018, University of Queensland, Queensland, Australia

Metabolic Tracing of NAD+ Precursors Using Strategically Labelled Isotopes. Lynn-Jee Kim, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu Cell Symposia Meeting: Aging and Metabolism 2018, Sitges, Spain.

Metabolic Tracing of NAD+ Precursors Using Strategically Labelled Isotopes. Lynn-Jee Kim, Lake-Ee Quek, David A. Sinclair, Lindsay E. Wu Cold Spring Harbor Laboratory 2018 Meeting: Mechanisms of Aging, Cold Spring Harbor, New York, USA.

The Role of NAD+ Biology in Chemotherapy-Induced Cardiotoxicity. Lynn-Jee Kim, Abhirup Das, David A. Sinclair, Lindsay E. Wu Australian Biology of Ageing Conference 2016, Coogee, NSW, Australia

Table of Contents

Acknowledgements ...... vi Publications and Presentations ...... vii Abstract ...... xiii List of Figures ...... xiv List of Tables ...... xvi List of Abbreviations ...... xvii ...... 1 1.1 Adding Health to Years ...... 1 1.2 The Biological Significance of NAD+ ...... 5 1.2.1 NAD+ as a redox cofactor ...... 8 1.2.2 NAD+ as a substrate ...... 15 1.3 NAD+ deficiency is a critical pathological factor in ageing and disease ...... 39 1.3.1 NAD+ deficiency in ageing and disease ...... 39 1.3.2 Increased NAD+ consumption as a cause of NAD+ deficiency ...... 40 1.3.3 Decreased NAD+ biosynthesis as a cause of NAD+ deficiency ...... 42 1.4 Consequences of NAD+ deficiency in cell function and survival ...... 44 1.5 Therapeutic potential of NAD+ boosting strategies ...... 46 1.6 NAD+ biosynthesis pathways ...... 49 1.6.1 The de novo synthesis pathway ...... 49 1.6.2 The Preiss-Handler pathway ...... 50 1.6.3 The Salvage/Recycling pathway ...... 51 1.6.4 Additional pathways in yeast and bacteria, that do not exist in mammals ...... 53 1.7 Current gaps in the field of NAD+ biology ...... 55 1.7.1 Unexplored mechanisms of NAD+ precursor deamidation ...... 55 1.7.2 NAD+ precursor uptake mechanisms still unclear ...... 56 1.7.3 Quantifying the NAD+ metabolome remains a challenge ...... 57 1.8 Thesis goals ...... 59 ...... 61 2.1 Isotopic labelled compounds...... 61 2.1.1 Synthesis of labelled NMN compounds ...... 61 2.1.2 Labelled glutamine...... 63 2.2 Sample processing for mass spectrometry ...... 64 2.2.1 Preparation of extraction buffer ...... 64

2.2.2 Cell harvest and extraction...... 65 2.2.3 Further sample processing for mass spectrometry ...... 65 2.2.4 NAD+ metabolite standard curve preparation ...... 65 2.3 LC-MS/MS ...... 66 2.3.1 Liquid chromatography ...... 66 2.3.2 Mass spectrometry ...... 67 2.4 Data analysis ...... 67 2.5 Statistics ...... 68 ...... 69 3.1 Introduction ...... 69 3.2 Methods...... 77 3.2.1 Optimisation of mass spectrometry parameters ...... 77 3.2.2 Liquid chromatography (LC) separation ...... 78 3.2.3 Isolation of primary hepatocytes and treatment ...... 79 3.2.4 C2C12 and HEK293 Cell Culture...... 81 3.3 Results ...... 82 3.3.1 Compound optimisation of mass spectrometry parameters for internal standards. 84 3.3.2 Optimisation of mass spectrometry parameters for NAD+ metabolites ...... 85 3.3.3 Detecting labelled NMN isotopes using LC-MS/MS and MRM...... 89 3.3.4 Tracing the metabolic fate of NMN isotopes using LC-MS/MS to detect labelled NAD+ metabolites...... 90 3.3.5 Intracellular NR is detected following extracellular supplementation of NMN in primary hepatocytes...... 95 3.3.6 NMN does not lead to increases in deamidated metabolites, NaMN and NaAD, following supplementation in primary hepatocytes...... 98 3.3.7 Time-course tracing of NMN metabolism in C2C12 and HEK293 cells – amidated metabolites ...... 106 3.3.8 Time-course tracing of NMN metabolism in C2C12 and HEK293 cells – deamidated metabolites ...... 110 3.4 Discussion ...... 115 ...... 121 4.1 Introduction ...... 121 4.1.1 Bacterial de novo NAD biosynthesis pathway...... 123 4.1.2 NAD+ is salvaged by deamidation in bacteria ...... 125 4.1.3 Potential NMN uptake routes in bacteria ...... 127 4.1.4 Key differences compared to eukaryotic pathway ...... 129

4.2 Methods...... 131 4.2.1 Bacterial culture and NMN treatment ...... 131 4.2.2 Extracting NAD+ metabolites from E. coli cell lysates and culture supernatants. 134 4.3 Results ...... 139 4.3.1 Supplementation of NMN in E. coli – changes to NAD+ metabolites...... 139 4.4 Discussion ...... 147 ...... 152 5.1 Introduction ...... 152 5.1.1 Rationale for NMN isotope design ...... 157 5.2 Methods...... 164 5.2.1 Animal housing ...... 164 5.2.2 Antibiotic and NMN treatment regimen ...... 164 5.2.3 Plasma collection and metabolite extraction...... 165 5.2.4 Tissue collection, metabolite extraction and data analysis ...... 167 5.2.5 DNA extraction from faecal pellets ...... 168 5.3 Results ...... 170 5.3.1 Antibiotic ablation of the gut microbiome ...... 170 5.3.2 Antibiotic treatment increases NAD+ metabolite and NMN availability in the intestinal tissue of mice...... 176 5.3.3 Evidence for NMN uptake facilitated by dephosphorylation into NR ...... 186 5.3.4 NMN deamidation is independent of the microbiome...... 190 5.4 Discussion ...... 193 ...... 201 6.1 Quantifying the NAD metabolome using LC-MS/MS—So close, yet [not] so far . 203 6.2 Mechanisms of NMN uptake in gut epithelial tissue — ¿Por qué no los dos? ...... 206 6.3 Host-microbe interactions in NAD+ precursor metabolism—Friend or foe? ...... 208 6.4 Preliminary evidence for host NMN deamidation—it’s not you, it’s me ...... 210 6.5 Beyond NAD metabolism—blue-sky thinking ...... 211 Appendix ...... 213 References ...... 281

xii

Abstract

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. Strategies to restore intracellular NAD+ levels are emerging as promising therapeutics 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 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 into 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.

xiii

List of Figures Figure 1.1 Chemical structure of NAD+ ...... 5 Figure 1.2 The biological roles of NAD+ ...... 7 Figure 1.3 Functions of NAD(P)(H) as a redox cofactor...... 14 Figure 1.4 NAD+ biosynthesis pathways in mammals...... 54 Figure 2.1 Chemical structure and isotope labelling positions of NMN ...... 62 Figure 2.2 Chromatogram peaks of NMN isotopes using LC-MS/MS injected at 100 µM. ... 63 Figure 2.3 Chemical structure and isotope labelling of glutamine...... 63 Figure 2.4 Chromatogram peaks of unlabelled glutamine and isotope labelled glutamine using LC-MS/MS injected at 100 µM...... 64 Figure 3.1 Labelling strategy of labelled NMN isotopes...... 74 Figure 3.2 Detecting labelled NMN and NAD+ isotopologues using MRM (multiple reaction monitoring)...... 83 Figure 3.3 Representative chromatograms of unlabelled (M+0) and labelled NMN isotopes from injection of a 100 µM standard solution...... 89 Figure 3.4 Labelled NMN compounds are detected intact and contribute to an increase in NAD+ in primary hepatocytes...... 93 Figure 3.5 Labelled NAD+ is degraded and contributes to increases in metabolites in the NAD+ recycling pathway...... 94 Figure 3.6 An abundance of intact labelled NR is detected following supplementation with labelled NMN...... 97 Figure 3.7 Chromatogram peaks of NMN/NaMN isotopologue pairs sharing the same MRM transition...... 102 Figure 3.8 Chromatogram peaks from NAD+/NaAD isotopologues sharing the same MRM transition...... 105 Figure 3.9 Labelled NMN detected intact and contributes to increases in NAD+ and NR over time in cell...... 108 Figure 3.10 Degradation of NAD+ and nicotinamide salvage ...... 109 Figure 3.11 Detection of NMN/NaMN isotopologue pairs in C2C12 and HEK293 cells following supplementation with NMN...... 113 Figure 3.12 Detection of NAD+/NaAD isotopologue pairs in C2C12 and HEK293 cells following supplementation with NMN...... 114 Figure 4.1 NAD+ biosynthesis pathways in bacteria...... 126 Figure 4.2 E. coli growth curve and sample OD600 values...... 132 Figure 4.3 E. coli growth curve and sample OD600 values...... 133 Figure 4.4 Detection of amidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN...... 143

xiv

Figure 4.5 Detection of amidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN1...... 144 Figure 4.6 Detection of deamidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN...... 145 Figure 4.7 Detection of deamidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN1...... 146 Figure 5.1 Labelled isotope compounds ...... 158 Figure 5.2 Schematic of the NMN deamidation and re-amidation hypothesis using NMN1 and 15N-glutamine...... 161 Figure 5.3 Schematic of NMN deamidation hypothesis using NMN2...... 162 Figure 5.4 Schematic for the potential biosynthesis pathways and NAD+ metabolites involved in the utilisation of NMN2...... 163 Figure 5.5 The effect of antibiotic treatment in mice...... 173 Figure 5.6 Reduced diversity of the murine faecal microbiome upon antibiotic treatment in NMN2 cohort...... 174 Figure 5.7 Reduced diversity of the murine faecal microbiome upon antibiotic treatment in NMN1 cohort...... 175 Figure 5.8 An increase in amidated NAD+ metabolites detected with antibiotic treatment in the intestinal tissue after NMN2 administration...... 180 Figure 5.9 Detection of amidated NAD+ metabolites with antibiotic treatment in the liver after NMN2 administration...... 181 Figure 5.10 Detection of nicotinamide (NAM) with antibiotic treatment in the plasma after NMN2 administration...... 182 Figure 5.11 An increase in amidated NAD+ metabolites detected with antibiotic treatment in the intestinal tissue after NMN1 administration...... 183 Figure 5.12 Detection of amidated NAD+ metabolites with antibiotic treatment in the liver after NMN1 administration...... 184 Figure 5.13 Detection of nicotinamide (NAM) with antibiotic treatment in the plasma after NMN1 administration...... 185 Figure 5.14 Schematic for the direct or indirect transport of NMN in intestinal cells...... 188 Figure 5.15 The ratio of the labelled-to-unlabelled isotopologues of NAD+ metabolites ..... 189 Figure 5.16 Deamidation pathway in the intestinal tissue of antibiotic treated mice following administration with NMN2 or NMN1 and 15N-amide-glutamine (15N-gln)...... 192

List of Tables Table 3.1 Liquid chromatography (LC) separation gradient ...... 78 Table 3.2 MRM transitions for internal standards and mass spectrometry parameters...... 84 Table 3.3 MRM transitions and MS parameters for unlabelled NAD+ metabolites ...... 85 Table 3.4 MRM transitions and mass spectrometry parameters for isotopologues of NAM, NMN, NR and NAD+ ...... 86 Table 3.5 MRM transitions and mass spectrometry parameters for isotopologues of NA, NaR, NaMN and NaAD ...... 87 Table 3.6 LC-MS precision and accuracy parameters for each metabolite ...... 88 Table 4.1 MRM transitions and mass spectrometry (MS) parameters to detect NAD+ metabolites in E. coli (supernatant and lysates) following supplementation with unlabelled NMN using LC-MS/MS...... 136 Table 4.2 MRM transitions and MS parameters to detect NAD+ isotopologues of amidated metabolites in E. coli (supernatant and lysates) following supplementation with NMN1 using LC-MS/MS...... 137 Table 4.3 MRM transitions and MS parameters to detect NAD+ isotopologues of deamidated metabolites in E. coli (supernatant and lysates) following supplementation with NMN1 using LC-MS/MS...... 138

xvi

Chapter 1 Introduction

List of Abbreviations

3-HAO 3-hydroxyanthranolic acid oxygenase 6-PG 6-phosphogluconate Abx Antibiotics ACN Acetonitrile AD Alzheimer's Disease ADP Adenosine diphosphate ADPR Adenosine diphosphate ribose AK Adenosine kinase ALC1 Amplified in liver cancer protein 1 aNHEJ Alternative pathway for non-homologous end joining AOPCP Adenosine-5′-(α,β-methylene) diphosphate ART Adenosine diphosphate-ribosyltransferase ATP Adenosine triphosphate BER Base excision repair BRCA1 Breast cancer type 1 susceptibility protein BRCT BRCA1 C-terminus BRMS1 Breast cancer metastasis suppressor 1 BSA Bovine serum albumin C2C12 Mouse myoblast cell line cADPR Cyclic adenosine diphosphate ribose CD157 Cluster of differentiation 157 CD38 Cluster of differentiation 38 CD73 Cluster of differentiation 73 CE Collision energy CID Collision induced dissociation CK2 Casein kinase 2 cNHEJ Classical pathway for non-homologous end joining CobT Cobaltochelatase CPS1 Carbamoyl phosphate synthetase 1 CS Cockayne Syndrome CSA Camphorsulfonic acid (internal standard) CtBP C-terminal binding protein CtIP C-terminal interacting protein CXP Cell exit potential cyclin-B/cdk1 Cyclin B/cyclin-dependent kinase 1 CYP450 Cytochrome P450 DBC1 Deleted in breast cancer 1 DDB2 DNA damage-binding protein 2 DDR DNA damage response DHAP Dihydroxyacetone phosphate Dip Dipyridamole

xvii

Chapter 1 Introduction

DMEM Dulbecco's minimum essential medium DNA-PKcs DNA-dependent protein kinase catalytic subunit DP Declustering potential DSB Double-stranded breaks DYRK Dual specificity tyrosine phosphorylation-regulated kinases EGTA Ethylene glycol-bis(β-aminoethyl ether)-N,N,N′,N′- tetraacetic acid eNAMPT Extracellular nicotinamide phosphoribosyltransferase ENT Equilibrative nucleoside transporter ER Endoplasmic reticulum ETC Electron transport chain FAD Flavin adenine dinucleotide FADH2 Flavin adenine dinucleotide FBS Fetal bovine serum FOXO1 Forkhead box O 1 G6PD Glucose-6-phosphate dehydrogenase GAPDH Glyceraldehyde phosphate dehydrogenase GDH Glutamate dehydrogenase GIT Gastrointestinal tract Gln Glutamine GPR109a G-protein coupled receptor 109a GPx Glutathione peroxidase GR Glutathione reductase GSH Glutathione HBSS Hanks buffered salt solution HD Helical domain HEK293 Human embryonic kidney cell line HepG2 Human hepatocellular carcinoma cell line HIC1 Hypermethylated in cancer 1 HIF-1α Hypoxia-inducible factor 1 HMGCS2 3-hydroxy-3-methylglutaryl CoA synthase 2 HPLC High performance liquid chromatography HR Homologous recombination I/R Ischemia/reperfusion IDH Isocitrate dehydrogenase IFN-1 Interferon-1 JNK c-Jun N-terminal kinases KFA Kynurenine formamidase KMO Kynurenine 3-hydroxylase KYU Kynureninase LB Luria Bertani LCAD Long-chain acyl coenzyme A dehydrogenase LCFA Long chain fatty acid LC-MS/MS Liquid chromatography tandem mass spectrometry

xviii

Chapter 1 Introduction

LPS Lipopolysaccharide m/z mass to charge ratio MART Mono-ADP-ribosyltransferase MDC1 Mediator of DNA damage checkpoint protein 1 ME Malic enzyme MeOH Methanol MERIT40 Mediator of Rap80 interactions and targeting 40 kd MES Methionine sulfonate MnSOD Manganese superoxide dismutase MPT Mitochondrial permeability transition MRE11 Meiotic recombination 11 MRM Multiple reaction monitoring MRN complex MRE11-RAD50-NSB1 MS Mass spectrometry NA Nicotinic acid NaAD Nicotinic acid adenine dinucleotide NAADP Nicotinic acid adenine dinucleotide phosphate NAD+ Nicotinamide adenine dinucleotide (oxidised form) NADH Nicotinamide adenine dinucleotide (reduced form) NADK Nicotinamide adenine dinucleotide kinase NADSYN Nicotinamide adenine dinucleotide synthetase NAM Nicotinamide NaMN Nicotinic acid mononucleotide NAMPT Nicotinamide phosphoribosyltransferase NAPRT Nicotinic acid phosphoribosyltransferase NaR Nicotinic acid riboside NBS1 Nijmegen breakage syndrome 1 NBTI S-(4-nitrobenzyl)-6-thioinosine NER Nucleotide excision repair NF-κB Nuclear factor kappa B NHEJ Non-homologous end joining NK Natural killer NMN Nicotinamide mononucleotide NMNAT Nicotinamide mononucleotide adenylyltransferase NNT Nicotinamide nucleotide transhydrogenase NOX NADPH oxidase NPP1 Nucleotide pyrophosphatase/phosphodiesterase 1 NPSC Neural/progenitor stem cells NR Nicotinamide riboside NRH Nicotinamide riboside (reduced) NRK Nicotinamide riboside kinase NTPDase Nucleoside tri- and diphosphohydrolase OD Optical density PAR Poly-ADP-ribose

xix

Chapter 1 Introduction

PARP Poly-(ADP)-ribose polymerase PBMC Peripheral blood mononuclear cells PBS Phosphate buffered saline PD Parkinson's Disease PD-1 Programmed death 1 PDH Pyruvate dehydrogenase PD-L1 Programmed death 1 ligand PGC-1α PPARγ coactivator 1-alpha Pnc1 Nicotinamide deamidase in yeast PncA Nicotinamide deamidase in E. coli PncC Nicotinamide mononucleotide deamidase in E. coli PNKP Polynucleotide kinase 3′-phosphatase PNP Purine nucleoside phosphorylase PPARγ Proliferator-activated receptor gamma PPP Pentose phosphate pathway PRPP 5-phosphoribosyl-1-pyrophosphate PRS Phosphoribosyl synthetase Prx Peroxiredoxins Q1 First quadrupole Q2 Second quadrupole Q3 Third quadrupole QA Quinolinic acid QAPRT Quinolinic acid phosphoribosyltransferase R5P Ribose-5-phosphate rDNA ribosomal DNA ROS Reactive oxygen species RT Retention time SASP Senescence associated secretory phenotype SDH Succinate dehydrogenase SIL Stable isotopically labelled Sir2 Sirtuin 2 in yeast SLC Solute carrier SOD2 Superoxide dismutase SRM Selective reaction monitoring SSB Single-stranded break SUMO Small ubiquitin-like modifier TCA Trichloroacetic acid TD4 Thymine-d4 TDO Tryptophan 2,3-dioxygenase TPCN Two pore calcium channels TR Thioredoxin reductase TRF1 Telomeric repeat factor 1 Trp Tryptophan TRX Thioredoxin

Chapter 1 Introduction

TRX-S2 Thioredoxin (oxidised) UCP1 Uncoupling protein 1 UPR Unfolded protein response UV Ultraviolet WAT White adipose tissue Wlds Slow Wallerian degeneration XPA Xeroderma pigmentosum group A-complementing protein XPC Xeroderma pigmentosum group C-complementing protein XRCC1 X-ray repair cross-complementing protein 1

xxi

Chapter 1 Introduction

Introduction

1.1 Adding Health to Years

Human lifespan is increasing, but not necessarily in better health. Globally, the greatest causes

of mortality in the elderly are heart disease, stroke and chronic lung disease while the greatest

causes of disability involve sensory impairments, back and neck pain, chronic obstructive pulmonary disease, depressive disorders, falls, diabetes, dementia and osteoarthritis (WHO

2015). The incidence of these and many other age-related diseases and disabilities can be lowered through lifestyle interventions including exercise and moderating nutritional intake, though promoting the widespread uptake of these interventions has met with mixed results.

In the last two decades, the biology of ageing has emerged as an exciting field with the aim of understanding fundamental processes that cause physiological decline with age. Ageing is now recognised as a malleable process, which was first demonstrated through the finding

Chapter 1 Introduction that a simple reduction in food intake in rats extended overall lifespan (McCay, Crowell, and

Maynard 1935). Since then, work has been undertaken to separate the concepts of biological age versus chronological age. The physical and social aspects of an individuals’ environment can have a significant impact on lifespan, and more importantly, on healthspan—the years of life spent in the absence of disease or disability and maintained in good health (Crimmins

2015).

At a biological level, ageing is characterized by a gradual, lifelong accumulation of molecular and cellular damage that results in a progressive impairment in physiological function, an increased vulnerability to environmental challenges and a growing risk of disease and death (WHO 2015). Importantly, this definition of ageing is also applicable to a number of other disease states, where molecular and cellular damage accumulates with time. On a molecular level, the mechanisms involved in the pathogenesis of individual age related diseases are thought to drive ageing itself, and drawing on these parallels helped define the nine hallmarks of ageing (López-Otín et al. 2013). These hallmarks are genomic instability, telomere attrition, epigenetic alterations, loss of proteostasis, deregulated nutrient sensing, mitochondrial dysfunction, cellular senescence, stem cell exhaustion and altered intercellular communication. The ultimate goal of ageing research is to find interventions that target these hallmarks to increase healthspan during ageing.

Interestingly, the interventions that extend lifespan, through genetic or pharmacological manipulation in experimental models, often result in a broad-spectrum improvement in health and vice versa. The most consistent and robust intervention that demonstrates this is caloric restriction (CR), defined by a reduction in food intake without malnutrition (Speakman and

Mitchell 2011). First demonstrated in rats (McCay, Crowell, and Maynard 1935), CR-induced lifespan extension has been shown across multiple organisms from yeast to humans, revealing that the mechanisms regulating lifespan involve evolutionarily conserved pathways (Anderson

Chapter 1 Introduction

et al. 2003; Burger, Buechel, and Kawecki 2010; Civitarese et al. 2007; Colman et al. 2009;

Colman et al. 2014; Kaeberlein et al. 2004; Lee et al. 1999; Lee et al. 2006; Lin et al. 2004;

Mattison et al. 2017; Rogina and Helfand 2004; Witte et al. 2009; Zhang et al. 2012). A lifespan

extension of 2-3-fold was achieved in yeast, fruit flies and worms and up to 50% extension in

median and maximal lifespan in laboratory rodents following a 20-50% caloric reduction. In

addition to lifespan extension, CR delays the onset and progression of various pathological

conditions including cardiomyopathies (Cohen et al. 2017; Niemann et al. 2010), diabetes

(Baumeier et al. 2015; Hammer et al. 2008; Teeuwisse et al. 2012), cancer (Cadoni et al. 2017;

Lanza-Jacoby et al. 2013) and neurodegeneration (Gräff et al. 2013).

Due to the challenges of implementing CR in daily life, considerable effort has been invested to find interventions that mimic the benefits of CR. For example, the anti-diabetic drug metformin has demonstrated a longevity benefit independent of its effect in glycaemic control (Anisimov et al. 2011a; Cabreiro et al. 2013; Martin-Montalvo et al. 2013), with various improved aspects of health (Haak et al. 2012; Martin-Montalvo et al. 2013; Morin-Papunen et

al. 2012) including the risk of certain cancers (Mohammed et al. 2013; Shankaraiah et al. 2019), kidney disease (Neven et al. 2018), neurodegeneration (Lu et al. 2016) and cardiac dysfunction

(Pandey et al. 2019), among many others. In addition to metformin, other CR-mimetics that show similar benefits to healthspan and lifespan include resveratrol (Rascón et al. 2012), rapamycin (Anisimov et al. 2011b; Bitto et al. 2016; Harrison et al. 2009), spermidine

(Eisenberg et al. 2016; Eisenberg et al. 2009), quercetin (Alugoju et al. 2018), and intermittent fasting (Catterson et al. 2018; Honjoh et al. 2009; Mitchell et al. 2019), among others.

There are four key proteins that have been identified as mediating the benefits of CR.

These include the insulin/insulin-like growth factor 1 (insulin/IGF-1), adenosine monophosphate activated protein kinase (AMPK), mechanistic target of rapamycin (mTOR) and the sirtuins (SIRTs) (Henrique Mazucanti et al. 2015; Pan and Finkel 2017). Briefly,

Chapter 1 Introduction

insulin/IGF-1 is part of the insulin/IGF-1 signalling (IIS) pathway, a highly conserved nutrient

sensing pathway regulated by hormones such as insulin and growth hormone (GH) which

regulates IGF-1 levels. One of the mechanisms to explain CR-induced lifespan extension is

thought to be through reducing plasma and tissue levels of insulin and IGF-1 (Li et al. 2011;

Zaarur et al. 2019; Zarse et al. 2012). While this has sparked controversy in the past, with some

arguing that lifespan extension through impaired IIS signalling is independent of CR-mediated lifespan extension (Houthoofd et al. 2003; Min et al. 2008), recent studies continue to show that downregulation of insulin/IGF-1 levels are still relevant to the beneficial effects of CR in lifespan extension (Zaarur et al. 2019; Zarse et al. 2012). The nutrient and energy sensor AMPK is activated by metabolic stressors that decrease energy availability, such as CR. Once activated, it promotes compensatory utilisation of other energy substrates for ATP generation, increasing the beta oxidation of fatty acids, and activates other proteins such as the sirtuins and mTOR and the IIS pathway to mediate its protective effects (Greer et al. 2007; Hardie, Ross, and Hawley 2012; Palacios et al. 2009). mTOR is also an important cellular nutrient and energy sensor activated in response to an abundance of nutrients (amino acids) or stimulated by growth factors to promote growth and proliferation by enhancing anabolic processes such as translation and ribosomal biogenesis, while inhibiting catabolic processes such as autophagy. The inhibition of mTOR, through the clinically used immunosuppressive small molecule rapamycin, extends lifespan (Anisimov et al. 2010) and the benefits of CR are also considered to be mediated in part by the downregulation of mTOR (Dogan et al. 2011; Yang et al. 2014).

The sirtuins are a family of seven histone deacylases whose activity is dependent on the consumption of nicotinamide adenine dinucleotide (NAD+) (Frye 1999; Imai et al. 2000a;

Landry et al. 2000). They play a major role in response to injury and stress and regulate almost

all cellular functions such as energy metabolism, inflammation and neuronal function and play

a major role in mediating lifespan extension (Houtkooper, Pirinen, and Auwerx 2012; Howitz

Chapter 1 Introduction

et al. 2003; Jęśko et al. 2017; Vachharajani et al. 2016). In recent years, the redox and enzyme

cofactor NAD+ has emerged as an important determinant of late-life health and lifespan (Fang

et al. 2017; Johnson and Imai 2018; Zhang et al. 2016a). NAD+ levels decline with age or in

various pathological conditions, and strategies that elevate NAD+ levels have created

promising new avenues to combat age-related diseases (Aman et al. 2018; Rajman, Chwalek, and Sinclair 2018; Yoshino, Baur, and Imai 2018).

1.2 The Biological Significance of NAD+

NAD+ is a pyridine nucleotide first discovered in 1906 as a cofactor for enhancing alcohol

fermentation in yeast (Harden and Young 1906a, 1906b). Shortly after, NAD+ was successfully

isolated from yeast extracts by Hans von Euler-Chelpin in 1936 who characterised its chemical structure as a pyridine nucleotide made up of two mononucleotides – adenosine monophosphate (AMP) and nicotinamide mononucleotide (NMN) (Figure 1.1, below).

Figure 1.1 Chemical structure of NAD+ Nicotinamide adenine dinucleotide (NAD+) is made up of two mononucleotides, adenosine monophosphate (AMP) and nicotinamide mononucleotide (NMN) (Schlenk and Euler 1936).

Chapter 1 Introduction

In the same year, Otto Warburg discovered the function of NAD+, as a molecule capable

of transferring electrons between compounds in metabolic reactions. Its phosphorylated form,

NADP+, was also discovered (Warburg and Christian 1936) and this compound has important functions as a redox cofactor and in anabolic reactions, especially in its reduced form, NADPH

(Agledal, Niere, and Ziegler 2010). In 1950, Arthur Kornberg was the first to show that NAD+

was synthesised from the condensation of ATP and NMN via the enzyme nicotinamide

mononucleotide adenylyltransferase (NMNAT) (Kornberg 1950). Since these discoveries, the

diverse roles of NAD+ continue to expand and emerge as an molecule of central importance in

molecular and cell biology.

Today, NAD+ is considered one of the most fundamental components in life involved

in over 500 enzymatic reactions in the body (Ansari and Raghava 2010). NAD+ levels decline

with age (Braidy et al. 2011b; Clement et al. 2019; Gomes et al. 2013; Massudi et al. 2012a;

Mouchiroud et al. 2013; Stein and Imai 2014) and are also implicated in pathological conditions such as diabetes (Trammell et al. 2016c; Yoshino et al. 2011), obesity (Cantó et al. 2012), nonalcoholic fatty liver disease (Zhou et al. 2016), and neurodegeneration (Hou et al. 2018; Lu et al. 2014a). NAD+ levels can be increased either through genetic manipulation or through

pharmacological supplementation with NAD+ precursors, resulting in benefits that are

mediated by its critical role as an essential cofactor used in oxidation-reduction (redox) reactions, and secondly, as a crucial substrate and modulator of NAD+-consuming enzymes

that regulate biological processes from energy metabolism to DNA repair and cell survival

(Braidy et al. 2019; Kulkarni and Brookes 2019; Xiao et al. 2018) (Figure 1.2, below).

Interestingly, NAD+ levels oscillate according to the circadian clock, an autonomous molecular

programme that drives the expression of thousands of genes around a 24-hour cycle. This

circadian cycle regulates the expression of nicotinamide phosphoribosyltransferase (NAMPT),

Chapter 1 Introduction the rate-limiting enzyme involved in NAD+ biosynthesis (Ramsey et al. 2009) (Figure 1.2, below).

Figure 1.2 The biological roles of NAD+ NAD+ is involved in over 500 enzymatic reactions in the body. NAD+ is an important cofactor in (1) oxidation-reduction (redox) reactions involved in key metabolic processes such as (2) glycolysis, reactions in the tricarboxylic acid (TCA) cycle and oxidative phosphorylation (OXPHOS). NAD+ is also an important substrate for NAD+-consuming enzymes such as (3) poly-ADP-ribose polymerases (PARPs) involved in DNA damage repair and (4) the sirtuins (SIRTs), histone deacetylases that regulates gene expression and respond to cellular stress. NAD+ is also degraded by glycohydrolases (NADases) such as (5) cluster of differentiation number 38 (CD38) and its homologue, CD157, that play a role in calcium (Ca2+) mobilization and the immune response. (6) NAD+ is also degraded by a protein called sterile alpha and Toll/interleukin-1 receptor motif-containing 1 (SARM1) which is implicated in axonal degeneration. (7) NAD+ levels also oscillate around a 24-hour cycle governed by the circadian clock, an autonomous molecular programme that drives the expression of thousands of genes around a 24-hour cycle.

Chapter 1 Introduction

1.2.1 NAD+ as a redox cofactor

NAD+ plays a fundamental role as a redox cofactor to donate or accept electrons, rapidly

shuttling between its oxidised (NAD+) and reduced (NADH) forms, in its dephosphorylated

state as NAD+ and NADH, or in its phosphorylated state as NADP+ and NADPH (Figure 1.3,

below).

1.2.1.1 NAD+ and NADH roles and functions

Redox reactions are primarily carried out by dehydrogenase, hydroxylase and reductase

enzymes in the cell and involves the transfer of electrons between metabolites (Monti et al.

2011). NAD+ severs as an electron acceptor, which is reduced to form NADH. Similarly,

NADH serves as an electron donor which is oxidised to regenerate NAD+. It is important to

recognise that redox reactions between NAD+ and NADH simply involve the exchange of

electrons in a non-destructive way. In contrast, the role of NAD+ as a substrate, discussed in

the next section, involves the hydrolysis of NAD+, requires its resynthesis from precursors in

a process that also consumes energy in the form of ATP.

NAD+ is reduced in major energy production pathways such as glycolysis (via glyceraldehyde phosphate dehydrogenase; GAPDH) (Harris 1972), pyruvate oxidation to acetyl-CoA (via pyruvate dehydrogenase; PDH) (Wieland 1983), the tricarboxylic acid (TCA) cycle (via α-ketoglutarate, isocitrate and malate dehydrogenases) (Cupp and McAlister-Henn

1992; Hägele, Neeff, and Mecke 1978; Pettit et al. 1973; Seelig and Colman 1978) and fatty acid oxidation (via 3-hydroxyacyl-CoA dehydrogenase) (Uchida et al. 1992). Another reduced cofactor, flavin adenine dinucleotide (FADH2) is generated alongside NADH in the TCA cycle

by reducing the oxidised form, flavin adenine dinucleotide (FAD) via succinate dehydrogenase

(Kita et al. 1989). NADH and FADH2 are both important cofactors in energy metabolism as

they donate their electrons to complex I and II of the electron transport chain (ETC),

Chapter 1 Introduction

respectively, to generate adenosine triphosphate (ATP) during oxidative phosphorylation

(OXPHOS) in the mitochondria (Papa et al. 2012). NADH made in the cytoplasm is shuttled

into the mitochondria via two separate shuttle systems: the malate-aspartate shuttle which is

reversible and relies on high cytosolic NADH levels, or the glycerol phosphate shuttle which

is irreversible and can act in low cytosolic NADH levels (Luo, Li, and Yan 2015). Delivery of

electrons to the ETC is an important step for regenerating NAD+ to maintain intracellular NAD+

levels and the NAD+/NADH ratio, which has been recorded at approximately 700 in the cytoplasm and 7-8 in the mitochondria (Stubbs, Veech, and Krebs 1972). An imbalance in the

NAD+/NADH ratio, usually from an excess of NADH can lead to increased oxidative stress,

and has been implicated in various pathological conditions such as diabetes (Wu, Jin, and Yan

2017; Wu et al. 2017), acute kidney injury (Nechifor and Dinu 2011), and heart failure (Lee et

al. 2016a).

1.2.1.2 NADP+ and NADPH roles and functions

An estimated 10% of the total NAD+ and NADH pool exists in their phosphorylated forms,

NADP+ and NADPH. The non-phosphorylated cofactors NAD+ and NADH primarily participate in catabolic reactions while their phosphorylated forms, NADP+ and NADPH are

mainly required in anabolic reactions and cellular oxidative stress defence.

NADP+

The main source of NADP+ is from the phosphorylation of NAD+ via ATP-dependent NAD

kinase (NADK) in the cytoplasm (Lerner et al. 2001) or mitochondria (Ohashi, Kawai, and

Murata 2012). As NADP+ is a crucial substrate to generate NADPH, which in turn is a key

regenerator of antioxidants in the cell, activity of NADK is also crucial. The downregulation

of NADK is associated with increased sensitivity to reactive oxygen species (ROS) such as

hydrogen peroxide (H2O2) (Pollak, Niere, and Ziegler 2007) and can lead to hepatic steatosis

Chapter 1 Introduction in mice (Koh et al. 2004; Zhang et al. 2018b). Overexpression of NADK increases NADPH production 4-5-fold, and provides increased protection against oxidative stress (Pollak, Niere, and Ziegler 2007).

Under normoglycemia, glycolysis is the main pathway for glucose metabolism but a small amount filters into the polyol pathway where glucose is converted into sorbitol via the rate-limiting enzyme aldose reductase (AR), a step which oxidises NADPH to form NADP+

(Wermuth et al. 1982). The polyol pathway is also a source of NADH in the next immediate step where sorbitol is converted into fructose via sorbitol dehydrogenase, reducing NAD+ in the process. Under hyperglycaemic conditions, there is increased glucose flux through the polyol pathway which not only depletes NADPH but also generates NADH. As NADPH is an important cofactor in regenerating cellular antioxidants, increased NADPH oxidation through the polyol pathway will lead to impaired antioxidant regeneration and an accumulation of ROS.

Further, excess NADH will also contribute to more oxidative damage as NADH oxidation through complex I leads to increased leakage of electrons from the ETC, which react with oxygen to create superoxide anions and H2O2, which are sources of ROS stress. Oxidative stress induced by overactivation of the polyol pathway is implicated in pathological conditions including diabetes (Snow et al. 2015; Son et al. 2012; Tang et al. 2014). Consistent with this, treatment with AR inhibitors have prevented or delayed the progression of various disease conditions and diabetic complications (Hotta et al. 2012; Kawai et al. 2010; Qiu et al. 2011;

Sonowal et al. 2017; Srivastava et al. 2011).

NADP+ is also the precursor for the synthesis of nicotinic acid adenine dinucleotide phosphate (NAADP) (Chini and Dousa 1995), a vital second messenger for intracellular calcium mobilisation (Calcraft et al. 2009; Steen, Kirchberger, and Guse 2007). The release of calcium from intracellular stores is important for biological processes including lysosomal

Chapter 1 Introduction

function (Lu et al. 2013a; Zong et al. 2009), autophagy (Lu et al. 2013b; Pereira et al. 2011)

and activation of the mTOR pathway when cellular energy levels are low (Cang et al. 2013).

NADPH

The majority of NADPH is generated by the rate limiting enzyme glucose-6-phosphate

dehydrogenase (G6PD) in the oxidative pentose phosphate pathway (PPP) (Kirkman 1962;

Yoshida 1966). G6PD catalyses the rate-limiting step of the PPP, where its substrate glucose-

6-phosphate (G6P) is converted into 6-phosphogluconate (6-PG), reducing NADP+ to NADPH

in the process. The next immediate step also generates NADPH, where 6-phosphogluconate

dehydrogenase (6PGD) (Pearse and Rosemeyer 1974, 1975) catalyses the conversion of 6-PG

to form ribose-5-phosphate (R5P), an important building block for nucleotide biosynthesis

(Blakley and Vitols 1968). In addition to the PPP, NADPH can also be synthesised via

isocitrate dehydrogenase (IDH) and malic enzyme (ME) which have cytosolic (IDH1, ME1)

(Geisbrecht and Gould 1999; Loeber et al. 1994) and mitochondrial (IDH2, ME3) isoforms

(Loeber et al. 1991; Luo, Shan, and Wu 1996). IDH1/2 catalyses the conversion of isocitrate

to α-ketoglutarate while ME1/3 catalyses the conversion of malate to pyruvate, both reducing

NADP+ to generate NADPH.

Other NADP+-dependent mitochondrial enzymes such as glutamate dehydrogenase

(GDH) and nicotinamide nucleotide transhydrogenase (NNT) also contribute to the NADPH

pool in mitochondria. GDH is commonly upregulated in many cancers (Jin et al. 2015; Spinelli

et al. 2017), promoting tumour growth through increased antioxidant activation and redox

scavenging. Inhibition of GDH activity has been a promising new development in anti-cancer therapeutics (Hou et al. 2019). NNT is a nuclear-encoded protein located in the inner mitochondrial membrane (Arkblad et al. 2002), where it serves to maintain redox homeostasis in the mitochondria in an NADPH-manner, and its deficiency slows tumour growth and reduces

Chapter 1 Introduction

tumorigenicity in cells (Ho et al. 2017), also making them prime therapeutic targets for treating

cancer (Chortis et al. 2018; Li et al. 2018).

NADPH acts as an essential cofactor in thioredoxin and glutaredoxin systems to

manage ROS-induced oxidative stress (Jacquot and Zaffagnini 2019). Glutathione reductase

(GR) (Carlberg et al. 1975) and thioredoxin reductase (TR) (Luthman et al. 1982) use NADPH

as a reducing factor to regenerate glutathione (GSH) and thioredoxin (TRX) from their oxidised

forms, GSSH and oxidised thioredoxin (TRX-S2), respectively. Glutathione peroxidases (GPx)

and peroxiredoxins (Prx) then assist in the removal of ROS in the cell by transferring electrons

from GSH and TRX onto oxidative species such as H2O2 forming water. The major source of

ROS in the cell comes from the leakage of electrons out of ETC into the mitochondrial matrix

during oxidative phosphorylation (Vinogradov and Grivennikova 2016). These electrons are

accepted by molecular oxygen, as it is highly electronegative, producing ROS such as

superoxide or H2O2. The removal of these cytotoxic species requires rapid detoxification by

NADPH-GSH-Gpx and NADPH-TRX-Prx systems where an impairment in their coordinated activity is associated with increased oxidative stress and cellular damage (Esposito et al. 2000;

Jin et al. 2011; Oelze et al. 2014).

Another group of enzymes called NADPH oxidases (NOXs) catalyse the production of

- superoxide free radicals (O2 ) by using NADPH as a reducing agent to transfer electrons to

+ + - oxygen in the following reaction: NADPH + 2O2  NADP + H + 2O2 . Under basal

conditions, NOXs are considered inactive, but become activated in response to infection by

pathogens. NOXs are typically embedded in the plasma membrane of phagocytes such as

neutrophils, eosinophils, monocytes and macrophages. In the presence of a pathogen, they

release of large amounts of ROS into the phagosome in an important immune response process

known as a respiratory burst (El‐Benna et al. 2016; Thomas 2017). Unlike other instances

where the generation of ROS and free radicals is highly detrimental to cells, respiratory bursts

Chapter 1 Introduction act to destroy extracellular pathogens such as bacteria or fungi ingested by the phagosome, serving as a protective mechanism for the cell. NOXs can be highly detrimental as they generate large amounts of superoxides and their overaction can lead to increased oxidative stress and cellular damage (Almenara et al. 2015; Babu et al. 2015; Stanicka et al. 2015). This has been implicated in various age-related conditions involving the heart (Barman et al. 2014; Di Marco et al. 2014; Wang et al. 2014a), brain (Bruce-Keller et al. 2010; Cooney, Bermudez-Sabogal, and Byrnes 2013; Pal et al. 2014; Shen et al. 2016), liver (Choi et al. 2014), kidney (Alhasson et al. 2017; Djamali et al. 2009; Gao et al. 2016) and loss of bone (Chen et al. 2011a).

Modulating NADPH levels through the inhibition of NOX isoforms attenuates oxidative damage and pathological progression (Cha et al. 2017; Chen et al. 2011a; Di Marco et al. 2014;

Kwon et al. 2017; Lu et al. 2014b; Qin et al. 2017).

Overall, NAD in its oxidised, reduced and phosphorylated forms are involved in diverse redox roles and functions accounting for over 400 enzymatic reactions in the body, highlighting its role as a central regulator of energy metabolism and redox homeostasis in cells.

Chapter 1 Introduction

Figure 1.3 Functions of NAD(P)(H) as a redox cofactor. The oxidised form of nicotinamide adenine dinucleotide (NAD+) participates in oxidation- reduction (redox) reactions in processes such as glycolysis, β-oxidation, fatty acid oxidation and reactions in the TCA cycle to produce NADH. NADH is a major carrier of electrons and donates its electrons to the electron transport chain (ETC) during oxidative phosphorylation in the mitochondria, regenerating NAD+ in the process. NAD+ can be phosphorylated to form NADP+ via NAD kinase (NADK) which requires energy in the form of adenosine triphosphate (ATP) and forms adenosine diphosphate (ADP) as a by-product. NADK also catalyses the phosphorylation of NADPH from NADH in an ATP-dependent manner. The generation of NADP+ is important for generating its reduced form NADPH, which plays a key role in regeneration of antioxidants such as glutathione. The main enzymes that generate NADPH from NADP+ are dehydrogenases in the Pentose phosphate pathway (PPP). In the reverse reaction, NADPH can be oxidised to NADP+ by NADPH oxidases and NADPH- dependent reductases. Recently, the enzyme nocturnin, which regulates metabolism under the control of circadian clock was shown to catalyse the dephosphorylation of NADPH and NADP+ to generate NADH and NAD+ respectively, and is conserved from fruit flies to humans (Estrella et al. 2019).

Chapter 1 Introduction

1.2.2 NAD+ as a substrate

In addition to the role of NAD+ as a redox cofactor, in the last two decades it has emerged as a

key signalling molecule and substrate capable of modulating the activity of important NAD+-

consuming enzymes. These include 1) poly-ADP-ribose polymerases (PARPs), 2) the sirtuins,

3) NAD+ glycohydrolases (CD38/CD157) and 4) sterile alpha and Toll/interleukin-1 receptor

motif-containing 1 (SARM1) (Figure 1.2, above). All cleave NAD+ at the nicotinamide-ribose bond to form free nicotinamide (NAM) and an ADP-ribose (ADPR) moiety. NAM can be recycled back into the salvage pathway to synthesise more NAD+ molecules while ADPR is used by these enzymes to carry out their respective biological functions.

1.2.2.1 NAD+ and PARPs

In 1963, nearly three decades after discovering the role of NAD+ as a redox cofactor, NAD+

was discovered as the immediate substrate to a family of enzymes called poly-ADP-ribose

polymerases (PARPs) (Chambon, Weill, and Mandel 1963; Kraus 2020) or mono-ADP-ribose transferases (MARTs), when only one ADP-ribose molecule is transferred to proteins

(Bütepage et al. 2015; Ueda and Hayaishi 1985). PARPs are responsible for the addition of polymers of adenosine diphosphate (ADP)-ribose to proteins (PARylation) (Chambon et al.

1963). Specifically, they target the amino acid residues on proteins such as arginine,

asparagine, aspartate, cysteine, glutamate, lysine, and serine (Leidecker et al. 2016; Manning

et al. 1984; Martello et al. 2016; Moss et al. 1979; Ogata, Ueda, and Hayaishi 1980; West et

al. 1985; Zhang et al. 2013). PARylated proteins act as a signal and docking site for protein

interactions with proteins involved in processes such as DNA repair.

In humans, there are 17 members of the PARP family, each with unique function and

location. Briefly, the members of the PARP family that are involved in the addition of polymers

of ADP-ribose (PARylation) are PARP1, PARP2, PARP5a (also known as tankyrase 1) and

PARP5b (also known as tankyrase 2) while most other PARP members (PARP3, 4, 6-16)

Chapter 1 Introduction

remain as MARTs (Cohen 2020; Fehr et al. 2020; Vyas et al. 2014). PARPs are located in the

nucleus (PARP1-3, 6-10, 15), nucleolus (PARP1 and 2), nuclear envelope (PARP11),

cytoplasm (PARP4-6, 8-10, 14), Golgi (PARP12), and endoplasmic reticulum (PARP16)

(Bindesbøll et al. 2016; Carter-O’Connell et al. 2016; Carter-O’Connell et al. 2018; Catara et

al. 2017; Di Paola et al. 2012; Gomez et al. 2018; Jwa and Chang 2012; Vyas et al. 2013),

where their role is discussed in following sections.

PARPs in DNA damage repair

PARPs involved in DNA repair (mainly PARP1) are activated in response to DNA damage

derived from endogenous sources such as the production of ROS from oxidative

phosphorylation in the mitochondria, or from exposure to environmental toxins such as X-rays,

ultraviolet (UV) light, radiation, carcinogens and cigarette smoke (Lindahl 1993; Tubbs and

Nussenzweig 2017). PARP1 is the founding member of the PARP family, which is best known

for its role in DNA damage repair (DDR) (El‐Khamisy et al. 2003; Haince et al. 2008; Le Page

et al. 2003; Mortusewicz et al. 2007).

PARP1 consists of three major domains that mediate its catalytic activity. The first is

the DNA binding domain, which consists of an N-terminal three-zinc finger domain (Zn1, Zn2,

and Zn3) and the WGR domain, named after the most conserved amino acid sequence

(tryptophan–glycine–arginine) (Langelier et al. 2012; Langelier et al. 2018). This domain

allows PARP1 to bind to cruciform DNA, which are inverted repeats of nucleotide sequences

(Brázda et al. 2011; Chasovskikh et al. 2005; Potaman et al. 2005) as well as single stranded

DNA breaks (SSBs) (Heale et al. 2006; Le Page et al. 2003; Pines et al. 2012) and double stranded DNA breaks (DSBs) (Audebert, Salles, and Calsou 2004; Beck et al. 2014b; Wang et al. 2006a). The second domain is a central auto-modification domain which includes a BRCA1

C-terminus (BRCT) domain that allows the recruitment of downstream DDR components to

Chapter 1 Introduction

the DNA damage site and for protein-protein interactions to occur between PARP1 and DDR

machinery (Ahel et al. 2009; El‐Khamisy et al. 2003; Haince et al. 2008; Langelier et al. 2012;

Li and Yu 2013; Masson et al. 1998). This auto-modification domain also allows PARPs such

as PARP1 and PARP2 to auto-PARylate themselves, which helps them dissociate from DNA

when damage is repaired, demonstrating a self-regulated negative feedback mechanism (Liu et

al. 2017b; Masaoka et al. 2012; Muthurajan et al. 2014; Satoh and Lindahl 1992; Sukhanova

et al. 2016).

The final structural domain in PARPs is a C-terminal catalytic domain which consists

of two subdomains: a helical domain (HD) and an ADP-ribosyltransferase (ART) catalytic

domain (Dawicki-McKenna et al. 2015; Langelier et al. 2018). When not bound to DNA, the

HD acts as an autoinhibitory domain by sitting in a conformation that prevents NAD+ from binding to the ART catalytic domain (Dawicki-McKenna et al. 2015). When bound to DNA

breaks, the HD again changes conformation such that the autoinhibitory function is lost,

allowing NAD+ binding which activates the catalytic activity of PARP1 (Dawicki-McKenna et

al. 2015). The use of clinical PARP inhibitors to treat diseases such as cancer target and bind

to the ART catalytic site, preventing its release from DNA and trapping PARP1 at the DNA

damage site to induce cell death (Lord and Ashworth 2017; Murai et al. 2014; Murai et al.

2012).

PARP1-mediated SSB repair can occur via base excision repair (BER) (Dantzer et al.

1999) or nucleotide excision repair (NER) (King et al. 2012). BER involves the removal of a

single or short length of damaged or misplaced DNA base(s) (A-C-T-G). The main protein

interacting with PARP1 to perform BER is X-ray repair cross-complementing protein 1

(XRCC1) which recruits other important DNA intermediates including DNA polymerase β

(Dianova et al. 2004; Kubota et al. 1996; Sobol et al. 1996), DNA ligase III (Cappelli et al.

1997), bifunctional polynucleotide kinase 3′-phosphatase (PNKP) (Jilani et al. 1999; Mandal

Chapter 1 Introduction et al. 2012) and DNA glycohydrolases (Krokan, Standal, and Slupphaug 1997; Mandal et al.

2012). XRCCI and DNA ligase III are also indispensable during NER which seals breaks in the DNA following repair (Moser et al. 2007). NER removes larger sites of DNA damage called oligonucleotide fragments. These fragments are initially recognised by proteins xeroderma pigmentosum group C-complementing protein (XPC) (Maltseva et al. 2015; Robu et al. 2013;

Robu et al. 2017) and xeroderma pigmentosum group A-complementing protein (XPA)

(Fischer et al. 2014; King et al. 2012) which recruit other proteins to form a large multiprotein complex, called DNA damage-binding protein 2 (DDB2) complex (Purohit et al. 2016). PARP1 interacts with DDB2 at DNA damage sites, stimulating its PARylation activity to recruit other chromatin remodelling factors such as amplified in liver cancer protein 1 (ALC1) which stimulates the DNA repair process (Pines et al. 2012). PARP2 is also a key player in BER by binding to PARP1, which assists in the recruitment of core DDR proteins, XRCC1, DNA polymerase β and DNA ligase III for efficient BER (Hanzlikova et al. 2017; Schreiber et al.

2002).

DSBs occur after exposure to DNA damaging agents such as UV radiation (Mehta and

Haber 2014; Rothkamm et al. 2003). DSBs are repaired either via homologous recombination

(HR) (Schultz et al. 2003) or non-homologous end joining (NHEJ) (Audebert, Salles, and

Calsou 2008; Wang et al. 2006a). HR repairs DNA during the S and G2 phase of the cell cycle

(Mao et al. 2008) using the sister chromatid as a template for repair and replacing the damaged

DNA with an identical nucleotide sequence with a high level of fidelity. In contrast, NHEJ is most efficient in the G1 phase (Mao et al. 2008; Moore and Haber 1996), and is an error prone repair pathway as it does not use a complementary template but simply ligates the break ends of DNA.

In both HR and NHEJ, PARP1 is involved in the recruitment of DNA damage sensors meiotic recombination 11 (MRE11) and Nijmegen breakage syndrome protein 1 (NBS1 or

Chapter 1 Introduction

nibrin), which rapidly accumulates at DNA damage sites following DSBs (Haince et al. 2008).

PARP1 is also involved in the early recruitment of breast cancer type 1 susceptibility protein

(BRCA1), which recruits other DDR proteins such as RAD51 to the damage site during HR

(Chen et al. 1999; Zhao et al. 2017). When the sister chromatid is not available for HR, DSBs

are repaired via NHEJ which has two different pathways: the classical (cNHEJ) or alternative

(aNHEJ) pathway. cNHEJ is dependent on the presence of DNA repair dimer Ku70-Ku80 that

recruit the DNA-dependent protein kinase catalytic subunit (DNA-PKcs), which when

PARylated recruits XRCC4/DNA ligase IV complex for DNA ligation (Ruscetti et al. 1998;

Spagnolo et al. 2012). In the absence of Ku70-Ku80, the aNHEJ pathway is activated and relies on PARP1-mediated recruitment of MRE11 in the MRE11-RAD50-NSB1 (MRN) complex for

DNA end ligation (Cheng et al. 2011; Fattah et al. 2010; Mansour, Rhein, and Dahm-Daphi

2010; Wang et al. 2006a). PARP3, a MART, helps to recruit and stabilise Ku80 at DNA damage sites to encourage DSB repair by stimulating the cNHEJ pathway (Beck et al. 2014a).

Two others MARTs, PARP10 and PARP14 play roles in alleviating replication stress by coordinating a restart to stalled replication forks that occur as a result of breaks in DNA during replication, or by interacting with RAD51 to promote DNA ligation via HR (Nicolae et al. 2015; Nicolae et al. 2014; Shahrour et al. 2016). While the PARPs responsible for DNA repair processes are essential for the survival of normal cells, they may be undesirable in the context of cancer by promoting cell proliferation and tumorigenesis (Schleicher et al. 2018).

The tankyrases PARP5a and PARP5b indirectly play a role in DDR by binding to DDR proteins

such as mediator of DNA damage checkpoint protein 1 (MDC1) (Goldberg et al. 2003; Lou et

al. 2006; Lou et al. 2003; Stewart et al. 2003; Stucki et al. 2005), mediator of Rap80 interactions

and targeting 40 kd (MERIT40) (Feng, Huang, and Chen 2009; Shao et al. 2009), or telomeric

repeat factor 1 (TRF1) (Smith et al. 1998; Yang et al. 2017b) that are collectively responsible

Chapter 1 Introduction for the recruitment and stabilisation of other DDR proteins to the damage site for efficient DNA repair.

PARP1 is the main member involved in DNA damage repair, however, as mentioned above PARP2, 3, 5a, 5b, 9, 10 and 14 also play a role in DNA repair processes (Boehler et al.

2011; Cook et al. 2002; Dregalla et al. 2010; Haince et al. 2008; Nicolae et al. 2014; Schreiber et al. 2002; Yang et al. 2017a). In addition to repairing DNA, PARPs are involved in several other important biological processes. PARP5a and 5b are important in the regulation of telomere length (Cook et al. 2002) and the Wnt signalling, a pathway commonly implicated in cancers (Bao et al. 2012; Huang et al. 2009; Wu et al. 2016). PARP6 is involved in dendritic development in the hippocampus (Huang et al. 2016) and acts as a tumour suppressor in the development of colorectal cancer (Qi et al. 2016; Tuncel et al. 2012). PARP7, 10, 12, 13 and

14 are involved in RNA processing including rDNA synthesis, ribosomal biogenesis, mRNA transcription and protein translation (Bock, Todorova, and Chang 2015). PARP9, 10 and 14 regulate the transcription of genes involved in macrophage activation and inflammation (Iwata et al. 2015; Iwata et al. 2016; Iwata et al. 2014) while PARP1, 5a, 7, 9-14 all play a role in the host response to viral infections (Fehr et al. 2020; Kuny and Sullivan 2016). PARP9 was also found to be overexpressed in human breast cancer and promoted cancer cell migration (Tang et al. 2018). In contrast, PARP10 seems to play a protective role by controlling cell proliferation (Chou, Chou, and Lee 2006; Yu et al. 2005), suppressing tumour metastasis (Zhao et al. 2018) and is beneficial to mitochondrial function when downregulated (Marton et al.

2018). It has been revealed that PARP11 is important for the proper development of sperm

(Meyer-Ficca et al. 2015), organisation of the nuclear envelope (Kirby et al. 2018) and is a potent regulator of type 1 interferon (IFN-I) anti-viral efficiency (Guo et al. 2019). Though the functions of PARP15 remain unclear, a genetic association of PARP15 polymorphisms have been found in patients with acute myeloid leukemia (Lee et al. 2016b). Finally, PARP16 is

Chapter 1 Introduction

essential in the regulation of the unfolded protein response (UPR) (Jwa and Chang 2012) and

its localisation to the endoplasmic reticulum (ER), has also been targeted to enhance ER stress-

induced cancer cell apoptosis (Di Paola et al. 2012; Wang et al. 2017a).

The ADP-ribosylating functions of PARPs are highly dependent on hydrolysis of NAD+

molecules and hyperactivation of PARPs can lead to a significant decrease in intracellular

NAD+ (Alano et al. 2010; Du et al. 2003; Pillai et al. 2005; Satchell et al. 2003; Wang et al.

2013), as discussed in later sections. PARP-induced depletion of intracellular NAD+ has been associated with increased cell death (Du et al. 2003; Ha and Snyder 1999; Pillai et al. 2005;

Ying, Garnier, and Swanson 2003; Zhang et al. 2014a) and use of PARP inhibitors protected against PARP-induced NAD+ depletion and cell death (Martín-Guerrero et al. 2017).

1.2.2.2 NAD+ and the sirtuins

The sirtuins are another family of enzymes which regulate cellular stress response, metabolism

and promote healthspan and lifespan extension, consuming NAD+ in the process (Frye 1999;

Imai et al. 2000b; Kaeberlein, McVey, and Guarente 1999; Landry et al. 2000). Silent

information regulator 2 (Sir2) was the founding member of this family, first discovered in the

budding yeast Saccharomyces cerevisiae as a transcriptional repressor of the mating type loci

HML and HMR (Klar and Fogel 1979; Rine et al. 1979; Shore, Squire, and Nasmyth 1984).

Further studies into yeast Sir2 revealed that it prevented the toxic accumulation of senescence-

related ribosomal DNA (rDNA) circles by suppressing recombination at the rDNA locus

(Sinclair and Guarente 1997), with later work showing that it could protect telomeres by

maintaining their length and attenuating age-related telomere shortening (Moretti et al. 1994;

Strahl-Bolsinger et al. 1997; Xu et al. 2007).

In mammals, there are seven known sirtuins (SIRT1-7), of which SIRT1 is the closest mammalian homologue to yeast Sir2 (Brachmann et al. 1995). In 1999, Frye and colleagues

Chapter 1 Introduction

discovered that mammalian sirtuins are dependent on NAD+ (Frye 1999) and shortly after, their role in histone deacetylation was also revealed (Imai et al. 2000b; Landry et al. 2000).

The sirtuins are well known for their role in histone deacetylation and play a major role as epigenetic regulators of gene expression. In addition to removing acetyl groups, they also remove other acyl groups including malonyl, succinyl, crotonyl and propionyl from various protein substrates, most notably histones, consuming NAD+ in a reaction that yields NAM and

2’-O-acetyl-ADPR (Imai et al. 2000b; Tanner et al. 2000). NAM serves as a substrate to the

salvage pathway enzyme NAMPT, where it is rate-limiting for NAD+ biosynthesis (Revollo,

Grimm, and Imai 2004), while ADPR accepts the acyl groups that are removed by the sirtuins to form 2’-O-acetyl-ADPR (Tanner et al. 2000). Importantly, high concentrations of NAM inhibit the deacetylase activity of the sirtuins via a negative feedback loop (Bitterman et al.

2002), highlighting the importance of the salvage pathway in using NAM not just as a substrate

to restore NAD+ levels, but to potentially avoid the inhibition of sirtuin activity.

Importantly, the beneficial effects of this activity is only achieved if NAD+ is readily

bioavailable for their consumption, and a decline or depletion in intracellular NAD+ levels is

concurrent with decreased sirtuin activity and downregulation of the processes they control

(Braidy et al. 2011b; Cantó et al. 2009; Fang et al. 2014; Furukawa et al. 2007; Pillai et al.

2005; Scheibye-Knudsen et al. 2014). Consistent with this, the benefits of NAD+ repletion

through the upregulation of NAD+ biosynthetic enzymes, inhibition of NAD+-consuming

enzymes and pharmacological supplementation with NAD+ precursors can in part be ablated

through deletion of sirtuin activity (Bai et al. 2011; Jang, Kang, and Hwang 2012; Li et al.

2015; Scheibye-Knudsen et al. 2014; Zou et al. 2016). Increasing NAD+ levels may be a

promising strategy to treat some pathologies and age-related diseases (Cantó and Houtkooper

2016; Imai and Guarente 2016; Johnson and Imai 2018; Wątroba and Szukiewicz 2016).

Chapter 1 Introduction

The seven mammalian sirtuins each have unique locations and functions—SIRT1, 6

and 7 are localised to the nucleus, SIRT2 is largely a cytoplasmic protein and SIRT3, 4 and 5

are found in the mitochondria. These subcellular localisations are relevant to their activity and

biological function. As describing all the interactions of the sirtuins and their targets is beyond

the scope of this thesis, prominent targets for each of the sirtuins will be discussed below.

SIRT1

SIRT1 is the most well-studied member of the sirtuin family, being closest in homology to the

yeast Sir2 enzyme (Brachmann et al. 1995; Vaziri et al. 2001). SIRT1 is ubiquitously expressed

and is localised largely to the nucleus but can shuttle between the nuclear and cytosolic

compartments in response to cellular stress (Jin et al. 2007b; Michishita et al. 2005; Tanno et

al. 2007). As such, SIRT1 has a range of nuclear and cytosolic targets that favour cell survival

in response to cellular stressors such as nutrient deprivation, oxidative stress, inflammation and

DNA damage.

The first ever identified substrate of SIRT1 was p53 (Luo et al. 2001; Vaziri et al. 2001),

a pro-apoptotic transcription factor and tumour suppressor that, upon deacetylation, inhibits cell cycle arrest and apoptosis (Han et al. 2008; Kim et al. 2007). SIRT1 also plays a role in reducing inflammation by deacetylating nuclear factor kappa B (NF-κB) (Salminen et al.

2008), a family of transcription factors implicated in inflammation, cell death, proliferation and development. This same target is also deacetylated by another nuclear sirtuin, SIRT6, which also dampens inflammation (Kawahara et al. 2009). In response to fasting, SIRT1 acts as a repressor of proliferator-activated receptor gamma (PPARγ) (Picard et al. 2004), a critical regulator of fat storage in white adipose tissue (WAT) and adipocyte differentiation (Rosen and Spiegelman 2006), favouring fat mobilisation instead of storage. A similar effect is demonstrated by cytosolic SIRT2 which deacetylates the transcription factor forkhead box O

1 (FOXO1) (Jing, Gesta, and Kahn 2007), a major regulator of adipocyte differentiation,

Chapter 1 Introduction

promoting binding to PPARγ and subsequent repression of its transcriptional activity (Wang

and Tong 2009).

SIRT1 also plays a major role in energy metabolism by deacetylating the transcriptional

coactivator PPARγ coactivator 1-alpha (PGC-1α) (Rodgers et al. 2005), which stimulates

gluconeogenesis, increases fatty acid oxidation and enhances mitochondrial biogenesis

(Dominy Jr et al. 2010; Lagouge et al. 2006). This was also demonstrated in the mitochondrial

compartment where the deacetylation of PGC-1α by SIRT3 enhanced adaptive thermogenesis

and reduced oxidative stress (Giralt et al. 2011; Kong et al. 2010; Shi et al. 2005). SIRT1 is

also important for DNA repair by deacetylating DNA repair proteins such as Ku70 in HR

(Jeong et al. 2007), xeroderma pigmentosum group A (XPA) in NER (Fan and Luo 2010) and

Nijmegen breakage syndrome 1 (NBS1) in aNHEJ (Yuan and Seto 2007). SIRT6 also plays a

role in DNA damage repair by deacetylating DNA polymerase β, the major polymerase

involved in BER (Mostoslavsky et al. 2006). Interestingly, SIRT6, which has both deacetylase

and ADP-ribosyltransferase activities, promotes stress-induced DNA repair (Mao et al. 2011;

Van Meter et al. 2016) and rescues age-related defect in BER (Xu et al. 2015) in a PARP1- dependent manner.

SIRT2

Like SIRT1, the cytosolic SIRT2 can shuttle between the nucleus and cytoplasm in response

to cellular stress such as CR, oxidative stress and inflammation (Michishita et al. 2005).

Upregulation of SIRT2 counteracts stress-induced oxidative stress by binding to and deacetylating the transcription factor FOXO3a. This activates and increases the expression of manganese superoxide dismutase (MnSOD) also known as SOD2 (Kops et al. 2002; Wang et

- al. 2007), a mitochondrial antioxidant which converts superoxide (O2 ) to H2O2 and ultimately

reduces cellular ROS levels (MacMillan-Crow and Thompson 1999; Wang et al. 2007).

Chapter 1 Introduction

Deacetylated FOXO3a also promotes its ubiquitination and degradation in a SIRT2-dependent

manner (Wang et al. 2012).

SIRT2 also plays an important role in regulating inflammation by inhibiting the

expression of cytokines (TNF-α, IL-1β, and IL-6), NF-kB and mitogen-activated protein kinase

(MAPK) (Kim et al. 2013b; Rothgiesser et al. 2010). Inhibition of SIRT2 or a lack of SIRT2 activity often leads to detrimental health outcomes. It can cause meiotic arrest, mitochondrial dysfunction and cellular redox imbalance during oocyte maturation (Xu et al. 2019), mediated by an increase in FOXO3a acetylation which blocks nuclear translocation and transcriptional activity (Wang et al. 2017c; Xu et al. 2019). SIRT2 inhibition also exacerbates neuroinflammation and disrupts blood-brain barrier integrity in experimental traumatic brain injury (Yuan et al. 2016). Interestingly, in some cases and through mechanisms that are still unclear, the inhibition of SIRT2 leads to neuroprotection (Chen et al. 2015; Chopra et al. 2012;

Harrison, Smith, and Dexter 2018; Hasegawa et al. 2010; Luthi-Carter et al. 2010; Outeiro et al. 2007; She et al. 2018; Wang et al. 2016a), antidepressant-like action (Erburu et al. 2017;

Muñoz-Cobo et al. 2017), anticancer activity (Hoffmann et al. 2014; Jing et al. 2016; Ma et al.

2014; McCarthy et al. 2013) and anoxia-reoxygenation-induced cell death (Lynn et al. 2008).

SIRT3

The major mitochondrial deacetylase (Lombard et al. 2007), SIRT3 is upregulated in response

to cellular stresses such as CR (Palacios et al. 2009; Qiu et al. 2010; Shi et al. 2005), fasting

(Hirschey et al. 2010), oxidative stress (Qiu et al. 2010) and cold exposure (Shi et al. 2005).

Upregulation of SIRT3 enhances the deacetylation of proteins that protect and regulate the

function and integrity of mitochondria. For example, SIRT3 deacetylates isocitrate

dehydrogenase 2 (IDH2), a mitochondrial enzyme that generates NADPH which is responsible

for regenerating antioxidants GSH (Schlicker et al. 2008; Someya et al. 2010; Yu, Dittenhafer-

Reed, and Denu 2012). Similarly, SIRT3-mediated deacetylation of FOXO3 (Tseng et al. 2014)

Chapter 1 Introduction

and MnSOD enhances ROS scavenging activity to reduce oxidative stress in the mitochondria

(Chen et al. 2011b; Qiu et al. 2010; Tao et al. 2010).

SIRT3 is also a major regulator of mitochondrial energy homeostasis by deacetylating

proteins involved in electron transport chain complexes (e.g. NDUFA9 in Complex I) and ATP

synthesis (e.g. ATP synthase F1 proteins) in response to cellular stress (Ahn et al. 2008; Shi et

al. 2005; Vassilopoulos et al. 2014). In a fasted state, SIRT3 stimulates the utilisation of fatty

acids by deacetylating long-chain acyl coenzyme A dehydrogenase (LCAD) and mitochondrial

3-hydroxy-3-methylglutaryl CoA synthase 2 (HMGCS2) generating energy by increasing fatty acid oxidation (Bharathi et al. 2013; Hallows et al. 2011; Hirschey et al. 2010) and ketone body production, respectively (Shimazu et al. 2010). SIRT3 is also important for regulating heat production, stimulating non-shivering thermogenesis in brown adipose tissue by enhancing uncoupling protein (UCP1) expression in the inner mitochondrial membrane. UCP1 is activated by long-chain fatty acids (LCFAs) (Fedorenko, Lishko, and Kirichok 2012) and induces proton leakage-mediated respiration to generate heat instead of ATP (Rossato et al.

2014; Shi et al. 2005; Srivastava et al. 2013).

In the context of cancers, metabolic reprogramming occurs such that there is a shift away from oxidative phosphorylation towards anaerobic glycolysis, known as the Warburg effect (Warburg 1956). SIRT3 represses this by destabilising the transcription factor hypoxia- inducible factor 1 (HIF-1α) (Bell et al. 2011; Finley et al. 2011) which maintains mitochondrial integrity and metabolism under cellular stress (Kim et al. 2010).

SIRT4 SIRT4 is another mitochondrial sirtuin enzyme with ADP-ribosylation and de-lipoylation

(removal of lipoyl group) activity in addition to its deacetylation activity (Ahuja et al. 2007;

Haigis et al. 2006; Laurent et al. 2013; Mathias et al. 2014). One major target of SIRT4 is repressing the activity of enzyme glutamate dehydrogenase (GDH) by ADP-ribosylation which

Chapter 1 Introduction prevents insulin secretion in response to amino acids (Ahuja et al. 2007; Haigis et al. 2006).

Interestingly, knock down of SIRT4 increased fatty acid oxidation and mitochondrial function in muscle cells (Nasrin et al. 2010) and consistent with this, another study showed a loss of

SIRT4 increased exercise capacity and protected against diet-induced obesity (Laurent et al.

2013).

In contrast, expression of SIRT4 seems to exhibit a tumour-suppressive function by orchestrating a metabolic block in glutamine metabolism (Jeong et al. 2014; Jeong et al. 2013;

Miyo et al. 2015) with therapeutic potential in the treatment of cancers such as colorectal cancer. There are also discrepancies as to whether SIRT4 activity mediates protection or harm in cardiac function. One study shows SIRT4 protects against hypoxia-induced apoptosis in cardiomyoblast cells (Liu et al. 2013) whereas another study revealed the knockout of SIRT4 offered protection again angiotensin-II induced cardiac hypertrophy and fibrosis in mice (Luo et al. 2017). The role of SIRT4 in response to oxidative stress remains unclear.

SIRT5 In contrast to most other sirtuins, SIRT5 has very weak deacetylase activity (Du et al. 2011). It instead exhibits stronger demalonylation, desuccinylation and deglutarylation activity

(Nakagawa et al. 2009; Nakamura et al. 2012; Park et al. 2013; Polletta et al. 2015). The first of its targets to be identified was carbamoyl phosphate synthetase 1 (CPS1) (Nakagawa et al.

2009), the enzyme catalysing the first step of the urea cycle and plays an important role in detoxification of ammonia. Under nutrient deprived conditions, SIRT5 activity is stimulated and leads to increased desuccinylation of CPS1 and urea formation (Du et al. 2011). SIRT5 has also been shown to improve the antioxidant activity of SOD1 through its desuccinylation, resulting in reduced ROS levels (Lin et al. 2013).

Other targets of SIRT5 are not well understood, though SIRT5 knockout mice exposed to cardiac ischemia exhibited larger infarct volume, increased oxidative stress and fibrosis as

Chapter 1 Introduction

well as reduced shortening fraction and ejection fraction (Boylston et al. 2015). An increase in

succinate dehydrogenase (SDH) activity was observed in this study, suggesting that SIRT5 also

desuccinylates SDH and may play a cardioprotective role. Recent studies using metabolomics-

assisted proteomics report SIRT5-deficient mice exhibit defects in fatty acid metabolism,

decreased ATP production and hypertrophic cardiomyopathy, suggesting that SIRT5-mediated

desuccinylation plays a major role in the regulation of heart metabolism and protection

(Sadhukhan et al. 2016).

SIRT6 SIRT6 is a nuclear sirtuin with deacetylation, ADP-ribosylation, and de-myristoylation activity. SIRT6 is required for the maintenance of glucose and lipid homeostasis by co- repressing the transcription factor hypoxia-inducible factor 1-alpha (HIF-1α) (Zhong et al.

2010) which in turn supresses glucose uptake and glycolysis. SIRT6 also plays a major role in genomic stability and DNA integrity. One of its targets is the histone H3 lysine 9 (H3K9) which modulates telomeric chromatin (Michishita et al. 2008). Further, activation of SIRT6 promotes

DNA repair under oxidative stress by binding to and ADP-ribosylating PARP1, hence

stimulating its DNA repair capacity (Mao et al. 2011). SIRT6 deacetylates C-terminal binding

protein (CtBP) interacting protein (CtIP), which promotes resection on either side of the DNA

break which is critical for DNA repair via HR (Kaidi et al. 2010). Interestingly, the

overexpression of Sirt6 demonstrated significantly longer lifespans in male, but not female

transgenic mice with lower serum IGF1 levels and altered IGF1 signalling, a key pathway in

the regulation of lifespan (Kanfi et al. 2012).

SIRT7 Finally, the last nuclear sirtuin, SIRT7 is found in the nucleolus and interacts with RNA

polymerase I (RNA Pol I), increasing RNA Pol I-mediated transcription involving cell proliferation and apoptosis. SIRT7-deficient mice experience a reduced median and maximum

Chapter 1 Introduction

lifespan and develop heart complications including cardiac hypertrophy, inflammatory

cardiomyopathy and extensive fibrosis that was related to hyperacetylation of p53

(Vakhrusheva et al. 2008). The same study showed that SIRT7 deficient cells also exhibit a

~200% increase in apoptosis and severely diminished resistance to oxidative stress, indicating

that SIRT7 plays a major role in the regulation of stress response and cell death in the heart.

Another target of SIRT7 is the transcription factor Myc which plays a role reducing

endoplasmic reticulum (ER) stress upon SIRT7-mediated deacetylation and can prevent fatty

liver disease (Shin et al. 2013).

Regulation of sirtuin activity

A prominent hypothesis in the field is that maintaining sirtuin activity may be needed to

maintain health and protection against conditions such as type 2 diabetes and metabolic

syndrome (Banks et al. 2008; Caron et al. 2014; Halperin-Sheinfeld et al. 2012; Herranz et al.

2010). Understanding the mechanisms that regulate their levels of expression and transcriptional activity may be relevant to the study of age-related diseases. Beyond their

activation in response to cellular stress or during conditions that compromise nutrient and

energy availability in the cell such as CR (Cohen et al. 2004; Nemoto, Fergusson, and Finkel

2004; Noriega et al. 2011), the sirtuins are also regulated at the level of transcription, post-

translational modification, through protein-protein interactions or by levels of its substrate

NAD+.

At the level of transcription, CR increases the protein expression and activity of SIRT1

(Cohen et al. 2004; Civitarese et al. 2007), SIRT3 (Lombard et al. 2007; Schwer et al. 2009)

and SIRT5 (Nakagawa et al. 2009). Exercise also increases SIRT1 mRNA levels in skeletal

muscle and SIRT3 protein levels increase in the triceps of inactive young men (Radak et al.

2011). Multiple transcriptional binding sites have been identified where several transcription

factors bind to and regulate SIRT1 expression and transcription in response to low energy status

Chapter 1 Introduction such as CR and exercise. For example, under normal energy status, the pro-apoptotic transcription factor and tumour suppressor p53 is bound to the SIRT1 promoter, repressing

SIRT1 gene expression. During situations of bio-energetic stress, p53 bound to the SIRT1 promoter is removed, a process that relies on the translocation of FOXO3a from the cytoplasm

(inactive) to the nucleus (active) to form a complex with p53 (Nemoto, Fergusson, and Finkel

2004). Interestingly, p53 is deacetylated directly by SIRT1, destabilising and inactivating its tumour suppressing functions and also promoting the transcription of SIRT1 that is normally repressed under basal conditions (Chen et al. 2005; Yamakuchi and Lowenstein 2009).

The expression of SIRT1 can also be regulated and repressed by another tumour suppressor, hypermethylated in cancer 1 (HIC1) in response to DNA damage (Chen et al.

2005). HIC1 deletion results in an increase in SIRT1 transcription and subsequent deacetylation and inactivation of p53, maintaining cell survival during DNA damage (Chen et al. 2005).

HIC1 can also repress SIRT1 by forming a complex with CtBP, which is activated by elevated

NADH levels and in turn leads to higher binding affinity with HIC1. Changes to cellular redox status, such as when glycolysis is blocked, alters the binding of CtBP to HIC1 which in turn reduces HIC1 binding and repression of SIRT1, inducing SIRT1 expression and transcription during CR (Zhang et al. 2007).

The DNA damage repair enzyme PARP2 is also a transcriptional repressor of SIRT1, where SIRT1 mRNA expression is increased in PARP2-deficient myotubes, suggesting

PARP2 is a direct negative regulator of the SIRT1 promotor (Bai et al. 2011). Consistent with the increase in sirtuin expression and activity under low cellular energy status, exposure to conditions that overload the cell with nutrients and energy, such as through the consumption of a high-fat diet, reduces mRNA and protein levels of SIRT1 in adipose tissue of mice

(Chalkiadaki and Guarente 2012) and these levels are also implicated in the subcutaneous adipose tissue of obese individuals (Pedersen et al. 2008).

Chapter 1 Introduction

A potential reason for low SIRT1 expression and activity could be the reduced

bioavailability of its substrate NAD+, which declines in livers and white adipose tissues of

high-fat-diet-fed mice (Yoshino et al. 2011). This same study showed that supplementation

with NAD+ precursor NMN restored NAD+ levels and ameliorated the pathophysiology associated with the consumption of a high-fat-diet in a SIRT1-dependent manner (Yoshino et

al. 2011). These studies highlight the importance of the coordination between energy-sensing

enzymes and transcription factors in regulating sirtuin expression, transcription and activation

in response to cellular stress.

One of the ways in which the sirtuins can be regulated at the post-translational level is through phosphorylation (Sasaki et al. 2008). SIRT1 has 15 phosphorylation sites, which affects their activity and regulates their own protein levels by influencing protein degradation pathways (Sasaki et al. 2008; Zschoernig and Mahlknecht 2009). SIRT1 can be phosphorylated by protein kinases including c-Jun N-terminal kinases (JNKs), casein kinase 2 (CK2), Cyclin

B/cyclin-dependent kinase 1 (Cyclin-B/Cdk1), and the dual specificity tyrosine phosphorylation-regulated kinases (DYRKs). Together, these phosphorylation events increase

SIRT1 deacetylation activity to counteract oxidative stress (Nasrin et al. 2009), reduce DNA damage (Kang et al. 2009), allow cell cycle progression and proliferation (Sasaki et al. 2008) and promote cell survival by inhibiting apoptosis (Guo et al. 2010).

SIRT2 can also be phosphorylated by kinases such as Cyclin E/Cdk2 which inhibits its deacetylase activity, blocking cell cycle progression and arresting cells in the G1 phase

(Pandithage et al. 2008). Another post-translational modification to the sirtuins is through the addition of small ubiquitin-like modifier (SUMO), otherwise known as sumoylation (Yang et al. 2007b). Under basal conditions, sumoylated SIRT1 can deacetylate and inactivate p53 whereas in response to oxidative stress and DNA damage, SIRT1 is desumoylated, and p53 is activated to induce cell death (Yang et al. 2007b).

Chapter 1 Introduction

The protein deleted in breast cancer 1 (DBC1) directly binds to and suppresses SIRT1

activity. When treated with the chemotherapeutic drug etoposide, DBC1 associates with

SIRT1, inhibiting its activity and leading to increased acetylation of p53 and its pro-apoptotic

functions (Kim, Chen, and Lou 2008; Zhao et al. 2008). The expression of DBC1 is associated

with a poorer prognosis in certain cancers (Cha et al. 2009; Kim et al. 2013a; Lee et al. 2011;

Noh et al. 2013), suggesting its inhibition, rather than expression, as a new therapeutic target

in the treatment of cancer. A recent study showed that the metastasis suppressor breast cancer

metastasis suppressor 1 (BRMS1) interferes with DBC1-SIRT1 interaction by binding to

DBC1 and inducing SIRT1-mediated p53 deacetylation under genotoxic stress (Liu et al.

2016). More studies are needed to understand the role of BRMS1 in DBC1-mediated inhibition

of SIRT1 deacetylation activity and how it can be used in the treatment of cancers.

As the activity of the sirtuins is dependent on the binding of NAD+ to their catalytic site, NAD+ levels are another factor regulating sirtuin activity. NAD+ is used as a cofactor in

redox reactions and are also consumed by other enzymes such as PARPs and CD38/CD157,

and increased NAD+-consuming activities can cause a rapid decline in NAD+ levels (Camacho-

Pereira et al. 2016; Wang et al. 2013), potentially compromising the activity of the sirtuins and

other NAD+ dependent enzymes. Raising NAD+ levels by increasing the expression of NAD+

biosynthetic enzymes (Yang et al. 2007a), pharmacologically inhibiting NAD+-consuming enzymes (Bai et al. 2011; Cerutti et al. 2014; Tarragó et al. 2018) or pharmacologically supplementing with NAD+ (Wang et al. 2013) or NAD+ precursors (Cerutti et al. 2014; Das et

al. 2018; Wang et al. 2018; Zou et al. 2016) leads to increased activity of the sirtuins, and is

associated with an improvement in health and disease parameters.

The sirtuins are also regulated by NAM, the by-product of NAD+ hydrolysis, creating

a negative feedback loop (Bitterman et al. 2002), though this inhibition only occurs at very

high doses, in the millimolar range. Binding of NAM is non-competitive and occurs at a

Chapter 1 Introduction

conserved pocket adjacent to NAD+, which blocks the binding of NAD+ and subsequent hydrolysis. Supplementation with exogenous NAM can inhibit SIRT1-mediated deacetylation of p53, which inhibits apoptosis and in the context of cancers has been shown to block proliferation and promote apoptosis in cancerous cells (Audrito et al. 2011). The application of exogenous NAM has also been shown to mediate anti-viral effects by suppressing SIRT1- mediated enhancement of viral DNA replication (Li et al. 2016).

1.2.2.3 NAD+ and CD38/CD157

Cluster of differentiation 38 (CD38) is a cell surface enzyme highly expressed in immune cells

such as lymphocytes, monocytes, macrophages, dendritic cells, granulocytes and natural killer

(NK) cells (Quarona et al. 2013). Together with its homologue CD157 (also known as BST1;

bone marrow stromal cell antigen 1), CD38 has several different enzymatic activities in various

biological roles.

CD38 and CD157 act as ADP-ribose cyclases which hydrolyse NAD+ to form NAM

and ADPR and cyclic ADPR (cADPR). NAM can be recycled as a substrate for synthesis back

into NAD+. ADPR is a source of extracellular adenosine, an important molecule generated in

response to cellular stress and plays an essential role in almost all aspects of physiology (Borea

et al. 2018). cADPR plays an important role as a secondary messenger that mobilises the release

of intracellular calcium (Ca2+) to regulate physiological processes including the immune

response, nervous system excitability, muscle contraction, enzyme activity and circadian

rhythm (Dodd et al. 2007; Wang et al. 2004; White, Kannan, and Walseth 2003).

CD38 is also responsible for the generation of another secondary messenger known as

nicotinic acid adenine dinucleotide phosphate (NAADP), which is produced through a base-

exchange reaction between NADP+ and nicotinic acid (NA) in an acidic environment (Aarhus

et al. 1995; Chini and Dousa 1995; Liang et al. 1999). NAADP regulates calcium/sodium

Chapter 1 Introduction

(Ca2+/Na+) channels (known as two pore calcium channels; TPCN1 and 2) (Morgan et al. 2015;

Ruas et al. 2015) which promotes calcium release from lysosomes (Fang et al. 2018) and inhibits autophagy (Lu et al. 2013b). Further, TPCN channels are important regulators of the mTOR pathway (Cang et al. 2013). As both autophagy and mTOR are dysregulated in ageing, it is possible that NAADP, through its regulation of TPCN channels, plays an important role in the ageing process through autophagy-/mTOR-mediated pathways.

For many years, it was thought that the main function of CD38 was to generate the secondary messengers cADPR and NAADP, though this remains controversial for several

reasons. First, the generation of cADPR is a highly inefficiency process, requiring nearly 100

molecules of NAD+ to generate 1 molecule of cADPR (Zielinska et al 2004). Second, though

NAADP synthesis has been shown to occur in cells overexpressing CD38 (Cosker et al. 2010),

it is unknown whether NAADP is generated in cells naturally expressing CD38 or in vivo.

Further, NAADP can be generated in the absence of CD38 expression (Soares et al. 2007),

indicating there are CD38-independent pathways to synthesise NAADP (Kim et al. 2008).

CD38 is a major consumer and key regulator of intracellular NAD+ levels (Aksoy et al.

2006; Barbosa et al. 2007). Pharmacokinetic studies have shown the NAD+-hydrolysing

efficiency of CD38 is several-fold higher than its homologue CD157 (Hussain, Lee, and Chang

1998) and compared to other NAD+-consuming enzymes, CD38 has one of the lowest binding

+ affinities for NAD with a Michaelis-Menten constant (Km) of 15-25 µM versus SIRT1-7

(range ~13-888 µM), PARP1 (20-97 µM), PARP2 (130 µM) and SARM1 (24 µM) (Katsyuba et al. 2020), though other work has suggested that these Km values are well below physiological

NAD+ levels of 400-700 µM, and may not be sensitive to physiological changes in NAD+ (Hara et al. 2019). Inhibition of CD38 could therefore increase overall NAD+ levels (Aksoy et al.

2006; Tarragó et al. 2018).

Chapter 1 Introduction

Interestingly, while the majority of CD38 acts as an ecto-NADase, hydrolysing NAD+

in the extracellular compartment, NAD+ is mostly intracellular. This topological paradox is

explained by recent discoveries that CD38 can be expressed in two opposing membrane

orientations with one catalytic site facing the outside (type II) and another facing the cytoplasm

(type III) (Liu et al. 2017a; Zhao, Lam, and Lee 2012). And, in addition to NAD+, CD38 also hydrolyses the extracellular NAD+ precursors NMN and nicotinamide riboside (NR) prior to

their transport into the cell (Grozio et al. 2013; Camacho-Pereira et al. 2016; Preugschat et al.

2014). These findings add weight to the notion that CD38 plays a critical role in the

biosynthesis and metabolism of intracellular NAD+ levels by hydrolysing its extracellular

precursors. Further, pools of intracellular CD38 have been found embedded in the inner

membranes of the nucleus and mitochondria and likely as a soluble form in the cytoplasm

(Chini 2009; Hogan, Chini, and Chini 2019; Malavasi et al. 2008; Shrimp et al. 2014),

demonstrating multiple routes for CD38 to access and degrade intracellular NAD+.

CD38 also plays a role in the generation of extracellular adenosine, a nucleoside

involved in regulating processes such as inflammation and the immune response, neuronal

activity, vascular function, platelet aggregation and blood cell regulation (Borea et al. 2018).

CD38 forms an axis with the ecto-nucleotide pyrophosphatase/phosphodiesterase 1 (NPP1,

also known as CD203a or PC-1) and the ecto-5’-nucleotidase CD73, independent of the

nucleoside tri- and diphosphohydrolase (NTPDase) CD39, which generates extracellular adenosine from ATP (Horenstein et al. 2013). CD38 hydrolyses extracellular NAD+, NMN or

NR forming ADPR which in turn is broken down into adenosine monophosphate (AMP) via

NPP1, followed by CD73 which catalyses the conversion of AMP to adenosine. Adenosine

mediates its effects by activating G protein-coupled receptors expressed on the cell membrane

(Borea et al. 2018). It is generated in response to cellular stresses such as hypoxia, metabolic stress or injury and promotes downstream processes required to prevent further cellular

Chapter 1 Introduction

damage. The upregulation of CD38-mediated adenosine receptor signalling causes

immunosuppression during cancer therapy by inhibiting CD8+ T-cell function, and was reported as a mechanism for tumour cells to escape detection through immune checkpoints such as programmed death 1 (PD-1) and its ligand, PD-L1 (Horenstein et al. 2013; Nijhof et

al. 2016). This has also been implicated in other cancers such as multiple myeloma (Horenstein

et al. 2016) and CD38 inhibition has emerged as an effective target to overcome resistance to

immune checkpoint inhibitors in cancer immunotherapy (Chen et al. 2018).

CD38 activity is also upregulated in normal biological ageing, which is has been

correlated with declining NAD+ levels (Camacho-Pereira et al. 2016; Schultz and Sinclair

2016). Preventing CD38-induced NAD+ depletion through the generation of CD38-deficient

mice or supplementing with CD38 inhibitors increased NAD+ levels up to 30-fold (Aksoy et

al. 2006; Barbosa et al. 2007; Young et al. 2006) and similar studies show it protects against

pathological conditions such as age-related metabolic dysfunction (Tarragó et al. 2018), postischemic heart dysfunction (Boslett et al. 2017) and the progression of cancers such as glioma (Blacher et al. 2015).

The cellular distribution of CD38, taken together with its various topological forms and cellular location suggests that this enzyme may play a role in NAD homeostasis. While upregulating CD38 activity depletes the cell of the crucial metabolite NAD+, it is also a key generator of important secondary messengers such as cADPR, ADPR, NAADP and adenosine.

Under certain conditions, inhibiting CD38 activity may have detrimental outcomes (Xu et al.

2012; Zhang et al. 2014b). Understanding the conditions under which the activation or inhibition of CD38 activity is beneficial, versus detrimental, is therefore important when considering how it may affect its downstream pathways including adenosine signalling, calcium mobilisation and NAD+ metabolism.

Chapter 1 Introduction

2.2.4 NAD+ and SARM1

Finally, the central executioner of the axonal degeneration pathway, sterile alpha and

Toll/interleukin-1 receptor motif-containing 1 (SARM1) (Gerdts et al. 2013; Osterloh et al.

2012) was recently shown to possess intrinsic NAD+ cleavage activity causing injury-induced

loss of NAD+ in neurons (Essuman et al. 2017; Gerdts et al. 2015; Summers et al. 2016).

SARM1-induced depletion of NAD+ triggers axon degeneration which can be blocked by

overexpressing NAD+ biosynthetic enzymes such as NAMPT and NMNAT (Gerdts et al. 2015;

Sasaki et al. 2016), or through pharmacological supplementation with NAD+ precursors such

as NR (Gerdts et al. 2015). Preventing the loss of NAD+ through SARM1 deletion (Gilley et

al. 2015; Gilley, Ribchester, and Coleman 2017; Turkiew et al. 2017) or supplementation with

the SARM1 inhibitor XAV939 (Gerdts et al. 2015) rescued these neuronal defects and axon

degeneration.

Interestingly, accumulation of NMN, the direct precursor for NAD+ biosynthesis via

NMNAT, seems to promote axon degeneration and the neuroprotective effects of NMNAT

enzymes may be due to its consumption of NMN, rather than its synthesis of NAD+ (Di Stefano

et al. 2015). This was first noted in the Wallerian degeneration Slow (WldS) mutant mouse

model, which encodes an intact and functional NMNAT1 enzyme and resists axon

degeneration but showed no difference in NAD+ levels in a WldS mouse brain (Mack et al.

2001). Recent studies have supported the notion that the protective effect of WldS mice and

NMNAT overexpression is not due to increasing NAD+ levels by showing transient expression of bacterial NMN deamidase in mice, which has NMN-consuming without NAD+-synthesising

activity, delays axon degeneration (Di Stefano et al. 2015; Di Stefano et al. 2017). Further, the

degenerative effects of NMN accumulation was recently shown to stimulate SARM1 activity

by ~3.5-fold, causing NAD+ depletion-induced non-apoptotic cell death (Zhao et al. 2019), and

more studies on this are warranted.

Chapter 1 Introduction

As the first member of a new class of NAD+-consuming enzymes, loss of SARM1

activity has emerged as an attractive therapeutic target to treat various neuropathies including

axon degeneration (Geisler et al. 2019) and traumatic brain injury (Henninger et al. 2016;

Marion, McDaniel, and Armstrong 2019), as well as neurodegenerative diseases such as

Alzheimer’s, Parkinson’s and Huntington’s disease (Krauss et al. 2020).

Chapter 1 Introduction

1.3 NAD+ deficiency is a critical pathological factor in ageing and disease

1.3.1 NAD+ deficiency in ageing and disease

There is mounting evidence from rodent and human studies showing NAD+ levels decline with

age in skeletal muscle (Mendelsohn and Larrick 2014; Yoshino et al. 2011), brain (Braidy et

al. 2014; Stein and Imai 2014; Zhu et al. 2015), heart (Braidy et al. 2011a; Pillai et al. 2005),

lung (Braidy et al. 2011a), liver (Braidy et al. 2011a; Zhou et al. 2016), kidney (Braidy et al.

2011a), pancreas (Yoshino et al. 2011), white adipose tissue (Yoshino et al. 2011), skin

(Massudi et al. 2012a), endothelial cells (Das et al. 2018) and oocytes (Bertoldo et al. 2020).

NAD+ deficiency is a common pathological factor implicated in a number of disease models. These include cardiometabolic diseases such as obesity (Choi et al. 2013), type 2 diabetes (Yoshino et al. 2011), non-alcoholic fatty liver disease (Gariani et al. 2016), myocardial I/R injury (Yamamoto et al. 2014a) and cardiac hypertrophy (Pillai et al. 2005).

Preclinical efficacy has also been shown in neurological disorders such as cerebral

ischemia/reperfusion (I/R) injury (Liu et al. 2009; Park et al. 2016), traumatic brain injury

(Clark et al. 2007; Satchell et al. 2003), Ataxia Telangiectasia (AT) (Fang et al. 2016),

Parkinson’s disease (PD) (Lu et al. 2014a), and Alzheimer’s disease (AD) (Gong et al. 2013).

Finally, this strategy has also been implicated in skeletal muscle degeneration in a mouse model of Duchene’s muscular dystrophy (Ryu et al. 2016) and chemotherapy- and inflammation- induced tissue injury (Tateishi et al. 2017; Umapathy et al. 2012). Collectively, these studies show that NAD+ deficiency is a critical pathological factor in ageing and disease and is a

promising strategy to potentially treat age-related diseases and the overall physiological decline with age.

Chapter 1 Introduction

1.3.2 Increased NAD+ consumption as a cause of NAD+ deficiency

Declining NAD+ levels in ageing and disease is attributed to increased NAD+ consumption and/or decreased NAD+ biosynthesis. The major consumers of NAD+ in the cell are the PARPs

(Alano et al. 2010) and CD38 (Camacho-Pereira et al. 2016), whose activities also increase in

an age-dependent manner and correlate with declining NAD+ levels with age (Camacho-Pereira et al. 2016; Massudi et al. 2012b). Activation of PARP1 in rodent and human models of ageing is predominantly triggered by increased DNA damage (Braidy et al. 2011b; Braidy et al. 2014;

Fang et al. 2014; Massudi et al. 2012b) which, when extensive or ineffectively repaired, lead to the hyperactivation of PARP1 followed by the rapid depletion of NAD+ (Fang et al. 2014;

Zhang et al. 2014a; Zhou et al. 2006).

NAD+ deficiency impedes proper functions of the sirtuins and PARPs, leading to

mitochondrial dysfunction (Fang et al. 2014; Scheibye-Knudsen et al. 2014; Zhou et al. 2006),

a major hallmark of ageing (López-Otín et al. 2013), and cell death (Alano et al. 2010; Muñoz-

Gámez et al. 2009; Zhang et al. 2014a). This is demonstrated in a recent study using a mouse

model of the accelerated ageing disorder Cockayne syndrome (CS) which is characterised by

progressive neurodegeneration due to mutations in genes encoding the DNA repair proteins CS

group A (CSA) or CS group B (CSB) (Scheibye-Knudsen et al. 2014). CSB deficiency in these

mice led to PARP1 activation, NAD+ depletion and attenuation of SIRT1 activity causing

mitochondrial dysfunction, inflammation and cell death. In this model, treatment with PARP1

inhibitors could rescue CS-associated phenotypes by restoring NAD+ and activating SIRT1

(Scheibye-Knudsen et al. 2014).

NAD+ deficiency can also be caused by increased CD38 activity with age, which attenuates SIRT3 activity and leads to mitochondrial dysfunction, characterised by abnormal mitochondrial morphology, reduced mitochondrial oxygen consumption rate and a significant reduction in ATP synthesis (Camacho-Pereira et al. 2016). CD38 gene expression may be

Chapter 1 Introduction

upregulated by inflammatory cytokines secreted by senescent cells as part of the senescent

associated secretory phenotype (SASP) (Chini et al. 2019), another major hallmark of ageing

(López-Otín et al. 2013) or bacterial endotoxins such as lipopolysaccharide (LPS) (Lee et al.

2012; Shu et al. 2018), contributing to cellular senescence and inflammation frequently

observed during ageing and age-related diseases (Franceschi and Campisi 2014; He and

Sharpless 2017).

Treatment with CD38 inhibitors (PMID 25828863)or genetic deletion of CD38 can

raise NAD levels, and have been effective in the treatment of pathological conditions such as

acute kidney injury (Shu et al. 2018), ischemic brain damage (Long et al. 2017)and cardiac

hypertrophy (Guan et al. 2017a). A note of caution, however, is warranted as CD38 catalyses

the synthesis of cADPR and its inhibition or genetic deletion may have deleterious impacts on

other cADPR-mediated roles. For example, cADPR is involved in the regulation of the immune

response (Frasca et al. 2006) as well as the release of oxytocin which controls many social

behaviours (Jin et al. 2007a). Inhibiting CD38 may increase susceptibility to bacterial

infections, weaken the immune response and induce mental impairments in individuals.

In contrast to the increase in activity of PARPs and CD38 with age, the activity of the

sirtuins decline with age (Braidy et al. 2011b; Massudi et al. 2012b), which has been attributed

to PARP- and CD38-mediated NAD+ depletion (Camacho-Pereira et al. 2016; Pillai et al. 2005;

Wang et al. 2013). Controversy surrounds this idea, as the affinity of sirtuins for NAD+ is below

the concentrations seen in the age-related decline in NAD+ availability, and it is not clear

whether declining NAD+ is related to declining sirtuin activity. Despite their extensive roles to

maintain cellular homeostasis, the activity of the sirtuins contribute very little to the depletion

of intracellular NAD+ stores, likely due to their tight regulation in the cell by several different mechanisms (Section 2.2.2 NAD+ and the sirtuins) including the generation of NAM from

NAD+ hydrolysis, which is an inhibitor of the sirtuins.

Chapter 1 Introduction

1.3.3 Decreased NAD+ biosynthesis as a cause of NAD+ deficiency

In addition to increased NAD+-consumption, NAD+ biosynthetic activity declines with age.

The rate-limiting enzyme for NAD+ biosynthesis in the salvage pathway, NAMPT, catalyses

the conversion of NAM to NMN (Revollo, Grimm, and Imai 2004) and is an important

regulator of the intracellular NAD+ pool (Garten et al. 2015). The decline in NAD+ with ageing has been heavily associated with the downregulation of this enzyme in several bodies of work.

For example, NAMPT levels decline with age in the hippocampus, where NAMPT is the main source of NAD+ biosynthesis for adult neural/progenitor stem cells (NPSC), crucial for proper cognitive function (Stein and Imai 2014). NSPC-specific loss of NAMPT was associated with a reduced capacity to proliferate, self-renew and differentiate into oligodendrocytes, leading to a decline in cognitive function (Stein and Imai 2014). Similarly, NAMPT expression was

decreased in mesenchymal stem cells obtained from aged rats (Ma et al. 2017) and in retinal

pigment epithelium from aged mice (Jadeja et al. 2018) compared to young controls. Reduced

NAD+ levels in these cells was associated with decreased SIRT1 activity and enhanced cellular

senescence, a major hallmark of ageing (López-Otín et al. 2013).

The consequences of NAD+ deficiency due to a loss of NAMPT expression were also

demonstrated in mouse models where the genetic deletion of Nampt in adult projection neurons

led to motor dysfunction and death (Wang et al. 2017b). Similarly, adipocyte-specific loss of

Nampt led to the development of multi-organ insulin resistance and impairment in adipose tissue functions (Stromsdorfer et al. 2016) whereas in skeletal muscle, Nampt deletion caused

muscle fibre degeneration, loss of muscle strength and reduced treadmill endurance (Frederick

et al. 2016). These studies demonstrate the importance of NAMPT in preserving the proper

functional capacity of tissues and preventing systemic metabolic complications by maintaining

adequate levels of NAD+ in cells. Supplementation with NAD+ precursors such as NMN and

NR, which synthesise NAD+ via NMNAT (Berger et al. 2005) and NR kinases (NRK1 or

Chapter 1 Introduction

NRK2), bypass the rate-limiting step (Tempel et al. 2007) and could ameliorate the functional

and metabolic defects in these studies by restoring NAD+ levels.

Apart from NAMPT, overexpression of enzymes responsible for NAD+ biosynthesis in the mitochondria, NMNAT3 and NNT (Section 2.1.2 NADP+ and NADPH roles and functions), restored age-dependent decline in mitochondrial NAD+ levels and increased the

activity of the mitochondrial sirtuin, SIRT3. This restored redox homeostasis, protected against

oxidative stress-induced cellular damage, enhanced reprogramming efficiency and led to the

extension of lifespan of mesenchymal stem cells by delaying replicative senescence (Son et al.

2016). Overall, these studies suggest that NAD+ deficiency, either due to increased

consumption or decreased biosynthesis, may be a critical factor in ageing and disease, and

strategies to restore NAD+ levels represent a potential therapeutic target to treat age-related

physiological decline and age-related diseases.

Chapter 1 Introduction

1.4 Consequences of NAD+ deficiency in cell function and survival

Due to the significant involvement of NAD+ in hundreds of reactions in the cell, declining

NAD+ levels can cause extensive impairments in cellular function and threaten cell survival.

A major consequence of NAD+ deficiency is mitochondrial dysfunction, a major

hallmark of aging (López-Otín et al. 2013). Mitochondrial dysfunction caused by low NAD+

levels, is commonly characterised by increased mitochondrial permeability transition (MPT),

release of proapoptotic factors such as apoptosis-inducing factor (AIF) and blockage of

glycolysis, leading to reduced ATP production (Camacho-Pereira et al. 2016; Di Lisa et al.

2001; Ha and Snyder 1999; Ying, Garnier, and Swanson 2003; Yu et al. 2002). This can cause the cell to enter an energy crisis and ultimately lead to cell death (Alano et al. 2010; Di Lisa et al. 2001; Wang et al. 2013; Ying, Garnier, and Swanson 2003).

Mitochondrial dysfunction may also induce cellular senescence, another major hallmark of ageing (López-Otín et al. 2013), and mitochondrial dysfunction-associated senescence reduced NAD+/NADH ratios and accelerated the ageing process (Wiley et al.

2016). The link between declining NAD+ levels and mitochondrial dysfunction has in part been

attributed to reduced activity of the sirtuins, which play a major role in the maintenance of

mitochondrial function. For example, the decline in NAD+ levels with age is associated with

reduced SIRT1 activity, impeding its ability to control the expression of mitochondria-encoded proteins, which are required for the coordination of and communication between the nuclear and mitochondrial genomes (Gomes et al. 2013).

Similarly, increased CD38 activity with age is correlated with a severe decline in NAD+

levels, reduced SIRT3 activity and mitochondrial dysfunction, whereas genetic deletion of

CD38 in mice led to an increase in NAD+ levels, increased SIRT3 activity and improved

mitochondrial function (Camacho-Pereira et al. 2016). These findings suggest that

Chapter 1 Introduction

mitochondrial function is highly dependent on SIRT1 and SIRT3 activity but require adequate

levels of intracellular NAD+ for proper function and protection. Restoring NAD+ levels by

inhibiting NAD+-consuming enzymes such as PARPs or supplementing with NAD+ precursors such as NMN and NR significantly attenuated oxidative stress-induced mitochondrial dysfunction, AIF release and neuronal cell death (Du et al. 2003; Sims et al. 2018), improving the pathophysiology of animal models of disease caused by mitochondrial dysfunction. This includes a mouse model of Ataxia Telangiectasia (Fang et al. 2016) and heart failure.

Another major consequence of NAD+ deficiency is reduced DNA repair capacity due

to reduced activity of the DNA repair enzymes such as PARPs and sirtuins (Braidy et al. 2011b;

Massudi et al. 2012a) which have been implicated in mouse models of disease such as Ataxia

Telangiectasia (Fang et al. 2016), xeroderma pigmentosum group A (XPA) (Fang et al. 2014),

Cockayne syndrome (Scheibye-Knudsen et al. 2014) and Alzheimer’s disease (Hou et al.

2018). Inhibition of PARPs and supplementation with NAD+ precursors enhanced the DNA damage repair capacity in these studies by stimulating SIRT1 and SIRT3, highlighting a role in NAD+ augmentation therapies to treat accelerated ageing syndromes and neurodegeneration

by enhancing activity of the sirtuins.

Finally, NAD+ deficiency may contribute to global cellular damage in the form of

increased oxidative stress (Braidy et al. 2011b; Massudi et al. 2012b), inflammation (Minhas

et al. 2019) and cell death (Zhou et al. 2015; Zhu et al. 2016). The accumulation of these process

has been associated with the development and progression of diabetes (Ogura et al. 2018;

Yoshino et al. 2011), acute kidney injury (Mehr et al. 2018) and neurodegeneration (Zhou et

al. 2015). NAD+ augmentation strategies, through administration of CD38 inhibitors or NAD+

precursors, significantly improved the pathophysiologies associated with diabetes, neuronal

and renal dysfunctions and highlight their therapeutic potential to treat age-related diseases by

reducing oxidative stress, inflammation and cell death.

Chapter 1 Introduction

1.5 Therapeutic potential of NAD+ boosting strategies

NAD+ is synthesised from precursors including NA, NAM, NR and NMN. These can be

sourced naturally from the dietary intake of vegetables, fruits, meat and milk (Krehl et al. 1946;

Mills et al. 2016; Trammell et al. 2016d; Ummarino et al. 2017) which is sufficient to prevent the development of pellagra, a disease caused by NAD+ deficiency and characterised by

dermatitis, diarrhea, and dementia (Hegyi, Schwartz, and Hegyi 2004). While dietary intake of

these precursors can prevent chronic NAD+ insufficiency, it is not clear that dietary sources

alone can correct the declining levels of NAD+ observed during biological ageing and disease.

Exogenous treatment with NAD+ precursors is an attractive strategy for treating age-related

disease, and treatment with NA is an FDA approved therapeutic for cardiovascular disease in

humans (Brown and Zhao 2008).

Studies in humans and rats have shown that NA is the most effective agent available in

the treatment of hypercholesterolemia (Davignon et al. 1994; Shah et al. 2013), though to avoid

painful flushing induced by its interaction with the G-protein coupled receptor GPR109a, is delivered clinically as a slow-release tablet (Benyó et al. 2005). While several rodent studies show NAM provides protection against conditions such as diabetes, glaucoma and renal dysfunction (John et al. 2012; Mitchell et al. 2018; Williams et al. 2017; Zheng et al. 2019), it is a sirtuin inhibitor (Bitterman et al. 2002). Supplementation with NAM at high doses can lead to undesirable or non-beneficial health outcomes, potentially through blocking sirtuin activity

(Li et al. 2013; Smith et al. 2019). In contrast to NA and NAM, the NAD+ precursors NMN

and NR can effectively raise NAD+ in cells without causing painful flushing or inhibiting the

sirtuins, and can be tolerated at high doses and administered for long periods without presenting

harmful side effects in mice for NMN (Mills et al. 2016) and in humans for NR (Martens et al.

2018).

Chapter 1 Introduction

There are, however, exceptions to these studies though they demonstrate largely

beneficial effects from NMN and NR treatment. One study using cultured superior cervical

ganglia (SCG) explants, neurites and cell bodies from mice showed that exogenous NMN can

promote axon degeneration, which can be rescued by inhibiting its synthesis via FK866

treatment or through over-expression of the NMN-consuming enzyme NMNAT3 (Di Stefano

et al. 2015). Another study showed that NR treatment reduced exercise performance in rats

(Kourtzidis et al. 2016), however this contrasts with findings from another study which show

that NMN treatment enhances physical endurance in aged mice (Das et al. 2018). The

mechanisms through which these contradictory findings occur are not completely understood

and a majority of preclinical rodent studies using NMN and NR have consistently reported

significant improvements in healthspan and lifespan (Rajman, Chwalek, and Sinclair 2018;

Yoshino, Baur, and Imai 2018).

NMN administration in preclinical rodent models of disease have shown to improve

pathological conditions including Alzheimer’s disease in mice (Yao et al. 2017), cerebral

ischemia in mice (Park et al. 2016), cognitive impairments in mice (Tarantini et al. 2019),

obesity in mice (Uddin et al. 2017), diabetes in mice (Yoshino et al. 2011), acute kidney injury

in mice (Guan et al. 2017b), myocardial I/R injury in mice (Yamamoto et al. 2014b), heart

failure in mice (Zhang et al. 2017), haemorrhagic shock in rats (Sims et al. 2018), age-related physiological decline in mice (Mills et al. 2016) and age-related infertility in mice (Bertoldo et al. 2020). Similarly, NR has demonstrated therapeutic potential in the treatment of preclinical disease models including obesity in mice (Cantó et al. 2012), diabetes in mice (Trammell et al.

2016c), peripheral neuropathy in mice (Trammell et al. 2016c), noise induced hearing loss in mice (Brown et al. 2014), dilated cardiomyopathy in mice (Diguet et al. 2018), mitochondrial myopathy in mice (Khan et al. 2014), liver fibrosis in mice (Pham et al. 2019), and nonalcoholic fatty liver disease in mice and humans (Zhou et al. 2016). In mice models, the ability

Chapter 1 Introduction

of NMN and NR to raise intracellular NAD+ levels has been reported consistently as around 2-

3 fold (de Picciotto et al. 2016; Trammell et al. 2016a; Yoshino et al. 2011). More recently, the

reduced form of NR, dihydronicotinamide riboside (NRH) (Giroud-Gerbetant et al. 2019; Yang

et al. 2019) was shown to increase NAD+ levels by up to 10-fold in cultured mammalian cells and multiple mouse tissues including liver, kidney, brain, muscle and adipose tissue (Giroud-

Gerbetant et al. 2019; Yang et al. 2019; Yang et al. 2020).

The mechanisms underlying the benefits of NAD+ precursor supplementation can be

largely attributed to the activation of the sirtuins and increased deacetylation of their protein

targets. For example, supplementation with NMN restored renal NAD+ content and SIRT1

activity in old mice, protecting against cisplatin-induced acute kidney injury through SIRT1-

mediated regulation of c-Jun N-terminal kinase (JNK) signalling (Guan et al. 2017b). Similarly,

administration of NR rescued alcohol-induced liver injuries by increasing NAD+ levels and

stimulating SIRT1-mediated deacetylation of PGC-1α, which promotes mitochondrial

biogenesis and function (Wang et al. 2018). There is mounting evidence to show that

stimulating the activity of the sirtuins and reducing the activity of other NAD+-consuming enzymes such as PARPs and CD38 protect against all major causes of disease and disability, from hearing and vision loss to cognitive and motor dysfunction as well as metabolic disorders, cardiovascular disease, infertility and immune deficiencies (Rajman, Chwalek, and Sinclair

2018). Increasing NAD+ levels through supplementation with NAD+ precursors, therefore, is a

promising therapeutic strategy to treat age-related diseases.

Chapter 1 Introduction

1.6 NAD+ biosynthesis pathways

The biosynthesis of NAD+ from dietary sources or following supplementation with NAD+

precursors is maintained through three main pathways—the de novo synthesis pathway, the

Preiss-Handler pathway, and the Salvage pathway (Figure 1.4, below). These pathways are

largely conserved in bacteria, yeast, and humans, with a few important exceptions which will

be discussed in the following sections below.

1.6.1 The de novo synthesis pathway

In yeast and humans, synthesis of NAD+ via the de novo pathway involves metabolism of the

amino acid, L-tryptophan which enters the cell via neutral or proton-coupled amino acid transporters SLC7A5 (Bhutia, Babu, and Ganapathy 2015) and SLC36A4 (Pillai and Meredith

2011; Thwaites and Anderson 2011), respectively. Once inside, L-tryptophan is converted to

N-formyl-kynurenine by tryptophan 2,3-dioxygenase (TDO, EC 1.13.11.11) (Lewis-Ballester et al. 2016; Meng et al. 2014). N-formyl-kynurenine is an unstable intermediate and is immediately hydrolysed into L-kynurenine by kynurenine formamidase (KFA, EC 3.5.1.9)

(Pabarcus and Casida 2002). L-kynurenine is then converted into 3-hydroxykynurenine via kynurenine 3-hydroxylase (KMO, EC 1.14.13.9) which requires NADPH (Breton et al. 2000).

Next, 3-hydroxykynurenine is converted into 3-hydroxyanthranilic acid via kynureninase

(KYU, EC 3.7.1.3) (Alberati‐Giani et al. 1996). From this, the intermediate, 2-amino-3- carboxymuconic semialdehyde, is formed via 3-hydroxyanthranolic acid oxygenase (3-HAO,

EC 1.13.11.6) which then undergoes non-enzymatic spontaneous condensation to form quinolinic acid (QA) (Malherbe et al. 1994). TDO, KFA, KYU and 3-HAO are all localised in the cytosol whereas KFA, in addition to the cytosol, is also found in the nucleus, and KMO is found in the outer membrane of mitochondria. The yeast homologues for TDO, KFA, KMO,

KYU and 3-HAO are mediated by Bna proteins, Bna2 (Iwamoto et al. 1995), Bna7 (Wogulis

Chapter 1 Introduction

et al. 2008), Bna4 (Panozzo et al. 2002), Bna5 (Panozzo et al. 2002) and Bna1 (Kucharczyk et

al. 1998), respectively, leading to production of QA.

1.6.2 The Preiss-Handler pathway

Quinolinic acid phosphoribosyltransferase (QAPRT, EC 2.4.2.19) catalyses the formation of

nicotinic acid mononucleotide (NaMN) from QA, requiring both 5-phosphoribosyl-1-

pyrophosphate (PRPP) (Fukuoka, Nyaruhucha, and Shibata 1998). NaMN may also be

synthesised from either nicotinic acid (NA) via nicotinic acid phosphoribosyltransferase

(NAPRT, EC 6.3.4.21) which requires ATP and PRPP (Galassi et al. 2012) or nicotinic acid

riboside (NaR) via nicotinamide riboside kinases (NRK1 or NRK2, EC 2.7.1.22) which also

requires ATP (Khan, Xiang, and Tong 2007; Tempel et al. 2007). NA is transported into the

cell via solute carriers SLC5A8 (Gopal et al. 2005) and SLC22A13 (Bahn et al. 2008) while

NaR may enter through equilibrative nucleoside transporters SLC29A1 and SLC29A2

(Baldwin et al. 2004).

NaMN is then converted into nicotinic acid adenine dinucleotide (NaAD) via

nicotinamide mononucleotide adenylyltransferases (NMNAT1-3, EC 2.7.7.18) in the presence

of ATP (Berger et al. 2005; Garavaglia et al. 2002; Raffaelli et al. 2002; Schweiger et al. 2001;

Zhang et al. 2003). Finally, NaAD is amidated by the glutamine-dependent enzyme, NAD+

synthetase (NADSYN1, EC 6.3.5.1) for the final synthesis of NAD+ which is also an ATP-

dependent reaction (Hara et al. 2003).

In mammals, QAPRT, NAPRT, NRK1/2, NMNAT2 and NADSYN1 are localised to

the cytosol whereas NMNAT1 is nuclear. NMNAT2, in addition to the cytosol, is also found

in the Golgi apparatus and NMNAT3 is mostly mitochondrial but also found in the cytosol.

The yeast homologues for QAPRT, NAPRT, NRK1/2, NMNAT1-3 and NADSYN1 are Bna6

(Di Luccio and Wilson 2008), Npt1 (Rajavel et al. 1998), Nrk1 (Bieganowski and Brenner

Chapter 1 Introduction

2004), Nma1/2 (Emanuelli et al. 2003; Emanuelli et al. 1999), Qns1 (Suda et al. 2003),

respectively.

1.6.3 The Salvage/Recycling pathway

The salvage pathway is considered the main NAD+ biosynthesis pathway in mammals. It

involves the “salvage” of nicotinamide (NAM) which is generated from cleavage of NAD+

from NAD+-consuming activities. This conserves the nicotinyl ring and lowers the need for

exogenous precursors such as tryptophan and NA to maintain intracellular NAD+ levels. NAM

may also enter the cell directly from external sources such as diet or supplementation with

exogenous nicotinamide through facilitated diffusion (Schuette and Rose 1983; Sofue et al.

1991). In the presence of PRPP, NAM is converted into nicotinamide mononucleotide (NMN)

via nicotinamide phosphoribosyltransferase (NAMPT, EC 2.4.2.12), the rate-limiting enzyme in this pathway (Revollo, Grimm, and Imai 2004). Interestingly, there is no yeast homologue for NAMPT, hence utilisation of NAM in yeast involves its deamidation into NA via Pnc1

(nicotinamide deamidase, EC 3.5.1.19) (Hu et al. 2007). NA may then synthesise NAD+

through the Preiss-Handler pathway, which is considered the preferred pathway for NAD+

biosynthesis in yeast (Bedalov et al. 2003; Raj et al. 2019; Sporty et al. 2009). The yeast

homologues for NMNAT enzymes include Nma1, Nma2 and Pof1 (Emanuelli et al. 2003;

Emanuelli et al. 1999; Kato and Lin 2014).

Exogenous NMN may enter the cell directly via NMN transporter SLC12A8 (Grozio

et al. 2019a) and converted to NAD+ via NMNAT1-3, the same enzymes in the Preiss-Handler pathway used to convert NaMN to NaAD (Berger et al. 2005; Emanuelli et al. 2001; Garavaglia et al. 2002; Raffaelli et al. 2002; Zhang et al. 2003). In mammamls, NAMPT is found in all nucleus and cytoplasm but is also detected in the circulation as a form known as extracellular

NAMPT (eNAMPT) (Revollo et al. 2007). This suggests NAM may be converted into NMN via eNAMPT outside the cell, however, some studies show that other NAMPT substrates such

Chapter 1 Introduction

as ATP and PRPP are not available in sufficient quantities in the extracellular space for this to

take place (Hara et al. 2011), warranting further investigations.

In mammals and in yeast, the salvage pathway also involves NAD+ synthesis from

another exogenous precursor, nicotinamide riboside (NR). NR may enter directly via

equilibrative nucleoside transporters (ENT) SLC29A1 and SLC29A2. Once inside, NR may be

phosphorylated to form NMN via NRK1 or NRK2 in an ATP-dependent manner (Khan, Xiang,

and Tong 2007; Tempel et al. 2007). Alternatively, NR can be hydrolysed to form NAM and

ribose-1-phosphate via purine nucleoside phosphorylase (PNP, EC 2.4.2.1) and requires

inorganic phosphate (Ealick et al. 1990). PNP is also responsible for the conversion of NaR to

NA which may then be converted to NaMN to synthesise NAD+ via the Preiss-Handler pathway. There are three yeast homologues for mammalian PNP, called Urh1, Pnp1 and Meu1

(Belenky et al. 2007), while the yeast homologue for NRK1/2 is Nrk1.

In yeast and mammals, NR and NaR metabolism via NRK1 or NRK2 is considered more physiologically relevant than their metabolism via purine nucleoside phosphorylases

(Pnp1, Urh1, Meu1 and PNP), which may be more condition-specific (e.g. if NRK-dependent pathways are inhibited). Purine nucleoside phosphorylases have greater catalytic efficiencies

(Kcat/Km) towards other substrates such as inosine than NR and NaR (Belenky et al. 2009).

Similarly, human NRK1 and NRK2 have greater catalytic efficiency towards NR and NaR than other substrates such as cytidine (Tempel et al. 2007). Hence, compared to NR metabolism via

PNP pathways, NRK-dependent pathways are considered more physiologically relevant in mammals.

Recently, the reduced form of NR, dihydronicotinamide riboside (NRH) was reported as another precursor for NAD+ biosynthesis, which increased NAD+ up to 10-fold in cells and

tissues (Giroud-Gerbetant et al. 2019; Yang et al. 2019; Yang et al. 2020), more than any other

Chapter 1 Introduction

known NAD+ precursor which typically increase NAD+ levels 2 to 3-fold (Mills et al. 2016;

Trammell et al. 2016a; Yoshino et al. 2011). These authors report that NRH can be converted

to NMNH, the reduced form of NMN, via adenosine kinase (AK), where NRH is in competition

with adenosine, another substrate for AK. They further show that NMNH is then adenylated to

NADH via NMNATs which is then oxidised to form NAD+, revealing a completely new

pathway for NAD+ biosynthesis.

1.6.4 Additional pathways in yeast and bacteria, that do not exist in mammals

In yeast and bacteria (E. coli), NAM can be deamidated via Pnc1 (Hu et al. 2007) and PncA

(Frothingham et al. 1996), respectively, to form NA, a pathway which does not seem to exist in mammals (Rongvaux et al. 2003). NA may then be converted into NaMN via Npt1 in yeast

(Rajavel et al. 1998) and PncB in bacteria (Wubbolts et al. 1990) to synthesise NAD+ via the

Preiss-Handler pathway. Bacteria also possess another deamidase enzyme called NMN deamidase, PncC (Galeazzi et al. 2011), forming NaMN which can also synthesise NAD+ via

the Preiss-Handler pathway. NAD+ biosynthesis pathways in bacteria and their contribution to mammalian NAD+ metabolism will be discussed in greater detail in Chapter 4.

Chapter 1 Introduction

Figure 1.4 NAD+ biosynthesis pathways in mammals. In mammals, NAD+ can be synthesised via three pathways. In the de novo synthesis pathway, L-tryptophan is transported into the cell via solute carriers SLC7A5 and SLC36A4 and converted into quinolinic acid (QA) via the kynurenine pathway in five enzymatic and one non-enzymatic steps. QA is then converted into nicotinic acid mononucleotide (NaMN) via quinolinic acid phosphoribosyltransferase (QAPRT) which requires 5-phosphoribosyl-1- pyrophosphate (PRPP). As part of the Preiss-Handler pathway, NaMN can also be synthesised from nicotinic acid riboside (NaR) and nicotinic acid (NA) via nicotinamide riboside kinases 1 and 2 (NRK1/2) and nicotinic acid phosphoribosyltransferase (NAPRT) respectively, which both require ATP (adenosine triphosphate). NaR is transported by equilibrative nucleoside transporters SLC29A1 and SLC29A2 while NA may enter via SLC5A8 and SLC22A13. NaMN may then be adenylated to form nicotinic acid adenine dinucleotide (NaAD) via nicotinamide mononucleotide adenylyltransferases 1, 2 and 3 (NMNAT1-3), which also requires ATP. NaAD is then amidated to nicotinamide adenine dinucleotide (NAD+) via the glutamine-dependent NAD synthetase (NADSYN1) and requires ATP. In the Salvage pathway, nicotinamide (NAM) is taken up via diffusion and converted into nicotinamide mononucleotide (NMN) via the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT) and requires PRPP. NMN can also be derived from dephosphorylation to nicotinamide riboside (NR) by ecto-‘5’-nucleotidase CD73 prior to entry. NR may then enter via equilibrative nucleoside transporters SLC29A1 and SLC29A2 and re-phosphorylated into NMN via NRK1/2 which requires ATP. NR may also be degraded into NAM and ribose-1-phosphate via purine nucleoside phosphorylase (PNP) which requires inorganic phosphate. NMN may also enter directly via SLC12A8 and is then converted to NAD+ via NMNAT1-3 in an ATP-dependent manner. NAD+ is cleaved by NAD+-consuming enzymes such as the sirtuins, poly-ADP-ribose polymerases (PARPs), NAD+ glycohydrolases CD38/CD157 and sterile alpha and Toll/interleukin-1 receptor motif- containing 1 (SARM1) generating ADP-ribose moiety and free NAM as by-products. NAM is recycled back into the Salvage pathway to synthesise more NAD+ and maintain intracellular NAD+ levels without being limited by the uptake of extracellular precursors.

Chapter 1 Introduction

1.7 Current gaps in the field of NAD+ biology

Despite extensive research in the field of NAD+ biology and its implications in health and

disease, there are major questions that are yet to be explored regarding NAD+ precursor metabolism.

1.7.1 Unexplored mechanisms of NAD+ precursor deamidation

Given the interest in using NAD+ precursors to improve healthspan and treat disease, it is

important to understand the metabolism of NAD+ precursors in mammalian systems and the pathways involved in their integration into the NAD+ metabolome. As described in Section

2.6.4 (above), NAM may be deamidated to form NA by Pnc1 in yeast and PncA in bacteria,

while NMN can be deamidated to form NaMN by PncC in bacteria. Mammals are not known

to have an sequivalent step, though some evidence suggests that deamidation of NR in

mammalian systems may exist.

Trammell and colleagues observed the appearance of the deamidated metabolite NaAD

in mouse liver and human peripheral blood mononuclear cells (PBMCs) following oral

administration of NR, an amidated NAD+ precursor. This was unexpected, as there is no

pathway in mammals by which NR can be integrated into NaAD. As part of the Preiss-Handler

pathway, NaAD is amidated to form NAD+ via the glutamine dependent enzyme NAD

synthetase (NADSYN1) (Hara et al. 2003), while NR is phosphorylated into NMN by the enzymes NRK1/2 (Tempel et al. 2007). One possible explanation for NR-derived NaAD formation could be deamidation of NMN to NaMN, the precursor to NaAD in the Preiss-

Handler pathway. Importantly, as there are no mammalian homologues of the bacterial NMN deamidase PncC, orally administered NMN may be deamidated by bacterial PncC in the gut.

This hypothesis, which is investigated in Chapter 4 and Chapter 5 of this thesis, is an important step in understanding the contribution of the microbiome to the metabolism of orally administered NAD+ precursors which may affect their therapeutic efficacy. Further, these 55

Chapter 1 Introduction

investigations may be important for the therapeutic development of NAD+ precursors, in terms

of dosing and routes of administration.

1.7.2 NAD+ precursor uptake mechanisms still unclear

With the exception of studies in neurons (Ying et al. 2007; Zheng et al. 2012) and

cardiomyocytes (Liu et al. 2014; Zhang et al. 2016b), NAD+ is thought to be unable to cross

the plasma membrane, due to its size (663.43 g/mol) and highly polar structure (Di Lisa and

Ziegler 2001). While an NAD+ transporter has been identified in bacteria (Haferkamp et al.

2004) as well as in the mitochondria of yeast (Todisco et al. 2006) and plants (Palmieri et al.

2009), it is unclear whether one exists in mammals. Dedicated NAD+ transporters are thought

to exist in mammalian cells as connexin 43 channels (Bruzzone et al. 2001) however, they may only be expressed in certain cell types (Billington et al. 2008; Pittelli et al. 2011; Wang et al.

2008; Ying et al. 2007) and supplementation with NAD+ precursors is more effective at raising intracellular NAD+ levels across different cell types and tissues.

In mammalian cells, tryptophan uptake occurs via solute carrier proteins that also

transport large uncharged amino acids (Palego et al. 2016). NA is also taken up by specific,

high affinity solute carrier proteins that involve an acidic pH, temperature and energy-

dependent anion antiporter or a proton cotransporter (Nabokina, Kashyap, and Said 2005; Said

et al. 2007; Takanaga et al. 1996). The exact mechanism of NAM is not completely understood,

but is considered to involve facilitated diffusion (Schuette and Rose 1983; Sofue et al. 1991) or conversion into NMN, as extracellular NAMPT is also present in the circulation (Revollo et al. 2007), though some evidence suggests that other NAMPT substrates such as ATP and PRPP are not available in sufficient quantities in the extracellular space (Hara et al. 2011). In the latter case, transport of NMN into the cell remains a controversial matter in the field of NAD+

biology and will be investigated in Chapter 5 of this thesis. Briefly, the debate is around

whether the uptake of exogenously supplemented NMN occurs directly, through transporter

Chapter 1 Introduction

SLC12A8 (Grozio et al. 2019a) or first requires dephosphorylation into its nucleoside form,

NR (Ratajczak et al. 2016). NR and NaR may be directly transported via equilibrative

nucleoside transporters (ENTs) SLC29A1 and SLC29A2 (Mangravite, Xiao, and Giacomini

2003) and re-phosphorylated into NMN and NaMN respectively, via NRK1 or NRK2

intracellularly (Sasiak and Saunders 1996; Tempel et al. 2007). While separate studies have

shown NMN and NR are both effective at raising intracellular NAD+ levels up to 2 to 3-fold

(Trammell et al. 2016a; Yoshino et al. 2011), understanding how these precursors are

transported and integrated into the NAD metabolome could improve their therapeutic potential.

1.7.3 Quantifying the NAD+ metabolome remains a challenge

Given the important role of NAD+ in health and disease, accurate measurement of NAD+ and

its metabolites is essential. These have been measured using a variety of methods. NAD+ may

be measured using enzymatic and colorimetric assays (Bernofsky and Swan 1973; Zhu and

Rand 2012), though these methods are often indirect, cannot detect low picomolar levels (thus

requiring more sample), and are easily influenced by minor changes in temperature and pH,

hence difficult to reproduce. To increase sensitivity, reverse-phase high-performance liquid

chromatography (HPLC) methods that use mobile phases with buffer salts and ion pairing

agents have been employed however, detecting changes in low picomolar ranges in complex

biological samples such as cell lysates and tissue homogenates remains a challenge (Casabona

et al. 1995). These techniques are also limited by their low throughput, measuring only one or

two metabolites at one time, and are unable to accurately separate metabolites that are similar

in biochemical properties and molecular structure in complex biological samples.

The ideal method to quantify the NAD metabolome must be rapid, reproducible, and

robust. Currently, the leading technique to quantify NAD+ metabolites is liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Its greatest advantage is being able to detect multiple metabolites in a single injection, which can be aided by using

Chapter 1 Introduction multiple reaction monitoring (MRM) mode to identify metabolites of interest as a function of time, which also increases the specificity of measurements. One major challenge, however, is the lack of standardised methods to extract, detect and analyse metabolites of interest, often requiring time-consuming optimisation to accurately measure metabolites of interest with good resolution while minimising signal-to-noise ratios. Separating metabolites with similar biochemical properties (e.g. mass, polarity, charge), also remains as a major challenge in quantification of the NAD metabolome as there are many closely related pairs of metabolites such as the amide/acid pairs, NAM/NA, NMN/NaMN, NAD+/NaAD, and NR/NaR, which all differ from each other by one Dalton. With more studies seeking to measure NAD+ and its metabolites in health and disease, these challenges need to be overcome and an improvement in methods to quantify the NAD+ metabolome is warranted.

Chapter 1 Introduction

1.8 Thesis goals

This thesis aims to investigate gaps in knowledge in the field of NAD+ precursor metabolism.

These studies focused on the metabolism of NMN as an emerging NAD+ precursor with

promising therapeutic potential, which has already started to undergo Phase I clinical trials in

humans (Irie et al. 2019, 2020). Important to the clinical translation of NMN, is an accurate

understanding of its metabolism through the oral route, which is the most common route of

administration in the clinical setting.

To explore the pathways involved in metabolism of orally administered NMN, we

strategically designed labelled isotopes of NMN at the ribose (13C) and nicotinamide moieties

(15N) (Chapter 2 Section 2.1) and developed an LC-MS/MS method to trace the incorporation of heavy labels into NAD+ metabolites (Chapter 3). This method was validated in mammalian cell lines to verify our labelling strategy and ensure utilisation of NMN occurred via the expected canonical routes. The overarching hypothesis was that the microbiome contributes to host NAD+ metabolism by metabolising orally delivered NMN via the deamidated route, giving

rise to the deamidated precursors NaMN and NaAD. To investigate this hypothesis, this project

first explored NMN utilisation in microbial systems, supplementing NMN into the culture broth

of E. coli bacteria and measuring changes to NAD+ metabolites in supernatants and cell lysates

(Chapter 4). As expected, deamidation of NMN via PncC was observed, measured by increases

in levels of NaMN in cell lysates and allowing us to explore our hypothesis further in vivo

(Chapter 5).

To investigate the contribution of the microbiome to the metabolism of NMN, labelled

NMN isotopes were orally delivered by gavage to antibiotic-treated mice, to ablate the

microbiome, and changes in the labelled NAD metabolome was detected in the intestinal

tissues, liver and plasma. These findings suggest the microbiome is in competition with the

host for orally administered NAD+ precursors such as NMN, consuming NAD+ metabolites for 59

Chapter 1 Introduction

their own growth and survival and making them less available to the host. Further, while there

was very little evidence to support the deamidation of NMN to NaMN, important factors such

as sampling at a single time point may have influenced these results. Interestingly, an

abundance of NR was observed following supplementation with NMN both in vitro and in vivo,

supporting the indirect mechanism of NMN transport which involves dephosphorylation to NR.

Overall, the findings from this thesis have important implications in the dosing requirements

and route of administration of NAD+ precursors such as NMN and NR, where their co- administration with antibiotics may improve therapeutic efficacy to delay ageing and treat age-

related diseases.

Chapter 2 General Methods

General Methods

2.1 Isotopic labelled compounds

2.1.1 Synthesis of labelled NMN compounds

Synthesis of our labelled NMN compounds employed a two-step process; 1) the initial synthesis of isotope labelled nicotinamide and ribose, followed by 2) an enzyme-mediated ligation of these isotope labelled constituents into NMN. The ribose moiety was 13C labelled

at all five carbon positions (purchased from Cambridge Isotope Laboratories, Cat. No. CLM-

3652) and was used to synthesise both NMN compounds. Nicotinamide was 15N labelled at either the base nitrogen of the nicotinyl ring or at both the base and the amide, following custom synthesis by Dr. Sebastian Marcuccio, Advanced Molecular Technologies, VIC. To combine the labelled ribose and labelled nicotinamide moieties an enzyme-based reaction was fuelled by two enzymes, recombinant phosphoribosyl synthetase (PRS) and recombinant nicotinamide

Chapter 2 General Methods phosphoribosyltransferase (NAMPT) (Wu, Sinclair, and Meetze 2019). NAMPT and PRS were added into a reaction buffer containing 1mM labelled ribose, 1mM labelled nicotinamide, 3mM

ATP, 1mM dithiothreitol, 1 mM MgCl2 and 50mM Tris-HCl (pH 7.5) and incubated at 37°C for 30 minutes. The reaction was terminated with the addition of 0.01% trichloroacetic acid

(TCA). The final compounds were name NMN1 and NMN2. NMN1 refers to the NMN isotope labelled at the ribose and base nitrogen of the nicotinyl ring (hereafter referred to as base) with a final mass shift of M+6 (MW: 340.2g/mol) (Figure 2.1 A) and NMN2 refers to the NMN isotope labelled at the ribose and at both the base and amide nitrogen positions with a final mass shift of M+7 (MW: 341.2g/mol) (Figure 2.1 B). Labelled isotopes were purified with size-exclusion and ion exchange columns and concentrated by lyophilization to yield >95% purity. Labelling was confirmed by mass spectrometry (Figure 2.2). The stability of labelled

NMN compounds were not tested, however, were stored either as powder or aliquoted at a concentration of 20mM, both preparations kept at -30℃.

Figure 2.1 Chemical structure and isotope labelling positions of NMN (A) NMN1 (M+6) labelled with five 13C at the ribose moiety and with 15N at the pyridine base of the nicotinamide moiety. (B) NMN2 (M+7) labelled with five 13C at the ribose moiety and with 15N at the pyridine base and amide of the nicotinamide moiety.

Chapter 2 General Methods

Figure 2.2 Chromatogram peaks of NMN isotopes using LC-MS/MS injected at 100 µM. (A) Unlabelled NMN (M+0) with MRM transition 335.15123.05. (B) Labelled NMN1 (M+6) with MRM transition 341.15124.05. (C) Labelled NMN2 (M+7) with MRM transition 342.1500125.05.

2.1.2 Labelled glutamine

15N-amide-glutamine (>98%) was purchased from Cambridge Isotope Laboratories (cat.no.

NLM-557-0) (Figure 2.3) and stored as powder at room temperature or in aliquots at a stock concentration of 200 mM at -30℃. Chromatograms of labelled glutamine peaks were confirmed by mass spectrometry and are shown in Figure 2.4.

Figure 2.3 Chemical structure and isotope labelling of glutamine.

Chapter 2 General Methods

Figure 2.4 Chromatogram peaks of unlabelled glutamine and isotope labelled glutamine using LC-MS/MS injected at 100 µM. (A) Unlabelled glutamine (M+0) with MRM transition 147.1444. (B) 15N-amide-labelled glutamine (M+1) with MRM transition 148.1445.

2.2 Sample processing for mass spectrometry

2.2.1 Preparation of extraction buffer

The extraction buffer for cell and tissue processing was acetonitrile (Ajax Finechem, cat.no.

2315), methanol (Ajax Finechem, cat.no. 2314) and deionized water (Merck MilliQ®) at a 2:2:1

(v/v) ratio. A mixture of the internal standards, methionine sulfonate (MES), camphorsulfonic acid (CSA) and thymine-d4 (TD4) was diluted into the acetonitrile:methanol:water mixture from a 200X concentrated stock to obtain a 0.1X concentration (targeting in-vial final concentration of 0.25 µM CSA, 5 µm MES and 5 µM TD4). The solution was pre-cooled to -

30℃ overnight before use (the freezing point of acetonitrile and methanol is -45℃ and -97.6℃ respectively) and maintained on ice during sample extraction.

Chapter 2 General Methods

2.2.2 Cell harvest and extraction

The following cell harvest protocol was applied to primary hepatocytes, C2C12 and HEK293

cells (see Chapter 3 for cell culture conditions). For cell harvest, media was removed before

rinsing the cells one with phosphate buffered saline (PBS) cooled to 4°C. Cells were extracted

based on a method previously published by Yuan and colleagues (Yuan et al. 2012). Five

hundred microliters of cold acetonitrile/methanol/water extraction buffer, as above, was added.

Using a cell scraper, the cells in extraction buffer were transferred into microcentrifuge tubes

and centrifuged at 16,000g for 10 minutes at 4°C. The supernatant was transferred into a clean

microcentrifuge tube and snap frozen immediately in liquid nitrogen for storage at -80°C until

further processing. The remaining pellet was used to determine protein concentration (Pierce™

BCA Protein Assay kit, Thermo Fisher Scientific, cat.no. 23225) for normalisation of data

during data analysis.

2.2.3 Further sample processing for mass spectrometry

All samples were thawed on ice and centrifuged briefly before being dried using a speed

vacuum system (Savant SpeedVac SPD140DDA Vacuum Concentrator, Thermo Fisher

Scientific) for approximately 2-3 hours at vacuum. The dried pellet was resuspended in 50 µL

of LC-MS-grade water (Fisher Healthcare, cat.no. PI51140) and centrifuged at 16,000g for 10 minutes at 4°C. The supernatant was transferred into HPLC vials and analysed promptly by

LC-MS/MS.

2.2.4 NAD+ metabolite standard curve preparation

Primary stock solutions of NAD+ metabolite standards were stored at -30℃ at concentrations

of 1 mM and above. Working standard solutions were stored at -30℃ at 100µM concentrations

in 100 µL aliquots to avoid multiple freeze-thaws. The standard calibration series of NAD+

metabolites were generated by performing two-fold serial dilutions of the 100 µM standard

Chapter 2 General Methods stock solution in HPLC-grade water. Standard calibration curves were generated using LC-

MS/MS for the following NAD+ metabolites; NAD+ (Sigma-Aldrich, cat.no. N7004), NAM

(Sigma-Aldrich, cat.no. N3376), NMN (GeneHarbour Biotechnologies, Hong Kong), NR

(synthesized by Dr. Hamish Toop, School of Chemistry, UNSW), NaR (synthesized by Dr.

Hamish Toop), NA (Sigma-Aldrich, cat.no. 72309), NaAD (Sigma-Aldrich, cat.no. N4256), and NaMN (GeneHarbour Biotechnologies, Hong Kong). The standard calibration series was prepared on the same day as further processing of samples for LC-MS/MS analysis.

2.3 LC-MS/MS

2.3.1 Liquid chromatography

High performance liquid chromatography (HPLC) was performed using the 1260 Infinity LC

System (Agilent) with a chilled autosampler set at 4-8℃. LC separation was accomplished on an amide capped ethylene-bridged hybrid (Amide XBridge BEH) HPLC column (100 mm x

2.1 mm, 3.5 µm particle size, Waters Corporation) at room temperature (Yuan et al. 2012). For the mobile phase, Buffer A, consisted of a 95:5% (v/v) mixture of water and acetonitrile

(CH3CN) containing 20 mM ammonium acetate (NH4CH3CO2) and 20mM acetic acid

(CH3COOH) at a final pH 5 which was stored at room temperature for up to 2 weeks. This acidic buffer was adapted based on the formulation described in Yuan et al. (2012), which contained 20mM ammonium hydroxide instead of acetic acid, and is a formulation that is commonly used (Anastasiou et al. 2011; Asara et al. 2009; Caroline et al. 2011; Gao et al. 2011;

Kelly et al. 2011; Locasale et al. 2011; Locasale et al. 2012; Vander Heiden et al. 2010). In this study, however, metabolites with the same MRM transitions eluted at similar retention times causing an overlap of peaks that were unable to be resolved during peak integration. In order to achieve separation of these peaks, the pH of Buffer A was lowered from 9.4 to 5 by replacing

20mM ammonium hydroxide with 20mM acetic acid. The acidic nature of the buffer interacts with the hydroxyl group from acidic NAD+ metabolites including NaMN, NaAD, NA and NaR,

Chapter 2 General Methods

separating them from NMN, NAD+, NAM and NR, respectively, to achieve better integration

of peaks. Buffer B was 100% acetonitrile (CH3CN) and was stored for up to 6 months at room

temperature. The flow rate was 200 µl per minute, and the buffers were flowed in gradient

mode with percentages of Buffer B set at 85% (0 min), 85% (0.1 min), 70% (10 min), 30% (13

min), 30% (17 min), 85% (17.5 min), and 85% (30 min). The injection volume taken from each

vial was 2.5μL.

2.3.2 Mass spectrometry

HPLC was coupled to the QTRAP® 5500 mass spectrometer controlled by Analyst® Software

(AB SCIEX, Toronto, Canada). The following MS settings were applied for MS/MS detection

of metabolites of interest: the ion source was set at 350°C and ionisation voltage at 4500 volts

with positive/negative polarity switching. Mass isotopologues of metabolites were acquired by

MS2, using the unscheduled multiple reaction monitoring (MRM) mode with a dwell time of

40 ms. The MS parameters consisting of the declustering potential (DP), collision energy (CE) and cell exit potential (CXP), and MRM transitions were optimised based on the monoisotopic mass of chemical standards.

2.4 Data analysis

For experiments using labelled compounds, MSConvert (version 3.0.18165-fd93202f5) and in-

house MATLAB scripts were employed by co-supervisor Dr. Lake-Ee Quek who kindly integrated the metabolite peaks and extracted the data. Deconvolution scripts were developed to resolve the overlapping pairs of closely related metabolites as in Table 3.2: NaAD

(665.03>428.06) & NAD+ (664.0>428.09), NaR (256.2>124.2) & NR (255.0>123.03) and

NaMN (336.10>123.78) & NMN (335.15>123.05) using MATLAB’s Optimisation Toolbox™

(version R2018a, The MathWorks, Natick, MA, USA). The integrated peak areas were

normalised first, by dividing by the integrated peak areas of the internal standards, to account

Chapter 2 General Methods for any sample loss during preparation, and then normalised again by dividing by the amount of protein (for cells), optical density values (for E.coli) or frozen tissue weight (for mouse tissue).

2.5 Statistics

All data are represented as mean ± s.d. and made using GraphPad Prism 8 software (version

8.3.0). Statistical analysis tests included testing for outliers using the ROUT method with

ROUT coefficient Q set at 1% and two-way ANOVA with Sidak’s multiple comparisons test to compare whether there were differences between two or more treatment groups. A p-value less than 0.05 was considered statistically significant.

Chapter 3 Quantifying the NAD metabolome

Development of an LC-MS/MS method to quantify the labelled NAD+ metabolome

3.1 Introduction

There is mounting evidence for the importance of NAD+ levels in health and disease. This

abundant cofactor is vital for maintaining redox homeostasis and plays an important role as a

substrate for enzymes involved in processes of cell survival including energy metabolism,

DNA damage repair, gene expression and cell signalling in response to cellular stress (Braidy

et al. 2018; Canto, Menzies, and Auwerx 2015; Chini, Tarragó, and Chini 2017; Connell,

Houtkooper, and Schrauwen 2019; Demarest et al. 2019; Johnson and Imai 2018; Kulkarni and

Brookes 2019). As NAD+ reportedly declines with age and in various pathological conditions

(Camacho-Pereira et al. 2016; Clement et al. 2019; Das et al. 2018; Frederick et al. 2016;

Gomes et al. 2013; Massudi et al. 2012b; Schultz and Sinclair 2016), the use of NAD+

precursors such as NMN and NR are being pursued to raise NAD+ levels to restore the cell

Chapter 3 Quantifying the NAD metabolome back into a youthful and healthy state (Caton et al. 2011; Chambon, Weill, and Mandel 1963; de Picciotto et al. 2016; Grozio et al. 2019a; Lu et al. 2014a; Mills et al. 2016; Park et al. 2016;

Rajman, Chwalek, and Sinclair 2018; Tarantini et al. 2019; Yamamoto et al. 2014b; Yao et al.

2017; Yoshino et al. 2011; Zhang et al. 2017).

The efficacy of NAD+ precursor supplementation is largely assessed by measurement of NAD+ itself, and less frequently, its metabolites. The therapeutic benefits of NMN are largely attributed to an increase in intracellular NAD+, however the precise mechanisms of

NMN transport and utilisation are controversial. Unanswered questions include the effect of

NMN on the overall NAD+ metabolome, including its impact on other metabolites. Other

NAD+ precursors such as nicotinic acid (NA) and nicotinamide (NAM) (collectively termed niacin) as well as nicotinamide riboside (NR) have been shown to increase levels of metabolites in the Preiss-Handler pathway such as nicotinic acid adenine dinucleotide (NaAD) and nicotinic acid mononucleotide (NaMN) in mouse studies (Trammell et al. 2016a). NA is a deamidated metabolite in the Preiss-Handler pathway, which can be converted to NaMN via

NA phosphoribosyltransferase (NAPRT) (Niedel and Dietrich 1973; Preiss and Handler

1958b). NaMN is then converted to NaAD via NMN adenylyltransferase (NMNAT) (Berger et al. 2005; Raffaelli et al. 2002; Schweiger et al. 2001; Zhang et al. 2003) before NaAD is amidated via the glutamine-dependent NAD+ synthetase (NADSYN1) in the final step of

NAD+ synthesis (Hara et al. 2003). As expected, NA treatment increases the formation of the deamidated metabolites NaMN and NaAD (Preiss and Handler 1958a). Surprisingly, these deamidated metabolites are also increased following supplementation with the amidated precursors NAM and NR, which are metabolised in the NAD+ salvage pathway (Trammell et al. 2016a). Evidence for the existence of deamidase enzymes in mammals is limited, with papers investigating this dating 4-5 decades ago failing to identify a mammalian enzyme that could mediate this activity, despite trace levels of nicotinamide deamidation being detected

Chapter 3 Quantifying the NAD metabolome

(Petrack et al. 1963; Petrack et al. 1965; Sarma, Rajalakshmi, and Sarma 1961; Tanigawa et al.

1971). Given recent findings reporting the appearance of deamidated metabolites following

administration with the amidated precursor NR, this pathway is worthy of further investigation.

Isotopic labelling studies allow for tracing the metabolic fate of compounds, which

could be used here to may enhance our understanding of how various NAD precursors are

incorporated into the NAD metabolome. After the initial report by Trammel and colleagues,

recent studies have used labelled versions of NMN to investigate the mechanisms of transport,

utilisation and bioavailability based on different routes of administration in mice including oral

gavage and intraperitoneal injections (Grozio et al. 2019a; Liu et al. 2018; Ratajczak et al.

2016). In these studies, the incorporation of labelled ribose and nicotinamide moieties from

NMN into NAD+ was traced, but their incorporation into other members of the NAD+

metabolome such as deamidated metabolites, NaMN and NaAD was lacking. The ability to

trace labelled ribose and nicotinamide moieties from isotope labelled versions of NAD+

precursors as they are incorporated into NAD+ metabolites is an important opportunity to

investigate the pathways involved in NMN uptake and utilisation, and its impact on other metabolites contributing to NAD+ biosynthesis in mammalian cells.

Liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) is the

gold-standard for the reliable analysis of NAD+ metabolites in mammalian cells (Bustamante

et al. 2018; Liang et al. 2014; Martino Carpi et al. 2018; Trammell and Brenner 2013; Yaku,

Okabe, and Nakagawa 2018b). In a typical mass spectrometer, quadrupoles made up of four

parallel metal rods allow the transmission of a voltage to be applied along their axis, to act as

mass filters for ionised compounds. Tandem mass spectrometers comprise two quadrupoles

flanking a collision cell, a third quadrupole, that enable mass spectrometry to be performed one

after another (MS/MS). The samples are first ionised through a process called electrospray

ionisation (ESI) (Bruins 1998; Ho et al. 2003) which gives each compound an additional

Chapter 3 Quantifying the NAD metabolome

charge. As these ions move through the electrical and magnetic fields of voltage-applied

quadrupoles, their movement is affected such that they may be selectively filtered based on

their mass to charge ratio (m/z). The first quadrupole selects the complete target metabolite,

called the precursor ion (or Q1). It then enters the collision cell (second quadrupole) where it

is fragmented into smaller ions using an inert gas, such as nitrogen or argon, in a process called

collision induced dissociation (CID) (Wells and McLuckey 2005). These small ions, called

product ions (or Q3) then enter the third quadrupole for selection and detection to generate a

mass spectrum with chromatogram peaks whose area can be quantified and analysed for a

metabolite of interest. This process is called selective reaction monitoring (SRM), which when

applied simultaneously to select and detect multiple precursor and product ion pairs, is called

multiple reaction monitoring (MRM).

An important aspect of using MRM is identifying the MRM transition value for each

metabolite, which refers to the m/z values of each precursor/product ion pair as a unique

identifier of each metabolite. This increases the specificity and the capacity to multiplex for

multiple metabolites which results in the high-throughput nature of this technique. In targeting

NAD+ metabolites in this study, the mass to charge (m/z) for each parent ion was based on the

monoisotopic mass of each compound derived from PubChem identity searches

(https://pubchem.ncbi.nlm.nih.gov). Nicotinamide adenine dinucleotide (NAD+), nicotinamide riboside (NR) and nicotinic acid adenine dinucleotide (NaAD) are the only NAD+ metabolites

from our metabolites of interest with a formal positive charge. As such, there is no change in

the parent ion m/z ratio and electrospray ionisation predominantly functions to produce gas phase ions from the solution phase ions to pass it through the quadrupoles. All other NAD+

metabolites analysed, namely, NMN, NaMN, NaR, NAM and NA carry a formal charge of

zero, and therefore the ESI adds an additional charge to their monoisotopic mass as a unique

identifier of their respective MRM transitions. In order to optimise the measurement of each

Chapter 3 Quantifying the NAD metabolome

metabolite, it is necessary to optimise a series of mass spectrometry parameters including

depolarisation potential (DP), collision energy (CE), and cell exit potential (CXP).

The retention time (RT), defined by the time from injection to detection of a metabolite,

adds another level of specificity to target each metabolite of interest and is another parameter

that needs to be determined during the optimisation process. Once established, this list serves

as a set of instructions for the mass spectrometer to identify and detect metabolites of interest

in experimental samples to generate chromatogram peaks and mass spectral data. As these

parameters are non-transferable and vary significantly between different mass spectrometers, buffers and LC columns, the optimisation of methods between studies is warranted.

The first aim of this study was to develop an optimised LC-MS/MS method to measure unlabelled and labelled NAD+ isotopologues (molecules that have the same chemical formula

and structural arrangement but differ by incorporation of isotope labels). These would be

measured following supplementation with two stable isotope labelled versions of NMN, which

are designated here as NMN1 and NMN2. NMN1 was isotopically labelled at five carbons

(13C) of the ribose moiety (M+5) and at the nicotinyl ring base (15N) of the nicotinamide moiety

(M+1), providing a total mass shift of M+6 (Figure 3.1 B, below). NMN2 was labelled at the same positions as NMN1 with an additional label at the amine functional group (15N) of the nicotinamide moiety (M+2), providing a total mass shift of M+7 (Figure 3.1 C, below). These

isotopically labelled versions of NMN were strategically designed to trace the incorporation of

labelled moieties in NAD+ metabolites to investigate the metabolic fate of NMN and explore

the mechanisms of NMN transport. Following optimisation of LC-MS/MS parameters to

analyse the NAD+ metabolome, we sought to validate these measurements and trace these

isotope-labelled metabolites using well-characterised cell lines, including primary hepatocytes,

C2C12 myoblasts and HEK293 cells.

Chapter 3 Quantifying the NAD metabolome

Figure 3.1 Labelling strategy of labelled NMN isotopes. The biochemical structure and molecular weight of (A) unlabelled (M+0), (B) (M+6) labelled NMN1 or (C) (M+7) labelled NMN2 is shown above. The ribose moiety was labelled at five positions with 13C and the nicotinamide moiety was labelled either at the base or base and amide position with 15N. The ribose and nicotinamide moieties were synthesised separately before being combined to synthesise the labelled NMN compounds through an enzyme-based reaction (Wu, Sinclair, and Meetze 2019) (see Chapter 2, Section 2.1.1 for more details on the enzymatic synthesis of labelled NMN compounds).

When validating our measurement system using these cell lines, these experiments

could address a third aim of this investigation regarding the mechanism by which NMN is

transported into cells, currently a topic of debate in the field of NAD+ biology. Understanding

this mechanism could provide important insights into how cells access extracellular NMN and

in turn, assess its effectiveness as an NAD+ precursor compared to other precursors such as

nicotinamide riboside (NR).

The first described mechanism for NMN transport into cells was demonstrated by its

dephosphorylation to NR outside the cell via the ecto-5’-nucleotidase CD73 (cluster of

differentiation number 73) (Grozio et al. 2013). NR could then be taken up into the cell via equilibrative nucleoside transporters (ENTs) (Nikiforov et al. 2011; Sociali et al. 2016), to be re-phosphorylated back into NMN via NR kinases (NRK1/2) (Bieganowski and Brenner 2004;

Ratajczak et al. 2016). While it was speculated that an NMN transporter may potentially exist, a recent study described a role for the transmembrane protein SLC12A8 as an NMN transporter. This transporter belongs to the SLC12 gene family of electroneutral cation-chloride

Chapter 3 Quantifying the NAD metabolome

cotransporters, with high levels of expression in the small intestine and pancreas, and moderate

expression in the liver and white adipose tissue (Grozio et al. 2019).

Studies conducted by Ratajzcak and colleagues hypothesised that if primary

hepatocytes take up intact NMN as a substrate for NAD+ synthesis, exposure to double labelled

NMN (2H-13C-NMN), labelled at the nicotinamide (13C) and ribose (2H) moieties, would

produce both double-labelled intracellular NMN and NAD+ (Ratajzcak et al. 2016).

Interestingly, neither doubly labelled NMN or NAD+ were detected in cells following

supplementation with double labelled NMN, unless double labelled NR was used instead. This

indicated NMN dephosphorylation to NR is a key step when cells assimilate NMN. The

detection of double labelled NR following supplementation with double labelled NMN was not

reported, which could have provided further evidence to support these claims. This was,

however, measured in a recent study by Grozio and colleagues to investigate NMN transport

via the putative transporter SLC12A8, by using NMN labelled at the nicotinamide moiety (18O-

D-NMN). In contrast to the study conducted by Ratajzcak and colleagues, Grozio and

colleagues provided evidence for intact NMN transport by detecting 18O-D-NMN from primary

hepatocytes isolated from wild-type mice but not from primary hepatocytes isolated from

SLC12A8 knock out mice (Grozio et al. 2019). The absence of 18O-D-NR also led researchers

to rule out the possibility of NMN transport via dephosphorylation into NR to further support

their claims for NMN transport via SLC12A8. The short harvest time point used in this study

(5 minutes) however, may not have been long enough to observe whether dephosphorylation of NMN to NR may occur, and further studies investigating this at later time points are warranted.

Another important consideration regarding the administration of NAD+ precursors is

whether there are physiological differences in their bioavailability based on their route of

administration. The bioavailability of orally versus intravenously delivered NMN was

Chapter 3 Quantifying the NAD metabolome

investigated by Liu and colleagues, and its subsequent ability to raise NAD+ in various tissues

was measured by administering double labelled NMN (2H-13C-NMN) in mice (Liu et al. 2018).

Interestingly, intravenous delivery of labelled NMN led to an increase in labelled NAD+ in the liver and kidney, however this was absent when delivered by oral gavage, suggesting the gastrointestinal tract may play a significant role in NMN metabolism and bioavailability in mammals. The role of the gut microbiome on the metabolism of labelled NMN isotopes will be explored in subsequent chapters, Chapter 4 (exploration of microbial NMN metabolism using E.coli) and Chapter 5 (oral administration of labelled NMN in mice), while the metabolic fate of NMN and its transport into the cell using labelled NMN isotopes in in vitro models will be explored in this chapter.

The specific aims of this study were to:

1. Develop a reliable and robust LC-MS/MS method to detect the incorporation of labelled

moieties in NAD+ metabolites.

2. Trace the incorporation of NMN into the NAD+ metabolome in well-characterised in

vitro models.

3. Investigate the mechanism for NMN transport into cells by tracing the incorporation of

labelled moieties into NMN and NR.

Chapter 3 Quantifying the NAD metabolome

3.2 Methods

3.2.1 Optimisation of mass spectrometry parameters

Adopting a targeted approach, NAD+ metabolites were measured using multiple reaction monitoring (MRM), where the mass spectrometer is empirically tuned, using chemical standards, to measure an analyte of a desired mass to charge ratio (m/z) with maximum selectivity and sensitivity. The Analyst® Software (AB SCIEX, Toronto, Canada), which controls the QTRAP® 5500 mass spectrometer, can automatically select for the best MRM, i.e., the m/z transition for a pair of precursor/parent and product/fragment ion, and correspondingly, the voltages for mass spectrometry parameters, declustering potential (DP), collision energy

(CE) and collision cell exit potential (CXP). These MS parameters are analyte dependent; optimum DP, CE and CXP are required to maximise the generation of a precursor ion with the expected m/z (DP), its subsequent conversion (CE) into product ion and enrichment (CXP).

Together, these MS parameters maximise the signal of a given MRM transition.

To optimise each NAD+ metabolite, pure metabolite at 1 µM was infused at a rate of

10 µL per minute into the ion source using a syringe pump (1 mL). The Analyst® software begins the optimisation once the monoisotopic mass and number of charges for the metabolite of interest are entered. Optimisations were repeated 3 times and the average value of each parameter was calculated.

Chapter 3 Quantifying the NAD metabolome

3.2.2 Liquid chromatography (LC) separation

LC (see Section 2.3.1) was coupled to tandem mass spectrometry (MS/MS) (see Section 2.3.2)

to quantify NAD+ metabolites using the optimised MS parameters (CE, DP and CXP). The LC separation gradient is presented in Table 3.1 with a flow rate set at 200 µL per minute and the percentage of each buffer shown over the course of 30 minutes.

Table 3.1 Liquid chromatography (LC) separation gradient Time (mins) Flow rate (µl/min) Buffer A (%) Buffer B (%) 0 200 15 85 0.1 200 15 85 10 200 30 70 13 200 85 30 17 200 85 30 17.5 200 15 85 30 200 15 85

Buffer A: 95:5 (v/v) HPLC H2O:Acetonitrile (CH3CN) with 20 mM ammonium acetate

(NH4OAc) + 20 mM acetic acid (CH3COOH), pH 5. Buffer B: 100% Acetonitrile (CH3CN).

One of the challenges faced during LC was the ability to separate peaks of closely

related metabolites such as NaMN (336 m/z) from NMN (335 m/z), and NaAD (665 m/z) from

NAD+ (664 m/z). To overcome this challenge without changing the LC column, the pH of the

mobile phase Buffer A was decreased from pH 9.4, as originally described in Yuan and

colleagues (Yuan et al. 2012), to pH 5 by replacing 20 mM ammonium hydroxide with 20 mM

acetic acid. In LC-MS, adjusting the pH of the mobile phase buffers can be used as a powerful

tool to change the ionisation state of ionisable metabolites and, in turn, influence their retention

times. NA, NaR, NaMN and NaAD are all acidic metabolites characterised by a carboxylic

acid functional group which is strongly ionised at neutral pH. Under low pH conditions,

interactions between the acid buffer and the carboxylic acid will create deionised (or more

neutral) species with greater retention that ultimately allow better chromatographic separation

Chapter 3 Quantifying the NAD metabolome

with their amidated counterparts. Note the original (basic) Buffer A system did not lead to peak

separation of the acid-amide complements despite changing the LC programme/gradient.

3.2.3 Isolation of primary hepatocytes and treatment

The isolation of primary hepatocytes was based on previous work by Montgomery and

colleagues (Montgomery et al. 2015) which is described here.

Before perfusion, Hanks Buffered Salt Solution (HBSS, 1X) containing 138mM sodium chloride (NaCl), 50mM HEPES, 5.6mM glucose, 5.4mM potassium chloride (KCl),

0.34mM disodium phosphate (Na2HPO4), 0.44mM monopotassium phosphate (KH2PO4), and

® 4.17mM sodium bicarbonate (NaHCO3) was prepared in 1L of deionised water (MilliQ ,

Merck) and pH adjusted to 7.4. This solution was sterile filtered and 0.5mM ethylene glycol-

bis(β-aminoethyl ether)-N,N,N′,N′-tetraacetic acid (EGTA) was added in 500mL of this buffer

while 2mM calcium chloride (CaCl2) was added into another 500mL of this buffer. Both

solutions were pre-warmed in a water bath set at 39-40℃ with carbogen.

Fifteen-week-old female C57BL/6J mice were anaesthetised using a cocktail of

ketamine (125mg/kg) and xylazine (25mg/kg) made up in saline. For perfusion, a cannula was

inserted into the inferior vena cava to initiate liver perfusion in situ. The portal vein was

immediately cut to allow excess buffer solution to drain out of the liver. Perfusion was

maintained at 5mL per minute for 15 minutes, after which the liver was digested with

collagenase H (50mL of 1mg/mL; Roche, cat.no. 11074032001) in warm HBSS solution

supplemented with 2mM CaCl2 for 9 minutes. Once perfusion was complete, the liver was

excised, and the digested liver capsule was removed using fine forceps on a glass petri dish

containing HBSS solution. The hepatocytes were then strained through a 100µM cell strainer

and centrifuged 3 times in ice cold CaCl2-HBSS buffer at 50g for 3 minutes at 4℃. Under aseptic conditions, the final cell pellet was resuspended in warm Medium 199 (Sigma-Aldrich,

Chapter 3 Quantifying the NAD metabolome cat.no. M4530) before seeding onto 6-well plates which were pre-coated with Type 1 rat collagen from tail (Gibco Life Technologies, cat.no. A1048301).

Cells were incubated for 4 hours in a 37℃, 5% CO2 incubator in the following adherence medium: Medium 199 (Sigma-Aldrich, cat.no. M4530) supplemented with penicillin-streptomycin solution (1X p/s) (10,000U/mL, Gibco cat.no. 15140), bovine serum albumin (BSA) (10%, Gibco, cat.no. 15260), UltroSer G (Pall Biosepra, cat.no. 15950-017), dexamethasone (10mM in ethanol, 100nM final concentration, Sigma cat.no. D-4902), and insulin (690µM, 100nM final concentration, Actrapid®). After 4 hours, the adherence media was changed to the following basal media: Medium 199 (Sigma-Aldrich, cat.no. M4530) supplemented with 1% p/s (10,000U/mL, Gibco cat.no. 15140) and dexamethasone (10mM in ethanol, 100nM final concentration, Sigma, cat.no. D-4902). After an overnight incubation in basal media, it was supplemented with the following treatment groups and incubated for 24 hours in a 37℃, 5% CO2 incubator: 1) Control (water), 2) NMN1 (0.2mM), and 3) NMN2

(0.2mM). NMN1 represents labelled NMN with a mass shift of M+6 (labelled at ribose and base of the nicotinyl ring from the nicotinamide moiety) while NMN2 represents labelled NMN with a mass shift of M+7 (labelled at ribose, base of the nicotinyl ring and amide group of the nicotinamide moiety) (Section 2.1.1) Cells were harvested after 24 hours for metabolite extraction (Section 2.2.2), and processing (Section 2.2.3) before undergoing analysis by LC-

MS/MS (Section 2.3) and data analysis (Section 2.4).

Chapter 3 Quantifying the NAD metabolome

3.2.4 C2C12 and HEK293 Cell Culture

C2C12 and HEK293 cells were grown and maintained in DMEM (Dulbecco's Modified Eagle

Medium) supplemented with 10% v/v fetal bovine serum (FBS) (Life Technologies Australia,

cat.no. 10099141) and 1% v/v penicillin-streptomycin (p/s) solution (Sigma Aldrich, cat.no.

P0781). Cells were seeded at a density of 0.1x106 cells per well (2mL/well) in 6-well plates and incubated for 24 hours in a 37℃, 5% CO2 incubator. Media was then removed and washed

once with warmed 1X phosphate buffered saline (PBS) solution before adding fresh DMEM

media supplemented with either vehicle (water) control or 0.1mM labelled NMN1 (Section

2.1.1). Cells treated at time zero were supplemented with an equivalent amount of unlabelled

NMN as the amount of M+6 labelled NMN1 isotope was limited. Cells were harvested at time points 0, 1, 2, 4, 8, and 24 hours in duplicate and NAD+ metabolites were extracted (Section

2.2.2), and processed (Section 2.2.3) before undergoing analysis by LC-MS/MS (Section 2.3)

and data analysis (Section 2.4). Data is represented as ion intensities normalised to the internal

standard (thymine-d4) and normalised again to the protein concentration of each sample and

represented either as normalised intensities or picomoles per mg protein (pmol/mg protein).

Chapter 3 Quantifying the NAD metabolome

3.3 Results To optimise detection by tandem mass spectrometry, the MRM transition (mass to charge [m/z] ratios for the precursor [Q1] and product [Q3] ion) for each metabolite,and their respective isotopologues need to be determined. The precursor ion m/z ratio was based on the monoisotopic mass of the unlabelled metabolite, while the fragment/product ion m/z ratio was determined by performing an automatic compound optimisation of mass spectrometry (MS) parameters—declustering potential (DP), collision energy (CE) and cell exit potential (CXP)— to select the m/z ratio with the highest signal intensity. The MRM transition was verified using an online MS/MS spectrum database (pubchem.ncbi.nlm.nih.gov).

Optimising MS parameters is an important process in maximising signal to noise ratios, to obtain well-resolved peaks for each metabolite. The de-clustering potential is the voltage applied to prevent ions in a sample from clustering, which assists the separation of ions of interest in the first quadrupole (Q1) of a triple quadrupole mass spectrometer. The collision energy is the voltage applied to increase the fragment ion intensity during collision induced dissociation (CID) in the second quadrupole, where smaller product ions are generated. The cell exit potential is the voltage applied to assist the transition of the fragmented product ions from the second quadrupole to the third quadrupole (Q3), where the metabolites of interest can be filtered and detected to generate chromatogram peaks.

The greatest advantage of MRM is being able to detect both intact and cleaved versions of the labelled nicotinamide and ribose moieties from NMN. The incorporation of intact labelled nicotinamide and ribose moieties into NAD+ can be measured by detecting a mass shift of either M+6 (from NMN1) or M+7 (from NMN2), while detection of M+1 (labelled nicotinamide from NMN1), M+2 (labelled nicotinamide from NMN2) or M+5 (labelled ribose from either NMN1 or NMN2) indicates incorporation of the cleaved labelled moieties after the glycosidic bond between the nicotinamide and ribose moieties of NMN is broken (Figure 3.2).

Chapter 3 Quantifying the NAD metabolome

Further, the ability to detect the incorporation of these labelled entities in other NAD+ metabolites, either completely or after the glycosidic bond is broken, allows their contribution to NAD+ biosynthesis to be explored in the context of NMN supplementation.

Figure 3.2 Detecting labelled NMN and NAD+ isotopologues using MRM (multiple reaction monitoring). NAD+ (M+6) and NAD+ (M+7) indicates “complete” incorporation of labelled moieties from M+6 labelled NMN1 and M+7 labelled NMN2, respectively. NAD+ (M+1), NAD+ (M+2) and NAD+ (M+5) indicate “partial” incorporation of labelled moieties after the glycosidic bond between the ribose and nicotinamide is broken. The MRM transitions for each NMN and NAD+ isotopologue are shown in brackets. Red asterisks denote isotope labels and blue dotted lines denote the point of fragmentation. The m/z ratio of the fragment ion detected is shown.

While this is not the first LC-MS/MS method to use MRM to investigate NMN metabolism, it is important to optimise these conditions between LC-MS instruments, buffers and protocols as these MS parameters can vary significantly under various laboratory conditions. Further, the labelled NMN isotopes used in this study were designed specifically to

Chapter 3 Quantifying the NAD metabolome

trace the metabolic fate of NMN and investigate its influence on the NAD+ metabolome. As

such, the differences in isotope design and application between studies requires optimisation

of MS parameters relevant to the specific metabolites of interest.

3.3.1 Compound optimisation of mass spectrometry parameters for internal standards.

An important component of quantifying analytes by mass spectrometry is the use of internal

standards, which provide a reference point for concentrations. Internal standards are either

structural analogues or stable isotopically labelled (SIL) analogues of the compound of interest.

Suitable candidates are not always available or easily accessible and SIL analogues can often

be expensive. As such, the internal standards, camphorsulfonic acid (CSA), methionine

sulfonate (MES) and thymine-d4 (deuterated), used here, were chosen due to availability, ease

of detection, and because they are not endogenously present in biological samples. Table 3.2

provides details of the optimised compound parameters for these three internal standards.

Table 3.2 MRM transitions for internal standards and mass spectrometry parameters. Internal standard Q1 (m/z) Q3 (m/z) DP (V) CE (V) CXP (V) RT (mins) CSA 230.92 80.00 -170.00 -40.00 -13.00 1.86 MES 196.12 100.00 140.00 31.00 25.00 4.00 Thymine-d4 129.03 42.10 -115.00 -52.00 -11.00 2.00 MRM: multiple reaction monitoring Q1: parent ion, Q3: fragment ion, m/z: mass to charge ratio DP: declustering potential, CE: collision energy, CXP: collision cell exit potential V: volts, RT: retention time CSA: camphorsulfonic acid, MES: methionine sulfonate

Chapter 3 Quantifying the NAD metabolome

3.3.2 Optimisation of mass spectrometry parameters for NAD+ metabolites

Following the internal standards, the optimisation of MRM parameters was repeated for

analytes of the NAD+ metabolome that were relevant to this investigation. The optimised

compound parameters for unlabelled (M+0) NAD+ metabolites are detailed in Table 3.3 for the following eight compounds - NAM, NMN, NR, NAD+, NA, NaR, NaAD and NaMN. Note

that isotopologues (isotope labelled versions) of a given metabolite have different Q1 and Q3

depending on the labelled state (i.e., mass shift), but share the same depolarisation potential

(DP), collision energy (CE), cell exit potential (CXP) and retention time (RT). The MRM

transitions and their mass spectrometry parameters are detailed in Table 3.4 for amidated

metabolites and in Table 3.5 for deamidated metabolites.

Table 3.3 MRM transitions and MS parameters for unlabelled NAD+ metabolites Isotopologue Q1 (m/z) Q3 (m/z) DP CE CXP RT (precursor_product) ion (V) (V) (V) (mins) mass shift NAM_0_0 122.80 79.80 80.00 30.00 25.00 1.99 NMN_0_0 335.15 123.05 48.00 24.00 11.00 22.50 NR_0_0 255.00 123.03 64.00 30.00 13.00 12.50 NAD+_0_0 664.00 428.09 33.00 36.00 31.00 16.70 NA_0_0 123.80 77.80 70.00 25.00 10.00 4.30 NaR_0_0 256.20 124.20 41.00 27.00 6.00 10.50 NaAD_0_0 665.00 428.10 139.00 35.00 38.00 17.05 NaMN_0_0 336.10 123.80 66.00 28.00 11.00 20.90 Abbreviations: NAM: nicotinamide, NMN: nicotinamide mononucleotide, NR: nicotinamide riboside and NAD+: nicotinamide adenine dinucleotide (oxidised), NA: nicotinic acid, NaR: nicotinic acid riboside, NaAD: nicotinic acid adenine dinucleotide, NaMN: nicotinic acid mononucleotide

Chapter 3 Quantifying the NAD metabolome

Table 3.4 MRM transitions and mass spectrometry parameters for isotopologues of NAM, NMN, NR and NAD+ Isotopologue Q1 (m/z) Q3 (m/z) DP (V) CE (V) CXP (V) RT (mins) (precursor_product) ion mass shift NAM_0_0 122.80 79.80 80.00 30.00 25.00 1.99 NAM_1_0 123.80 79.80 80.00 30.00 25.00 1.99 NAM_1_1 123.80 80.80 80.00 30.00 25.00 1.99 NAM_2_1 124.80 80.80 80.00 30.00 25.00 1.99 NMN_0_0 335.15 123.05 48.00 24.00 11.00 22.50 NMN_1_1 336.15 124.05 48.00 24.00 11.00 22.50 NMN_2_2 337.15 125.05 48.00 24.00 11.00 22.50 NMN_5_0 340.15 123.05 48.00 24.00 11.00 22.50 NMN_6_1 341.15 124.05 48.00 24.00 11.00 22.50 NMN_7_2 342.15 125.05 48.00 24.00 11.00 22.50 NR_0_0 255.00 123.03 64.00 30.00 13.00 12.50 NR_1_1 256.00 124.03 64.00 30.00 13.00 12.50 NR_2_2 257.00 125.03 64.00 30.00 13.00 12.50 NR_5_0 260.00 123.03 64.00 30.00 13.00 12.50 NR_6_1 261.00 124.03 64.00 30.00 13.00 12.50 NR_7_2 262.00 125.03 64.00 30.00 13.00 12.50 NAD+_0_0 664.00 428.09 33.00 36.00 31.00 16.70 NAD+_1_0 665.00 428.09 33.00 36.00 31.00 16.70 NAD+_2_0 666.00 428.09 33.00 36.00 31.00 16.70 NAD+_5_0 669.00 428.09 33.00 36.00 31.00 16.70 NAD+_6_0 670.00 428.09 33.00 36.00 31.00 16.70 NAD+_7_0 671.00 428.09 33.00 36.00 31.00 16.70 NAD+_1_1 665.00 429.09 33.00 36.00 31.00 16.70 NAD+_2_2 666.00 430.09 33.00 36.00 31.00 16.70 NAD+_5_5 669.00 433.09 33.00 36.00 31.00 16.70 NAD+_6_1 670.00 429.09 33.00 36.00 31.00 16.70 NAD+_6_6 670.00 434.09 33.00 36.00 31.00 16.70 NAD+_7_0 671.00 428.09 33.00 36.00 31.00 16.70 NAD+_7_1 671.00 429.09 33.00 36.00 31.00 16.70 NAD+_7_7 671.00 435.09 33.00 36.00 31.00 16.70 NAD+_8_1 672.00 429.09 33.00 36.00 31.00 16.70 NAD+_10_5 674.00 433.09 33.00 36.00 31.00 16.70 NAD+_11_5 675.00 433.09 33.00 36.00 31.00 16.70 NAD+_11_6 675.00 434.09 33.00 36.00 31.00 16.70 NAD+_12_5 676.00 433.09 33.00 36.00 31.00 16.70 NAD+_12_6 676.00 434.09 33.00 36.00 31.00 16.70 NAD+_13_6 677.00 434.09 33.00 36.00 31.00 16.70 Abbreviations: NAM: nicotinamide, NMN: nicotinamide mononucleotide, NR: nicotinamide riboside and NAD+: nicotinamide adenine dinucleotide (oxidised).

Chapter 3 Quantifying the NAD metabolome

Table 3.5 MRM transitions and mass spectrometry parameters for isotopologues of NA, NaR, NaMN and NaAD Isotopologue Q1 (m/z) Q3 (m/z) DP (V) CE (V) CXP (V) RT (mins) (precursor_product) ion mass shift NA_0_0 123.80 77.80 70.00 25.00 10.00 4.30 NA_1_1 124.80 78.80 70.00 25.00 10.00 4.30 NaR_0_0 256.20 124.20 41.00 27.00 6.00 10.50 NaR_1_1 257.20 125.20 41.00 27.00 6.00 10.50 NaR_5_0 261.20 124.20 41.00 27.00 6.00 10.50 NaR_6_1 262.20 125.20 41.00 27.00 6.00 10.50 NaAD_0_0 665.00 428.10 139.00 35.00 38.00 17.05 NaAD_1_0 666.00 428.10 139.00 35.00 38.00 17.05 NaAD_5_0 670.00 428.10 139.00 35.00 38.00 17.05 NaAD_6_0 671.00 428.10 139.00 35.00 38.00 17.05 NaAD_1_1 666.00 429.10 139.00 35.00 38.00 17.05 NaAD_2_1 667.00 429.10 139.00 35.00 38.00 17.05 NaAD_5_5 670.00 433.10 139.00 35.00 38.00 17.05 NaAD_6_1 671.00 429.10 139.00 35.00 38.00 17.05 NaAD_6_5 671.00 433.10 139.00 35.00 38.00 17.05 NaAD_6_6 671.00 434.10 139.00 35.00 38.00 17.05 NaAD_7_1 672.00 429.10 139.00 35.00 38.00 17.05 NaAD_7_6 672.00 434.10 139.00 35.00 38.00 17.05 NaAD_7_7 672.00 435.10 139.00 35.00 38.00 17.05 NaAD_10_5 675.00 433.10 139.00 35.00 38.00 17.05 NaAD_11_5 676.00 433.10 139.00 35.00 38.00 17.05 NaAD_11_6 676.00 434.10 139.00 35.00 38.00 17.05 NaAD_12_6 677.00 434.10 139.00 35.00 38.00 17.05 NaMN_0_0 336.10 123.80 66.00 28.00 11.00 20.90 NaMN_1_1 337.10 124.80 66.00 28.00 11.00 20.90 NaMN_5_0 341.10 123.80 66.00 28.00 11.00 20.90 NaMN_6_1 342.10 124.80 66.00 28.00 11.00 20.90 Abbreviations: NA: nicotinic acid, NaR: nicotinic acid riboside, NaAD: nicotinic acid adenine dinucleotide, NaMN: nicotinic acid mononucleotide.

Chapter 3 Quantifying the NAD metabolome

The LC-MS method was not optimised for recovery however, to compensate for

potential losses during sample handling and processing, samples and standards were subjected

to the same extraction method. R-squared regression (R2) values for each metabolite are shown

in Table 3.6 for accuracy, along with Relative Standard Deviation (RSD) for measures of precision. The Limit of Quantification (LOQ), the lowest concentration at which the metabolite can be reliably detected, is a measure of sensitivity of the LC-MS method. LOQ is calculated from the standard deviation of the calibration curve generated for each metabolite and also shown in Table 3.6. The LOQ and RSD for NAD+ metabolites, especially NR, is relatively high compared to previous studies (Evans et al. 2010; Trammell and Brenner 2013) however,

NMN and NR are known to be unstable metabolites in mouse serum and blood (Liu et al. 2018) contributing to the high variability detected in our samples in vivo (Chapter 5).

Table 3.6 LC-MS precision and accuracy parameters for each metabolite Metabolite Transition CE RT LOQ R2 RSD (m/z) (V) (mins) (pmol) (%) NMN 335.15>123.05 24.00 18.80 91.01 0.998 8.10 NaMN 336.10>123.80 28.00 18.50 92.92 0.997 10.10 NR 255.00>123.03 13.00 11.45 44529.13 0.989 24.80 NaR 256.20>124.20 6.00 10.20 180.77 0.999 7.00 NaAD 665.00>428.10 35.00 16.80 85.82 0.985 20.90 Nam 122.80>79.80 30.00 1.98 87.91 0.991 17.10 Na 123.80>77.80 25.00 4.20 91.50 0.999 5.80 NAD+ 664.00>428.09 36.00 16.95 89.35 0.989 11.50 Abbreviations: CE (V): collision energy (volts); RT (mins): retention time (minutes); LOQ (pmol): level of quantification (picomoles); R2: R-squared regression; RSD (%): relative standard deviation; NA: nicotinic acid, NaAD: nicotinic acid adenine dinucleotide, NAD+: nicotinamide adenine dinucleotide (oxidised), NAM: nicotinamide, NaMN: nicotinic acid mononucleotide. NaR: nicotinic acid riboside, NMN: nicotinamide mononucleotide, and NR: nicotinamide riboside.

Chapter 3 Quantifying the NAD metabolome

3.3.3 Detecting labelled NMN isotopes using LC-MS/MS and MRM.

Following the custom synthesis of strategically designed NMN isotopes (see Chapter 2, Section

2.1.1), the purity and mass of these compounds were verified to ensure proper labelling using

the optimised LC-MS/MS method and their predetermined MRM transitions. Representative

chromatograms are shown for unlabelled NMN (Figure 3.3 A) and the two labelled NMN isotopes, NMN1 (M+6) (Figure 3.3 B) and NMN2 (M+7) (Figure 3.3 C). These compounds appeared at their expected m/z, with the appearance of smaller peaks from other isotopologues that can be attributed to the natural abundance of 13C (1.1%) and 15N (0.37%) isotope labels

(Deléens, Cliquet, and Prioul 1994; Dijkstra et al. 2006; Yu et al. 2019) as well as some

impurity or degradation of the NMN isotopes over time, especially NMN2 (M+7) where there

is a significant M+6 peak visible (in yellow) which represents the substitution of amide-15N with amide-14N from the nicotinamide moiety.

Figure 3.3 Representative chromatograms of unlabelled (M+0) and labelled NMN isotopes from injection of a 100 µM standard solution. (A) Unlabelled NMN (M+0) with MRM transition 335.15>123.05. (B) NMN (M+6) or NMN1 with MRM transition 341.15>124.05. (C) NMN (M+7) or NMN2 with MRM transition 342.15>125.05.

Chapter 3 Quantifying the NAD metabolome

3.3.4 Tracing the metabolic fate of NMN isotopes using LC-MS/MS to detect labelled

NAD+ metabolites.

To test this LC-MS/MS method in a biologically relevant sample, primary hepatocytes were

isolated from fifteen-week-old female C57BL/6 mice and treated with the labelled NMN

isotopes NMN1 and NMN2 for 24 hours. As the liver contains one of the highest concentrations

of NAD+ and is highly active in the metabolism of orally delivered NAD+ precursors (Liu et

al. 2018), primary hepatocytes serve as a good in vitro model to explore NAD+ metabolism

following supplementation with NMN. The labelled nicotinamide (15N) and ribose (13C)

moieties were traced in NAD+ metabolites involved in the canonical pathway for NMN

metabolism, the salvage pathway.

NMN synthesises NAD+ via the three isoforms of nicotinamide mononucleotide

adenylyltransferase (NMNAT1-3) (Raffaelli et al. 2002; Schweiger et al. 2001; Zhang et al.

2003) before it is degraded by NAD+-consuming enzymes such as the sirtuins, poly-ADP-

ribose polymerases (PARPs) and glycohydrolases (CD38/CD157) into nicotinamide (NAM)

and ADP-ribose (ADPr) (Imai et al. 2000a; Kim, Jacobson, and Jacobson 1993; Kim, Zhang, and Kraus 2005).

ADPr is an important by-product of NAD+ consumption, where protein deacetylation

by sirtuin enzymes results in the transfer of the acetyl group from acetylated protein substrates

to form O-acetyl-ADP-ribose (OAADPr) (Tanny and Moazed 2001), acting as a signalling

molecule for various proteins such as the Ca2+ channel TRPM2 (Tanner et al. 2000; Tong and

Denu 2010; Zhao et al. 2004). Generation of ADPr is also important for PARPs in protein and

histone ADP-ribosylation during DNA damage repair (Palazzo et al. 2018) and by

glycohydrolases such as CD38/CD157 to regulate intracellular calcium signalling (Deshpande

et al. 2003; Howard et al. 1993) and the immune response (Partida-Sánchez et al. 2001). NAM

is on the other hand recycled back into the salvage pathway to synthesise NMN via

Chapter 3 Quantifying the NAD metabolome

nicotinamide phosphoribosyltransferase (NAMPT), the rate limiting enzyme in this pathway

(Revollo, Grimm, and Imai 2004; Wang et al. 2006b), which is then used to synthesise NAD+

via NMNAT1-3 enzymes (Berger et al. 2005; Garavaglia et al. 2002; Schweiger et al. 2001;

Zhang et al. 2003).

As expected, intact labelled NMN1 with an overall mass shift of M+6 (Figure 3.4 A)

and intact labelled NMN2 with an overall mass shift of M+7 (Figure 3.4 B) was detected in

cells treated with NMN1 and NMN2, respectively. To avoid any residual NMN1 or NMN2

supplemented in the cell culture media affecting the measurement of intracellular metabolites,

cells were rinsed with phosphate buffered saline (1XPBS) and discarded prior to cell harvest

in extraction buffer. As a direct precursor to NAD+ biosynthesis, supplementation with labelled

NMN compounds led to increased levels of intracellular NAD+ which incorporated the M+6

and M+7 mass shifts from NMN1 and NMN2, respectively (Figure 3.4 C-D). The presence of these labels in NAD+ indicates these metabolites incorporated the ribose and nicotinamide

moieties intact, as both moieties were labelled with 13C and 15N isotopes to contribute to the

overall increase in mass shift.

As expected, we also detected labelling of the NAD+ degradation product, nicotinamide

(NAM), as NAM (M+1) (Figure 3.5 A) and NAM (M+2) (Figure 3.5 B) following

supplementation with NMN1 and NMN2 respectively. These represent the cleavage activity of

NAD+ (M+6) and NAD+ (M+7), respectively, by NAD+-consuming enzymes. These data do not exclude the possibility that NMN undergoes intracellular cleavage at the glycosidic bond to release free NAM and ribose-5-phosphate, as this bond could be labile under biological conditions (Bell, Yeates, and Eisenberg 1997; Göckel and Richert 2015; Liu et al. 2008).

Following cleavage of NAD+, the ADP-ribose moiety is liberated through NAD+-consuming

activities while NAM is recycled back into NMN.

Chapter 3 Quantifying the NAD metabolome

The regeneration of NMN from NAM via the NAD+ salvage pathway involving the rate-limiting enzyme, NAMPT (Revollo, Grimm, and Imai 2004), was detected. This was inferred based on the presence of NMN (M+1) (Figure 3.5 C) and NMN (M+2) (Figure 3.5

D) which form the precursors for the synthesis of NAD+ (M+1) (Figure 3.5 E) and NAD+

(M+2) (Figure 3.5 F), respectively, via NMNAT1-3 (Raffaelli et al. 2002; Schweiger et al.

2001; Zhang et al. 2003).

These results largely verified that the labelling strategy using NMN isotopes to trace labelled nicotinamide and ribose moieties in NAD+ metabolites through the optimised LC-

MS/MS method developed here, are sound. These NMN isotopes demonstrated expected behaviour through the canonical salvage pathway in a biologically relevant in vitro model, ensuring the use of these strategies in subsequent experiments to explore NMN metabolism.

Chapter 3 Quantifying the NAD metabolome

Figure 3.4 Labelled NMN compounds are detected intact and contribute to an increase in NAD+ in primary hepatocytes. Primary hepatocytes were isolated from 15-week-old female C57BL/6J mice and treated with either 1) vehicle control, 2) NMN1 (0.2 mM), or 3) NMN2 (0.2 mM). Data are represented as mean ± s.d. (n=3) and normalised to the average of internal standards (MES, CSA and thymine-d4). (A) Increase in NMN (M+6) detected in cells after extracellular NMN1 supplementation. (B) Increase in NMN (M+7) detected in cells after extracellular NMN2 supplementation. (C) Increase in NAD+ (M+6) detected in cells after extracellular NMN1 supplementation. (D) Increase in NAD+ (M+7) detected in cells after extracellular NMN2 supplementation.

Chapter 3 Quantifying the NAD metabolome

Figure 3.5 Labelled NAD+ is degraded and contributes to increases in metabolites in the NAD+ recycling pathway. Primary hepatocytes were isolated from 15-week-old female C57BL/6J mice and treated with either 1) vehicle control, 2) NMN1 (0.2 mM), or 3) NMN2 (0.2 mM). Data are represented as mean ± s.d. (n=3) and normalised to the average of internal standards (MES, CSA and thymine-d4). (A) NAM (M+1) formed from cleavage of NAD+ (M+6) by NAD+-consuming enzymes. (B) NAM (M+2) formed from cleavage of NAD+ (M+7) by NAD+-consuming enzymes. (C) NMN (M+1) synthesised from NAM (M+1) via enzyme, NAMPT. (D) NMN (M+2) synthesised from NAM (M+2) via enzyme NAMPT. (E) NAD+ (M+1) synthesised from NMN (M+1) via enzyme isoforms NMNAT1-3. (F) NAD+ (M+2) synthesised from NMN (M+2) via enzyme isoforms NMNAT1-3.

Chapter 3 Quantifying the NAD metabolome

3.3.5 Intracellular NR is detected following extracellular supplementation of NMN in

primary hepatocytes.

The mechanism for transport of NMN into cells remains elusive. While there is evidence that

NMN is transported intact (Grozio et al. 2019a; Yoshino et al. 2011), others argue NMN

requires dephosphorylation into nicotinamide riboside (NR) for transport and utilisation inside

the cell (Ratajczak et al. 2016). To investigate mechanisms of NMN transport into cells, the

incorporation of the labelled nicotinamide (15N) and ribose (13C) moieties into NMN and NR isotopologues were traced in primary hepatocytes.

Initially, the intact labelling of NMN (M+6) (Figure 3.4 A) and NMN (M+7) (Figure

3.4 B) detected in primary hepatocytes after 24 hours exposure to NMN1 and NMN2, respectively, suggested these isotopes were transported directly into the cell. The concomitant detection of NR (M+6) (Figure 3.6 A) and NR (M+7) (Figure 3.6 B), also suggested that these

NMN isotopes underwent dephosphorylation into NR and were taken up by cells. Direct transport of NMN into cells via SLC12A8 has been reported to involve rapid uptake (within 1 minute) and immediate conversion (within 15 minutes) to NAD+ (Grozio et al. 2019a; Mills et

al. 2016). Compared to this minute-order uptake of NMN into the cell reported by Grozio and

colleagues, the extracellular dephosphorylation of NMN to NR followed by its intracellular re-

phosphorylation back into NMN reportedly occurs slowly over the course of 24 hours (Belenky

et al. 2007; Ratajczak et al. 2016) which is consistent with the detection of NMN at this time

point observed in this study.

In order to compare the intracellular labelled pools of NMN and NR, the fold change

increases in labelled NMN and NR species were calculated relative to the control group.

Following exposure to NMN1, the fold change increase in M+6 labelled NR relative to control

was 968.84, while the fold change increase in M+6 NMN relative to control was 639.22

(Figure 3.6 C). These data indicate NR is more abundant than NMN in the cell following

Chapter 3 Quantifying the NAD metabolome

supplementation with NMN1, which suggests that NMN transport may occur via

dephosphorylation to NR. Similarly, NMN2 supplementation led to a fold change increase in

M+7 NR of 1276.92 relative to control (Figure 3.6 D). Despite this being lower than the fold

change increase in M+7 NMN of 2503.17 relative to control, this still suggests some evidence

that NR is being produced abundantly following NMN2 supplementation. These data alone are

not enough evidence to prove this mechanism, nor does this exclude the possibility that NMN

may still achieve cellular entry through direct intact transport. This is especially considering

the fact that intracellular labelled NMN was observed following administration with labelled

NMN isotopes in this study, an observation which contradicts findings from a similar study by

Ratajczak and colleagues where labelled NMN was not detected in primary hepatocytes

supplemented with labelled NMN (2H-13C-NMN) over a 6-hour time course. Considering the longer time point (24 hours) captured in this study, the detection of labelled NMN could come from its conversion to NR inside the cell, which is a possibility supported by the abundant detection of labelled NR observed in this study. This is investigated more closely later in this chapter, in the mammalian cell lines C2C12 and HEK293, where labelled NMN is supplemented in the cell culture media and harvested at multiple time points.

Overall, without completely excluding the potential uptake of NMN directly through the NMN transporter, the abundance of labelled NR detected in primary hepatocytes following exogenous supplementation with labelled NMN isotopes suggest that NMN may be taken up through dephosphorylation into NR prior to its uptake and subsequent re-phosphorylation to

NMN once inside the cell.

Chapter 3 Quantifying the NAD metabolome

Figure 3.6 An abundance of intact labelled NR is detected following supplementation with labelled NMN. Primary hepatocytes were isolated from 15-week-old female C57BL/6J mice and treated with either 1) vehicle control, 2) NMN1 (0.2 mM), or 3) NMN2 (0.2 mM). Data are represented as mean ± s.d. (n=3) and normalised to the average of internal standards (MES, CSA and thymine-d4). (A) Intact labelled NR (M+6) following supplementation with labelled NMN (0.2 mM). (B) Intact labelled NR (M+7) following supplementation with labelled NMN (0.2 mM). (C) Fold change of M+6 NMN and NR relative to control following supplementation with labelled NMN1 or NMN2 (0.2 mM). (D) Fold change of M+7 NMN and NR relative to control following supplementation with labelled NMN1 or NMN2 (0.2 mM).

Chapter 3 Quantifying the NAD metabolome

3.3.6 NMN does not lead to increases in deamidated metabolites, NaMN and NaAD,

following supplementation in primary hepatocytes.

These findings thus far, demonstrate that NMN metabolism occurs through the expected

salvage pathway, primarily involving amidated NAD+ metabolites. That is, NAD+ is

synthesised inside the cell and subsequently degraded into nicotinamide (NAM) where it is

recycled back into the salvage pathway to synthesise NMN via NAMPT, and NAD+, via

NMNAT1-3. The effect of extracellular NMN on intracellular NR was also demonstrated in

primary hepatocytes through a higher abundance of labelled NR pool versus labelled NMN

detected inside the cell, suggesting the potential mechanism for the uptake of NMN may

involve dephosphorylation to NR.

It is unknown whether exogenous NMN treatment influences the deamidated NAD+

metabolite nicotinic acid mononucleotide (NaMN), nicotinic acid adenine dinucleotide

(NaAD), nicotinic acid (NA) and nicotinic acid riboside (NaR) as there are no established

mammalian pathways for this to be expected, let alone investigated. These deamidated

metabolites form another NAD+ biosynthesis pathway called the Preiss-Handler pathway.

NaMN is synthesised either from NA, via nicotinic acid phosphoribosyltransferase (NaPRT), or from NaR, via nicotinamide riboside kinases (NRK1/2), the same enzymes responsible for the conversion of NR to NMN in the salvage pathway (Bieganowski and Brenner 2004; Tempel et al. 2007). NaMN is then converted into NaAD via NMNAT1-3, which also convert NMN to

NAD+ in the salvage pathway. The glutamine-dependent reaction catalysed by NAD+

synthetase (NADSYN1) is then used to amidate NaAD to NAD+ in the final step of the Preiss-

Handler pathway.

It is important to recognise that none of the precursors from the salvage pathway, such

as NMN or NR, are involved in the biosynthesis of NAD+ in this pathway. Despite this, the

presence of the deamidated metabolite, NaAD, was strikingly detected following

Chapter 3 Quantifying the NAD metabolome

supplementation with the amidated metabolite, nicotinamide riboside (NR) (Trammell et al.

2016a), suggesting an unknown mammalian pathway may exist that forms NaAD from

metabolites in the salvage pathway such as NMN and NR.

To explore this possibility, the abundance of these deamidated metabolites were

explored following supplementation with NMN isotopes, focusing on NaMN and NaAD.

Despite our best efforts (details described in following paragraphs), our results showed that

levels of NaMN and NaAD was not increased, nor labelled, following exogenous

supplementation with labelled NMN. From these findings, it appears that the deamidation

pathway does not occur in primary hepatocytes.

Interpretation of mass spectrometry data obtained from NaAM and NaAD presented

some challenges due to the isobaric (differ by one Dalton) nature to their amidated counterparts,

NMN and NAD+, respectively. The monoisotopic mass of NaMN (335.04 g/mol; PubChem

CID 5288991) and NaAD (665.10 g/mol; PubChem CID 165491) is one Dalton higher than

that of NMN (334.06 g/mol; PubChem CID 14180) and NAD+ (664.11 g/mol; PubChem CID

5893), respectively. Consequently, the isotope labelling strategy, where an extra isotope label

is incorporated, will cause several pairs of NaMN/NMN and NaAD/NAD+ isotopologues to

share the same MRM transitions. For example, the MRM transitions for NMN labelled at the

nicotinyl ring base of the nicotinamide moiety, NMN (M+1), with m/z 336.15>124.05, will

have the same MRM transition as unlabelled NaMN (M+0), with m/z 336.10>123.80.

Similarly, the isotopologue pairs NMN (M+2)/NaMN (M+1), NMN (M+6)/NaMN (M+5) and

NMN (M+7)/NaMN (M+6) will also share the same MRM transitions (Figure 3.7) causing

them to be detected under the same chromatogram peak or in some cases overlap. This can be

observed in Figure 3.3 B and C where the smaller peaks to the left of the main NMN peaks represent the corresponding isotopologues from NaMN.

Chapter 3 Quantifying the NAD metabolome

To determine which isotopologue each peak truly represents, the chromatogram peaks

from primary hepatocytes supplemented with unlabelled NMN were explored. Figure 3.7 A

shows chromatogram peaks following supplementation with unlabelled NMN (0.2 mM) while

Figure 3.7 B shows chromatogram peaks following supplementation with M+6 labelled

NMN1 (0.2 mM) in primary hepatocytes. As shown in Figure 3.7 A, the larger main peak (in

dark blue) represents NMN (M+0) while the smaller peak (in light blue) represents both NMN

(M+1) and NaMN (M+0) under the same peak. As these cells were supplemented with

unlabelled NMN, the NMN (M+1) detected here represents the incorporation of isotope labels

that exist normally in nature, that is, the naturally occurring isotope 13C occurs ~1.1% and 15N occurs ~0.37% in natural abundance (Deléens, Cliquet, and Prioul 1994; Dijkstra et al. 2006;

Yu et al. 2019). The isotope distributions for NMN (C11H15N2O8P) can be calculated with the

isotope distribution calculator available at https://www.sisweb.com/mstools/isotope.htm. The natural abundance of NMN with an extra isotope (MW 335) is 13.2% (Figure 3.7 C), which is approximately one tenth (or 0.1) of the abundance of unlabelled NMN (MW 334). Any abundance detected above this ratio, therefore, represents the formation of unlabelled NaMN

(M+0) following supplementation with unlabelled NMN which was quantified in Figure 3.7

D. The ratio of NMN (M+1) (mean 37.26) to NMN (M+0) (mean 413.78) was 0.09 and the ratio of NaMN (M+0) (mean 25.20) to NMN (M+0) was 0.06. As both were less than 0.1, it was established that the combined NMN (M+1)/NaMN (M+0) peak most likely represented

NMN (M+1). Further, very little unlabelled NaMN was endogenously present or newly synthesised following supplementation with unlabelled NaMN. As such, this negligible influence of NMN on NaMN levels indicated that the shared peaks between pairs of NMN and

NaMN isotopologues in Figure 3.7 B most likely represent the NMN isotopologues rather than their NaMN counterparts.

100

Chapter 3 Quantifying the NAD metabolome

Further, following supplementation with the NMN isotope NMN1, the abundance of the NMN (M+7)/NaMN (M+6) peak was similar to the control (no NMN), indicating that not only was there very little NMN (M+7) detected, very little NaMN (M+6) was present in samples. Therefore, the abundance of labelled NaMN (M+6) observed after supplementing with the labelled NMN isotope NMN2 (M+7) most likely represents the abundance of NMN

(M+7), rather than NaMN (M+6), which shares a similar MRM transition.

These results show that levels of the deamidated metabolite NaMN are largely not affected by exogenous supplementation with NMN.

101

Chapter 3 Quantifying the NAD metabolome

Figure 3.7 Chromatogram peaks of NMN/NaMN isotopologue pairs sharing the same MRM transition. Primary hepatocytes were isolated from 15-week-old female C57BL/6J mice and treated with either unlabelled NMN (0.2 mM) or NMN1 (0.2 mM). Chromatogram peaks of unlabelled and labelled NMN/NaMN isotopologues after supplementation with (A) unlabelled, (B) M+6 labelled NMN1 and quantified in (D) and (E), respectively. (C) The isotope distribution of NMN (C11H15N2O8P) was calculated with the isotope distribution calculator available at https://www.sisweb.com/mstools/isotope/htm. Data are represented as mean ± s.d. (n=3) and normalised to the average of internal standards (MES, CSA and thymine-d4).

102

Chapter 3 Quantifying the NAD metabolome

In contrast to the chromatograms shared by NMN/NaMN pairs of isotopologues

described in Figure 3.7 (above), the normalised intensities calculated from the integration of

chromatogram peaks shared by NAD+/NaAD pairs of isotopologues showed discrepancies. The

natural abundance of M+1 labelled NAD+ is 26.2% (Figure 3.8 C, below) and any value above this percentage in an injected sample suggests the presence of unlabelled NaAD (M+0) as it shares the same MRM transition as M+1 labelled NAD+ (665.00>428.09 for NAD+ [M+1] vs.

665.00>428.10 for NaAD [M+0]).

This was the case observed in Figure 3.8 A, where the chromatogram peak shared by

NAD+ (M+1) and NaAD (M+0) was approximately half (~50%) of the unlabelled NAD+ (M+0)

peak (absolute percentage not quantified), suggesting NaAD is detected in cells following

supplementation with NMN. The same observation was made following supplementation with

M+6 labelled NMN1 (Figure 3.8 B), although this value was slightly lower (absolute

percentage not quantified) but still above the expected 26.2% from natural abundance. The

autogenerated values which were extracted following the integration of chromatogram peaks

suggest that NaAD is detected in very low amounts in samples following supplementation with

NMN, and is independent of the increases belonging to corresponding NAD+ isotopologues.

Following supplementation with unlabelled NMN, the combined normalised intensities

calculated from NAD+ (M+1) and NaAD (M+0) is approximately 18.1% of the intensity

calculated from NAD+ (M+0) (Figure 3.8 D), of which NAD+ (M+1) contributes the majority and most likely represents naturally abundant isotope. Following supplementation with M+6 labelled NMN1, M+6 labelled NAD+ increases but NaAD (M+5), which shares the same MRM

transition, does not (Figure 3.8 E). Similarly, NAD+ (M+1) increases slightly whereas NaAD

(M+0) does not, suggesting that NaAD is not present in samples. The isotopologue pairs NAD+

(M+7)/NaAD (M+6) and NAD+ (M+2)/NaAD (M+1) represent complete and partial

incorporation of labelled moieties from M+7 labelled NMN2 respectively, though as expected,

103

Chapter 3 Quantifying the NAD metabolome these isotopologues were largely unaffected following supplementation with M+6 labelled

NMN1. These data suggest that, like NaMN, levels of the deamidated metabolite NaAD is not detected in primary hepatocytes following treatment with exogenously supplemented NMN.

104

Chapter 3 Quantifying the NAD metabolome

Figure 3.8 Chromatogram peaks from NAD+/NaAD isotopologues sharing the same MRM transition. Primary hepatocytes were isolated from 15-week-old female C57BL/6J mice and treated with either unlabelled NMN (0.2 mM) or NMN1 (0.2 mM). Chromatogram peaks of unlabelled and labelled NMN/NaMN isotopologues after supplementation with (A) unlabelled or (B) M+6 labelled NMN1 + and quantified in (D) and (E), respectively. (C) The isotope distribution of NAD (C21H27N7O14P2) was calculated with the isotope distribution calculator available at https://www.sisweb.com/mstools/isotope/htm. Data are represented as mean ± s.d. (n=3 technical replicates) and normalised to the average of internal standards (MES, CSA and thymine-d4).

105

Chapter 3 Quantifying the NAD metabolome

3.3.7 Time-course tracing of NMN metabolism in C2C12 and HEK293 cells – amidated metabolites

The study conducted in primary hepatocytes achieved two things—first, it verified the labelling

strategy of the labelled NMN isotopes NMN1 and NMN2, and second, it validated the use of

LC-MS/MS in MRM mode optimized to detect unlabeled and labelled NAD+ metabolites in a

biologically relevant in vitro model. However, as we only used a single time point, we are

unable to evaluate the dynamics of NMN assimilation, such as how fast NMN is converted into

NAD+. As such, in this next study, samples were harvested at multiple time points—0, 1, 2, 4,

8, and 24 hours—to profile any potential meaningful incorporation of labelled moieties from

NMN into NAD+ metabolites over time. Based on differences in enrichment rates, we can infer which NAD+ metabolite pools are proximal to exogenous NMN and have faster enrichment.

Further, while primary cells are isolated directly from tissues, and are closer in

morphology and function than cells from cultured cell lines, they have limited proliferative

capacity and are overall more difficult to obtain and maintain. Thus, the mouse myoblast cell

line, C2C12, and the human embryonic kidney cell line, HEK293, were used. C2C12 were

used as muscle is an important site of action for NAD+ precursors (Agerholm et al. 2017; Ryu

et al. 2016), while HEK293 cells represent an easy-to-use non-differentiating cell line for investigating NAD+ metabolism (Cantó et al. 2012; Davila et al. 2018; Kulikova et al. 2019).

The labelled NMN isotope NMN1, was added to culture media at a final concentration of 0.1

mM, and harvested at time points, 1, 2, 4, 8, and 24 hours. For the time zero sample, we used

an equivalent dose of NMN, but unlabeled, to conserve our labelled substrate. NMN2 labelling

experiments were not performed due to limited amount of substrates available.

Intact, labelled NMN1 (M+6) was detected in C2C12 and HEK293 cells within the first

hour as shown in Figure 3.9 A and Figure 3.9 B respectively, indicating rapid uptake into both

cell lines. The levels of NMN detected were either maintained (in C2C12 myoblasts) or

106

Chapter 3 Quantifying the NAD metabolome increased (in HEK293 cells) by the 8-hour time point, but showed a steep decline by the 24- hour time point in both cell lines, suggesting that by this point, NMN in the media has been depleted. This was confirmed by observing the labelling pattern of (M+6) labelled NAD+, which increased over time in both cell lines as shown in Figure 3.9 C for C2C12 and Figure

3.9 D for HEK293 cells, suggesting that while transport of NMN into the cell may be rapid

(within 1 hour), conversion into NAD+ tends to occur gradually.

The consumption of NAD+ , which produces nicotinamide, was also observed over time indicated by the detection of labelled NAM (M+1) in C2C12 (Figure 3.10 A) and HEK293

(Figure 3.10 B). ). However, the appearance of NAM (M+1) could be attributed to cleavage of

NMN (M+6) or NR (M+6) through a labile glycosidic bond as NR levels were quite high compared to NAD+ (M+6) at the same time points. Interestingly, the formation of NMN (M+1) from its precursor NAM (M+1), was low in both C2C12 (Figure 3.10 C) and HEK293 (Figure

3.10 D) cells, with minimum changes observed over time which may suggest the NMN pool is rapidly converted to NAD+ by fast acting NMNAT enzymes (Berger et al. 2005; Garavaglia et al. 2002; Schweiger et al. 2001; Zhang et al. 2003), which may explain why very little fluctuation in this intermediate species was observed.

In contrast, synthesis of NAD+ (M+1) (Figure 3.10 E-F) from NMN (M+1) increased over time in both cell lines, indicating activation of the salvage pathway following exogenous supplementation with NMN to maintain endogenous NAD+ levels. Interestingly, M+6 intact labelling of NR was detected in C2C12 (Figure 3.9 E) and HEK293 (Figure 3.9 F) cells, similar to that observed in primary hepatocytes (Figure 3.6 A) following extracellular supplementation of NMN1, providing more evidence to suggest its dephosphorylation into NR via CD73 (Grozio et al. 2013).

107

Chapter 3 Quantifying the NAD metabolome

Figure 3.9 Labelled NMN detected intact and contributes to increases in NAD+ and NR over time in cell. C2C12 and HEK293 cells were supplemented with labelled NMN (M+6) (0.1mM) for 1, 2, 4, 8, and 24 hours. An equal volume of unlabelled NMN (0.1mM) was supplemented at time zero and harvested for LC-MS/MS analysis. Data represent picomoles normalised to mg protein (pmol/mg protein) with mean ± s.d. (n=2). (A)-(B) Labelled (M+6) NMN in C2C12 and HEK293 cells. (C)-(D) Labelled (M+6) NAD+ in C2C12 and HEK293 cells. (E)-(F) Labelled (M+6) NR in C2C12 and HEK293 cells.

108

Chapter 3 Quantifying the NAD metabolome

Figure 3.10 Degradation of NAD+ and nicotinamide salvage C2C12 and HEK293 cells were supplemented with labelled NMN (M+6) (0.1mM) for 1, 2, 4, 8, and 24 hours. An equal volume of unlabelled NMN (0.1mM) was supplemented at time zero and harvested for LC-MS/MS analysis. Data represent picomoles normalised to mg protein (pmol/mg protein) with mean ± s.d. (n=2). (A)-(B) Labelled (M+1) NAM in C2C12 and HEK293 cells. (C)-(D) Labelled (M+1) NMN in C2C12 and HEK293 cells. (E)-(F) Labelled (M+1) NAD+ in C2C12 and HEK293 cells.

109

Chapter 3 Quantifying the NAD metabolome

3.3.8 Time-course tracing of NMN metabolism in C2C12 and HEK293 cells – deamidated

metabolites

Generation of the deamidated metabolites NaMN and NaAD from the amidated precursor NR

observed by Tammell and colleagues was speculated to come from the deamidation of NMN

by the enzyme, NMN deamidase (Galeazzi et al. 2011). While NMN deamidase is a well-

known enzyme which actively participates in NAD+ metabolism in bacteria, evidence for NMN deamidase activity in mammalian cells is largely absent (Magni et al. 2008; Rongvaux et al.

2003), despite being reported in early studies (Kirchner, Watson, and Chaykin 1966; Petrack et al. 1963; Petrack et al. 1965; Tanigawa et al. 1971). It was hypothesized that the generation of the deamidated metabolites NaMN and NaAD following supplementation of NMN in the mammalian cell lines C2C12 and HEK293 is unlikely.

Consistent with this hypothesis, our study failed to detect significant production of these deamidated metabolites from exogenous NMN. Figure 3.11 A shows chromatogram peaks of

NMN/NaMN isotopologue pairs detected in C2C12 and HEK293 cells following NMN

supplementation. At time zero, cells were treated with an equivalent amount of unlabeled NMN

(due to the limited availability of labelled NMN isotope) and harvested immediately following

supplementation. The intensity of unlabeled NMN (in dark blue) detected at this time point

indicates rapid uptake of NMN into the cell. The smaller peak below this represents M+1

labelled NMN (in light blue) as well as unlabeled NaMN (M+0) which shares the same MRM

transition, that is, 336.15>124.05 for NMN (M+1) and 336.10>123.80 for NaMN (M+0). The

intensity of this smaller peak compared to the larger peak is close to the value expected from

natural abundance of M+1 labelled NMN (13.2%) (Figure 3.7 C) which suggests very little

NaMN is contributing to this peak and in turn suggests very little NaMN is generated in C2C12

and HEK293 cells following exposure to NMN. Even at the latest time point (24 hrs), this ratio

did not change significantly after cells were exposed to M+6 labelled NMN. The intensity of

110

Chapter 3 Quantifying the NAD metabolome

the peak representing M+6 labelled NaMN (in orange), which is also shared by M+7 labelled

NMN, is small in comparison to the intensity of the peak formed from M+6 labelled NMN (in

yellow) detected in cells. The intensities are quantified in Figure 3.11 B showing that even at

earlier time points, the level of M+6 labelled NaMN (in orange) in C2C12 (left) and HEK293

(right) cells after supplementation with M+6 labelled NMN1 (in yellow) is relatively low.

The deamidated metabolite NaAD, is synthesized from NaMN in mammals via the

enzyme NMNAT as part of the Preiss-Handler pathway (Preiss and Handler 1958b; Zhang et

al. 2003). As very little NaMN was detected in this study, the intracellular levels of NaAD in

C2C12 and HEK293 cells were also expected to be low. Supplementing C2C12 and HEK293

cells with unlabeled NMN led to an intensity of M+1 labelled NAD+ below the expected value

derived from natural abundance (26.2%) (Figure 3.8 C), suggesting very little contribution of

the unlabeled NaAD (M+0) isotopologue which shares the same MRM transition (Figure 3.12

A). The intensity of M+6 labelled NaMN following NMN1 supplementation also remained low at the late 24-hour time point in both C2C12 and HEK293 cells (in orange) compared to M+6 labelled NAD+ (in yellow), suggesting very little synthesis of NaMN in cells following

supplementation with NMN. The low levels of M+0 and M+6 labelled NaAD after

supplementation with unlabeled and M+6 labelled NMN, respectively, also suggests the high

intensity of the peak shared by M+1 labelled NAD+ and unlabeled NaAD (in light blue) most likely represents the former isotopologue, NAD+ (M+1), rather than NaAD (M+0).

Degradation of M+6 labelled NAD+ yields M+1 labelled nicotinamide, which may be salvaged to synthesize M+1 labelled NMN via NAMPT (Revollo, Grimm, and Imai 2004; Wang et al.

2006b), before final synthesis of M+1 labelled NAD+ via NMNAT (Berger et al. 2005;

Garavaglia et al. 2002; Schweiger et al. 2001; Zhang et al. 2003). The chromatogram peaks

shared by pairs of NAD+/NaAD isotopologues shown in Figure 3.12 A are quantified in Figure

111

Chapter 3 Quantifying the NAD metabolome

3.12 B where there is no significant detection of M+6 labelled NaAD (in orange), even at earlier

time points following supplementation with M+6 labelled NMN1.

Overall, these data show very little incorporation of the labelled moieties from M+6

labelled NMN1 into NaMN and NaAD in C2C12 and HEK293 cells. This is consistent with

the hypothesis that the appearance of deamidated metabolites observed by Trammell and

colleagues may be generated from the deamidation of NMN via NMN deamidase, an enzyme

which is present in bacteria, but not mammalian cells such as C2C12 and HEK293 used in this

study. Bacteria in the gastrointestinal tract of mammals could be contributing to the metabolism

and utilisation of orally administered NAD+ precursors such as NMN and NR, a hypothesis which is explored in the next two chapters.

112

Chapter 3 Quantifying the NAD metabolome

Figure 3.11 Detection of NMN/NaMN isotopologue pairs in C2C12 and HEK293 cells following supplementation with NMN. The media of C2C12 and HEK293 cells were supplemented with M+6 labelled NMN (0.1 mM) and harvested at time points 1, 2, 4, 8, and 24 hours after treatment. At time zero, cell media was supplemented with unlabelled NMN (0.1 mM) and harvested immediately. The intensity of NMN and NaMN isotopologue pairs were detected under the same peak due to shared MRM transitions. The data represents mean ± s.d. (n=2) with picomolar amounts normalised to protein concentration of each sample (pmol/mg protein). (A) Chromatogram peaks from NMN/NaMN isotopologue pairs at time zero and 24 hours after supplementation with NMN. (B) Quantification of chromatogram peaks generated from NMN/NaMN isotopologues.

113

Chapter 3 Quantifying the NAD metabolome

Figure 3.12 Detection of NAD+/NaAD isotopologue pairs in C2C12 and HEK293 cells following supplementation with NMN. The media of C2C12 and HEK293 cells were supplemented with M+6 labelled NMN (0.1 mM) and harvested at time points 1, 2, 4, 8, and 24 hours after treatment. At time zero, cell media was supplemented with unlabelled NMN (0.1 mM) and harvested immediately. The intensity of NAD+ and NaAD isotopologue pairs were detected under the same peak due to shared MRM transitions. The data represents mean ± s.d. (n=2) with picomolar amounts normalised to protein concentration of each sample (pmol/mg protein). (A) Chromatogram peaks generated from NAD+/NaAD isotopologues at time zero and 24 hours after supplementation with NMN. (B) Quantification of chromatogram peaks generated from NAD+/NaAD isotopologues

114

Chapter 3 Quantifying the NAD metabolome

3.4 Discussion

In this chapter, an LC-MS/MS method was developed to detect labelled NAD+ metabolites in

cells following supplementation with labelled isotopes of NMN. This involved optimising mass

spectrometry parameters such as the depolarisation potential (DP), collision energy (CE), cell

exit potential (CXP) and retention time (RT) for 8 different NAD+ metabolites (NMN, NR,

NAM, NAD+, NA, NaR, NaMN and NaAD). The multiple reaction monitoring (MRM) transitions for 64 different isotopologues were also pre-determined during this optimisation

process based on their respective monoisotopic masses. It was important to optimise these

parameters in order to establish the maximum signal-to-noise ratio and obtain high resolution peaks for the integration and quantification of these metabolites. These parameters will serve as an important resource for later studies in the field related to NAD+ metabolism using isotope

labelling strategies. This LC-MS/MS method was then validated using primary hepatocytes,

C2C12 and HEK293 cells as in vitro models. In doing so, the metabolic fate of NMN was

explored by tracing the incorporation of the labelled ribose and nicotinamide moieties into

NAD+ metabolites, which also provided some evidence for the mechanisms of NMN transport

into cells and its utilisation through NAD+ biosynthesis pathways.

In mammals, NMN is part of the salvage pathway where it can be converted into NAD+

in one enzymatic step via NMNAT1-3 (Berger et al. 2005; Garavaglia et al. 2002; Schweiger

et al. 2001; Zhang et al. 2003) or resynthesized from nicotinamide after NAD+ is degraded, via

NAMPT (Revollo, Grimm, and Imai 2004; Wang et al. 2006b). According to this canonical view of NAD+ metabolism in mammals, the results from this study show an increase in both

NAD+ and NAM following exogenous supplementation with NMN, as expected. Further, the

labelling of these metabolites indicated NAD+ turnover was occurring through the salvage of

nicotinamide, the by-product (along with ADP ribose) of NAD+ degradation via NAD+

115

Chapter 3 Quantifying the NAD metabolome

consuming enzymes (Imai et al. 2000a; Kim, Jacobson, and Jacobson 1993; Kim, Zhang, and

Kraus 2005).

One of the main interests of using labelled isotope versions of NMN was to explore the

generation of deamidated metabolites in mammalian cells following supplementation with an

amidated NAD+ precursor, as previously observed by Trammell and colleagues (Trammell et

al. 2016a). Whether this occurs through an unknown mammalian NMN deamidase or, as

previously speculated by Trammell and colleagues, through bacterial enzymes in the gut when

delivered orally, is unknown (Trammell et al. 2016a). In this study, very little labelling of

deamidated metabolites NaMN and NaAD was detected, which largely suggests that

mammalian NMN deamidase activity is unlikely, at least in primary hepatocytes and

mammalian cell lines (C2C12 and HEK293 cells) supplemented with NMN. Whether these

metabolites are generated from bacterial enzymes in the gastrointestinal tract following oral

delivery in mammals still remains a possibility and is explored in greater detail in Chapter 4

and Chapter 5.

Another highly debated topic in the field of NAD+ biology is the mechanism of NMN

transport to raise NAD+ levels inside the cell. One mechanism argues that the rapid increase of

NMN (within 1 minute) and synthesis of NAD+ (within 15 minutes in tissues) detected in cells and tissues is highly suggestive of a direct transport mechanism (Grozio et al. 2019a; Mills et

al. 2016; Yoshino et al. 2011). The elusive NMN transporter was recently suggested to come

from the family of solute carrier transporters, namely Solute Carrier Family 12 Member 8,

SLC12A8, and despite ongoing controversy in the field around the bioanalytical methods used

to determine this (Schmidt and Brenner 2019), is supposedly highly selective for NMN (Grozio

et al. 2019a). Another mechanism argues that NMN utilization by cells requires extracellular

dephosphorylation into NR before it can enter the cell (Ratajczak et al. 2016). NR then enters

through a family of equilibrative nucleoside transporters (ENTs), SLC29, comprising 4

116

Chapter 3 Quantifying the NAD metabolome

different types (ENT1-4) where ENT1 (SLC29A1), ENT2 (SLC29A2) and ENT3 transport

NR, while ENT4 transports adenosine (Baldwin et al. 2004). NR is then re-phosphorylated into

NMN via NR kinase enzymes, NRK1/2 (Bieganowski and Brenner 2004). Whether both

mechanisms co-exist or complement each other remains a matter of debate.

While intact labelling of NMN was observed inside the cell (Figure 3.5 A-B and

Figure 3.9 A-B), there was also an abundance of intact labelled NR detected in the in vitro models supplemented with NMN in this study (Figure 3.6 A-B, Figure 3.9 E-F). The labelled

NR pool inside the cell was determined to be higher than the labelled NMN pool (Figure 3.6

C-D) which supports the dephosphorylation of NMN to NR (Ratajczak et al. 2016). It is

important to note however, that the rapid incorporation (within first hour) of NMN detected in

C2C12 and HEK293 cells following supplementation of NMN in culture media, could provide

some evidence to support the NMN transporter mechanism in addition to the

dephosphorylation mechanism, which is a slower process reported to occur over a 24-hour time

period (Ratajczak et al. 2016; Yoshino et al. 2011). Further studies are warranted which

measure labelled NMN and NR at earlier time points (over several time points in minutes rather

than hours) to enhance our understanding of NMN transport mechanisms.

Study Limitations

One of the greatest challenges in the development of our LC-MS/MS method was the

separation of closely related pairs of NAD+ metabolites, in particular, NMN and NAD+ to their

acidic counterparts, NaMN and NaAD, respectively. Lowering the pH of the separation buffer

from pH 9 to pH 5 improved the detection of these closely related species in Chapter 4 and 5,

however this remained one of the main challenges in this study. To overcome this, the natural

abundance for the incorporation of an isotope label into NMN (13.2%) and NAD+ (26.2%) was used to assess whether unlabelled NaMN or NaAD, if present, contributed to the peak shared by M+1 labelled NMN or NAD+, respectively, following supplementation with unlabelled 117

Chapter 3 Quantifying the NAD metabolome

NMN in cells. Due to the nature of the isotope labelling strategy, where some closely related

isotopologues only differ by one mass unit, striving to achieve good chromatographic

separation is a common challenge in LC-MS based metabolomic studies (Bustamante et al.

2018). Despite this, the LC-MS/MS method developed here can detect 64 isotopologues from

8 different NAD+ metabolites following the exogenous supplementation with NMN isotopes, and was validated in three biologically relevant in vitro models which may be used to provide

future perspective on NMN transport and utilisation pathways.

Heavy labelled versions of the metabolites of interest are the ideal internal standards to

normalise sample output measures in LC-MS analysis to account for any variable loss during

sample handling and processing (Bustamante et al. 2018; Clement et al. 2019; Evans et al.

2002; Trammell and Brenner 2013). In our studies, however, the use of alternative non-heavy-

labelled internal standards, methionine sulfonate (MES), camphorsulfonic acid (CSA) and

thymine-d4 (TD4) were chosen as they were readily available, yielding consistent output values

for normalisation over multiple mass spectrometry runs and hence deemed suitable for the

investigations outlined in this thesis. They also provided an accessible option over more

expensive labelled isotope versions of compounds to study the NAD metabolome.

A sensitive component in the design of LC-MS based metabolomic studies is the time of sampling. In order to achieve the closest representation of the biological pathway being targeted, it is ideal to harvest samples rapidly to stop metabolic activity while also maintaining the stability of metabolites of interest. Recently, it was shown that metabolic activity continues to occur rapidly during and after cell harvest, and is difficult to prevent even when the extraction process is fast and performed under cold temperatures (Trefely et al. 2019). The concern is that these enzymatic activities may contribute to the artifactual detection of metabolites in whole cell extracts potentially leading to an unwanted misinterpretation of detected metabolites. One strategy to address post-harvest metabolism involves the

118

Chapter 3 Quantifying the NAD metabolome

introduction of a labelled substrate at the point of harvest which may be compared to samples

that were not introduced to the labelled substrate. The difference in incorporation of labelled

isotopes between these samples will be to assess the amount of post-harvest metabolism occurring in cells. This may be an important consideration for future studies as part of the data correction process in order to detect the presence and/or absence of certain metabolites with better accuracy and reliability.

Recently, it was shown that HEK293 cells grown in media supplemented with 10% fetal bovine serum (FBS) led to the degradation of nucleotides such as NMN to their corresponding nucleosides, namely NR, and in turn NR was cleaved to the corresponding bases, namely NAM (Kulikova et al. 2019). Further, this was also shown to occur even in the absence of cells, albeit to a lesser extent, indicating that FBS alone has the capacity to degrade

NAD+ intermediates. The cell culture conditions implemented in this study include

supplementation of cell culture media with 10% FBS which is the standard method for the

maintenance of growth in C2C12 and HEK293 cell lines, however this may also pose a

potential limitation in light of these FBS-degrading activities. As such, in future in vitro studies

involving supplementation with NAD+ precursors, it may be ideal to design experiments both

with and without FBS as well as with and without cells. Future studies should take into careful

consideration the influence of these culture conditions when investigating changes to NAD+

metabolites following supplementation with NAD+ precursors.

The use of inhibitors targeting enzymes involved in the synthesis or transport of NAD+

intermediates is another effective strategy which could be used in conjunction with labelled

NMN isotopes to investigate mechanisms of transport and utilisation pathways. Many of the

findings from this study were inferred purely from the detection of metabolites in samples,

however, the use of enzyme inhibitors may enhance our understanding of the pathways

involved in the utilisation and transport mechanisms associated with NMN metabolism. The

119

Chapter 3 Quantifying the NAD metabolome

direct utilisation of NMN for NAD+ biosynthesis in the salvage pathway is catalysed by the

enzyme, NMNAT, which can be inhibited by gallotannin (Berger et al. 2005). Mammalian

NMNATs are also responsible for the conversion of NaMN to NaAD, hence, can influence

NAD+ biosynthesis through the Preiss-Handler pathway (Berger et al. 2005; Preiss and Handler

1958b; Zhang et al. 2003). Salvage of nicotinamide, the by-product of NAD+-degradation, can

be prevented by the FK866 which inhibits NAMPT, the rate-limiting enzyme of the salvage

pathway (Revollo, Grimm, and Imai 2004; Wang et al. 2006b). To investigate the mechanism

for NMN transport through dephosphorylation into NR via the 5’-nucleotidase, CD73 (Grozio

et al. 2013), the CD73 inhibitor, adenosine-5′-(α,β-methylene) diphosphate (AOPCP), may be used (Grozio et al. 2019a). The NR transporter family of ENTs, SLC29 (ENT1-3) (Baldwin et al. 2004), which allows entry of NR into the cell can be blocked by the ENT inhibitors, S-(4-

nitrobenzyl)-6-thioinosine (NBTI) and dipyridamole (Dip), to investigate whether the

dephosphorylation of NMN to NR is required prior to its uptake and utilisation (Kulikova et

al. 2019; Ratajczak et al. 2016). The use of these inhibitors in conjunction with NMN isotopes

and LC-MS can further elucidate on the mechanisms involved in the utilisation and transport

of NMN inside the cell.

120

Chapter 4 Investigating microbial NAD+ metabolism

Investigating NAD+ metabolism following supplementation with NMN in bacteria

4.1 Introduction

The microbiome plays an important role in health, encompassing all organisms that live on the

external and internal surfaces of the gastrointestinal tract (GIT), skin, oral cavity and

conjunctiva. The GIT has the highest microbial load, with estimates of up to 1014 microbial cells consisting mainly of bacteria (~98%) and a small percentage (2%) of archaea, viruses and other eukaryotes (Berg 1996; Luckey 1972; Savage 1977; Zhernakova et al. 2016). As a collective whole, the microbiome (genomes of the microbiota) contain approximately 150 times more genes than the human genome (Qin et al. 2010), encoding proteins and enzymes that play important roles in metabolic processes such as digestion and metabolism of host- indigestible foods, the immune response and breakdown of xenobiotics (Carmody and

Turnbaugh 2014; Cha et al. 2010; Fukuda et al. 2011; Hooper, Midtvedt, and Gordon 2002;

Koppel, Rekdal, and Balskus 2017; Maslowski et al. 2009; Savage 1986; Sousa et al. 2008;

121

Chapter 4 Investigating microbial NAD+ metabolism

Spanogiannopoulos et al. 2016). These interactions represent a symbiotic relationship between

the host and the microbiome, which impacts metabolic processes that are important to health.

The importance of the microbiome in governing the metabolism and bioavailability of pharmaceuticals is emerging as an under-appreciated area, with drug-microbiome interactions that could impact clinical efficacy (ElRakaiby et al. 2014; Guthrie, Wolfson, and Kelly 2019;

Khalsa et al. 2017; Li and Jia 2013). While the liver has been traditionally studied as the main organ responsible for metabolising xenobiotics, recent studies emphasise the role of microbial drug metabolism in the GIT prior to interaction with the liver (Carmody and Turnbaugh 2014;

Guthrie, Wolfson, and Kelly 2019; Kang et al. 2013b; Koppel, Rekdal, and Balskus 2017;

Spanogiannopoulos et al. 2016; Sun, Chen, and Shen 2019). This is especially the case for orally delivered medicines which are not completely absorbed by the small intestines, as these compounds then encounter a far greater microbial density in the distal gut, or those which undergo metabolism through the enterohepatic circulation, where the liver and the intestines undergo the exchange of metabolites (Macfarlane and Macfarlane 2004).

Applying this phenomenon to the administration of NAD+ precursors, this study sought

to investigate how the gut microbiome alters the metabolic fate and bioavailability of orally

delivered NMN. Specifically, pathways leading to NMN incorporation by the host are poorly

understood because NMN is an atypical nutrient in our diet; nicotinic acid (NA), nicotinamide

(NAM) and nicotinamide riboside (NR) are the main constituents of vitamin B3, which the gut

microbiome and our body are accustomed to. For example, while Trammell et al. (2016)

reported a sharp spike in the appearance of the deamidated precursors nicotinic acid

mononucleotide (NaMN) and dinucleotide (NaAD) in mouse tissues following oral

administration of NR (Trammell et al. 2016a), it remains unclear how both deamidated

metabolites were raised, as mammalian deamidase enzymes are yet to be identified (Carson,

Seto, and Wasson 1987; Gazzaniga et al. 2009; Lin et al. 1972; Rongvaux et al. 2003; Shibata,

122

Chapter 4 Investigating microbial NAD+ metabolism

Hayakawa, and Iwai 1986). Nonetheless, deamidation of NAD metabolites in mammalian

systems have been reported elsewhere (Kirchner, Watson, and Chaykin 1966; Petrack et al.

1963; Petrack et al. 1965; Sarma, Rajalakshmi, and Sarma 1961). These earlier findings suggest that a clear description of the pathways that actively assimilate orally delivered NMN are still missing.

One theory is that upon exposure to a bolus of amidated metabolites such as NR or

NMN, bacterial enzymes in the GIT may contribute to the generation of NaMN and NaAD via bacterial deamidation enzymes, prior to the uptake of these metabolites by the host. This is because deamidation of NAM and NMN by the deamidase enzymes PncA and PncC is regarded as a necessary enzymatic route for the utilisation of these precursors for NAD+

biosynthesis in bacteria (Galeazzi et al. 2011). If true, this could suggest a role of the

microbiome in altering the metabolism and bioavailability of exogenously administered NAD+

precursors when delivered via the oral route. These potential host-microbiome interactions in

the metabolism of NAD+ precursors could impact therapeutic strategies that involve NMN

supplementation.

4.1.1 Bacterial de novo NAD biosynthesis pathway

Bacteria such as E. coli possess enzymes capable of NAD+ biosynthesis through de novo and

salvage pathways, which include enzymatic steps that are absent in eukaryotes (Figure 4.1,

below). The NAD+ biosynthesis pathway in E. coli is well characterised. With the exception

of bacterial NAMPT (NadV), E. coli possess all the genes necessary for the de novo (nadA,

nadB, nadC, nadD, nadE, pncB) and salvage (nadR) pathways (Galeazzi et al. 2011; Kurnasov et al. 2002), including those encoding NAM and NMN deamidase enzymes pncA (Frothingham et al. 1996) and ygdA (Galeazzi et al. 2011). The deamidation of NAM and NMN by the

123

Chapter 4 Investigating microbial NAD+ metabolism

deamidase enzymes PncA and PncC contribute to the utilisation of these precursors for NAD+

biosynthesis in bacteria (Galeazzi et al. 2011).

The universal precursor in the de novo NAD+ biosynthesis pathway is quinolinic acid

(QA), or quinolinate, which is essential in providing the pyridine ring for synthesis of NAD+.

In most bacteria, QA is generated from ʟ-aspartate, transported by a proton/glutamate-aspartate symporter encoded by gltP gene (Tolner et al. 1992). First, the enzyme NadB (ʟ-aspartate oxidase, EC 1.4.3.16, encoded by nadB) oxidises ʟ-aspartate forming the intermediate α- iminoaspartate in the cytoplasm (Flachmann et al. 1988; Nasu, Wicks, and Gholson 1982;

Seifert et al. 1990). Second, the enzyme NadA (quinolinate synthase, EC 2.5.1.72, encoded by nadA) catalyses the formation of QA from α-iminoaspartate in the presence of dihydroxyacetone phosphate (DHAP) in the cytoplasm (Ceciliani et al. 2000; Flachmann et al.

1988), thus completing the aspartate-to-quinolinate pathway.

NAD+ is then synthesised from QA via the Preiss-Handler pathway (Preiss and Handler

1958a, 1958b). First, QA undergoes phosphoribosylation and decarboxylation by NadC

(quinolinic acid phosphoribosyl transferase, EC 2.4.2.19, encoded by nadC) to form NaMN

(Bhatia and Calvo 1996; Gholson et al. 1964). NaMN can also be regenerated from another precursor, nicotinic acid (NA) by the enzyme PncB (nicotinic acid phosphoribosyltransferase,

EC 6.3.4.21, encoded by pncB) in the presence of phosphoribosyl pyrophosphate (PRPP) and

ATP (Imsande 1961). NA may be taken up by niacin transporters NiaX (Johnson et al. 2015;

Rodionov et al. 2008). NaMN is then adenylated into NaAD by the nicotinic acid mononucleotide adenylyltransferase enzyme NadD (EC 2.7.7.18, encoded by nadD) (Imsande

1961; Mehl, Kinsland, and Begley 2000; Zhang et al. 2002).

In the last step of bacterial NAD+ biosynthesis, NaAD undergoes amidation by the NAD synthetase NadE (EC 6.3.1.5, encoded by nadE) into NAD (Spencer and Preiss 1967). The

124

Chapter 4 Investigating microbial NAD+ metabolism

amidation of NaAD is dependent on free ammonia (NH3) as a source of the amide in most

prokaryotes, including E. coli (De Ingeniis et al. 2012; Spencer and Preiss 1967).

4.1.2 NAD+ is salvaged by deamidation in bacteria

It is important to note that NMN is not an intermediate of the de novo pathway. It is instead

part of the salvage pathway, used to regenerate NAD+ following its hydrolysis by NAD+

consuming enzymes, releasing free NAM, which can be recycled into NAD+ via NMN

(Andreoli et al. 1972; Olivera and Lehman 1967). NAM may enter through niacin transporters

NiaX (Johnson et al. 2015). Interestingly, in bacteria, NAM or NMN are utilised by first

converting them into their acid forms, NA and NaMN respectively by the nicotinamide deamidase PncA (EC 3.5.1.19, encoded by pncA) (Frothingham et al. 1996) and the NMN

deamidase PncC (EC 3.5.1.42, encoded by ygaD) (Foster and Baskowsky-Foster 1980;

Galeazzi et al. 2011; Imai 1973; Manlapaz-Fernandez and Olivera 1973).

125

Chapter 4 Investigating microbial NAD+ metabolism

Figure 4.1 NAD+ biosynthesis pathways in bacteria. The synthesis of NAD+ in bacteria can occur via three pathways. The de novo synthesis pathway initiates with the metabolism of L-aspartate in most bacteria, but L-tryptophan in some groups such as those in the taxonomic order Xanthomonodales, Flavobacteriales and Burkholderiales. Both L-tryptophan and L-aspartate ultimately form quinolinic acid (QA) which is converted into nicotinic acid mononucleotide (NaMN) via NadC in the Preiss- Handler pathway. NaMN may also be synthesised from the precursor nicotinic acid (NA) via PncB. NaMN is then adenylated into nicotinic acid adenine dinucleotide (NaAD) via NadD followed by amidation into NAD+ via NadE. In the salvage pathway, nicotinamide mononucleotide (NMN) may enter the periplasmic space via the porin OmpP2, where it may be metabolised by two separate routes to form NAD+. It may either be hydrolysed to nicotinamide (NAM) and a ribose moiety via NMN glycohydrolase or undergo dephosphorylation into nicotinamide riboside (NR) via periplasmic 5’-nucleotidase UshA. NAM may enter via the niacin transporters NiaX while NR may enter through PnuC where the latter is re-phosphorylated into NMN via the NR kinase activity of NadR. NAM and NMN may both undergo deamidation via deamidases PncA and PncC to form NA and NaMN respectively and synthesise NAD+ via the Preiss-Handler pathway. The nicotinamide mononucleotide adenylyltransferase (NMNAT) activity of NadR is weak in most bacteria, hence relying mostly on NMN deamidation for NAD+ biosynthesis. In some species such as Haemophilus influenzae however, NAD+ biosynthesis from NR via the activity of trifunctional NadR is essential as they lack genes that encode for de novo pathway and Preiss- Handler pathway enzymes.

126

Chapter 4 Investigating microbial NAD+ metabolism

There are two reasons for the deamidation of NAM and NMN. First, NMN can inhibit

NAD+-dependent DNA ligation in bacteria (Chen et al. 2002; Geider 1972; Zimmerman and

Oshinsky 1969). Intracellular NMN builds up quickly during the exponential growth of bacteria

due increased rate of DNA repair imposing a greater conversion of NAD+ into NMN (Lehnman

1974; Olivera and Lehman 1967). Deamidation therefore acts to promote growth and repair by

reducing the availability of NMN, which inhibits DNA ligase. NMN is salvaged through the

NMN deamidase PncC, yielding NaMN as a substrate for NAD+ synthesis by the Preiss-

Handler pathway (Galeazzi et al. 2011).

The second reason for NMN deamidation in bacteria is that NaMN is the preferred phosphoribosyl donor for the enzyme cobaltochelatase (CobT) (Agerholm et al. 2017; Warren et al. 2002). This enzyme catalyses the last step of adenosylcobalamin (vitamin B12) biosynthesis, an important cofactor involved in DNA synthesis, red blood cell production and various functions of the nervous system (Fenech 2001; Reynolds 2006). Even in bacterial species that are capable of NAM and NMN salvage via the non-deamidated route, such as in

E. coli, the deamidation route is preferred. These processes emphasise why deamidation to

NAD+ synthesis are preferred in bacteria, as the amidated intermediate NMN can impair DNA

ligase, and the deamidated intermediate NaMN is an important precursor for cofactor synthesis

(Maggio-Hall and Escalante-Semerena 2003).

4.1.3 Potential NMN uptake routes in bacteria

Gram-negative bacteria such as E. coli have a bi-dermal membrane consisting of an inner and outer membrane separated by an area known as the periplasmic space. NMN enters the periplasmic space through a porin (channel-forming protein) known as OmpP2 (Andersen et

al. 2003; Pullen et al. 1995). Here, the assimilation of NMN into the cytoplasm can occur via

two different routes.

127

Chapter 4 Investigating microbial NAD+ metabolism

The first route involves the dephosphorylation of NMN to nicotinamide riboside (NR)

by the 5’nucleotidase UshA (EC 3.1.3.5, encoded by ushA) (Burns and Beacham 1986).

Transport of NR across the inner membrane involves the transporter PnuC (Jaehme, Guskov,

and Slotboom 2014) and once in the cytoplasm, NR is then re-phosphorylated into NMN by

NadR (NR kinase, EC 2.7.7.1, encoded by nadR).

The second route involves degradation of NMN by the enzyme, NMN glycohydrolase

(EC 3.2.2.14), which is embedded in the inner membrane, forming NAM and ribose 5- phosphate (Andreoli et al. 1972). As NAM is a relatively small molecule, it can permeate the inner and outer membranes, entering the cytoplasm where it may undergo deamidation through

PncA, forming NA. As in de novo biosynthesis from aspartate, NA is used as a substrate for

NAD+ synthesis by the Preiss-Handler pathway enzymes, PncB (NA to NaMN), NadD (NaMN

to NaAD) and NadE (NaAD to NAD+).

Interestingly, NadR is a uniquely tri-functional enzyme which can carry out the following

roles:

1. Transcriptional downregulation of the genes pncB (salvage pathway), nadA and nadB

(de novo pathway) when NAD+ levels in the cell are replete,

2. Interaction with the NR transporter PnuC to promote the uptake of NR into the cell

when NAD+ levels are scarce,

3. Dual NR kinase (NRK) and NMNAT enzymatic activity for the substrates NR and

NMN, respectively (Foster and Baskowsky-Foster 1980; Grose, Bergthorsson, and

Roth 2005; Penfound and Foster 1999; Raffaelli et al. 1999).

NadR is therefore an important regulatory enzyme for coordinating the utilisation and

transport of NMN and NR through both transcriptional and direct enzymatic control. NadR

activity is essential for some species of bacteria, such as Haemophilus influenzae, that lack

128

Chapter 4 Investigating microbial NAD+ metabolism

genes that encode for the enzymes involved in de novo NAD+ biosynthesis (Singh et al. 2002).

In most other bacteria however, NadR has weak NMNAT activity and its activity is insufficient

to prevent the accumulation of NMN, and cells instead rely on PncC to for this activity (Grose,

Bergthorsson, and Roth 2005).

4.1.4 Key differences compared to eukaryotic pathway

There are three main features which bacterial and eukaryotic pathways differ. First, de novo synthesis in higher eukaryotes starts with ʟ-tryptophan to derive the pyridine ring of QA (refer to Chapter 1, Section 2.6.1), instead of condensing ʟ-aspartate. Certain taxonomic groups, such

as Xanthomonadales (Order), Flavobacteriales (Order) and Burkholeriales (Order) do not

express genes encoding nadA and nadB, and therefore rely on QA synthesis from ʟ-tryptophan

(Kurnasov et al. 2003; Lima, Varani, and Menck 2009) which can be transported into the cell

via three transporters, Mtr, TnaB and AroP (Yanofsky, Horn, and Gollnick 1991), however,

most bacterial species rely on QA synthesis from ʟ-aspartate.

Importantly, the deamidation of NAM and NMN which occurs in bacteria does not

occur in mammals. In eukaryotes, NAM is converted to NMN by the nicotinamide

phosphoribosyltransferase NAMPT (EC 2.4.2.12, encoded by NAMPT) (Preiss and Handler

1957). NMN is then used as a substrate by nicotinamide mononucleotide adenylyltransferase

enzymes (NMNAT, EC 2.7.7.1 encoded by Nmnat) to generate the final step of NAD+

(Emanuelli et al. 2001; Raffaelli et al. 2002; Zhang et al. 2003). Phylogenetic analysis of

bacterial genomes reveals an absence of NAMPT (NadV) and NMNAT (NadM and NadR)

(Galeazzi et al. 2011), and bacteria must instead rely on PncA and PncC to convert the amidated

metabolites nicotinamide and NMN to their acid forms of nicotinic acid and NaMN before they

become substrates for NAD+ biosynthesis. In eukaryotes the canonical pathway for NAD+

biosynthesis from NR involves its phosphorylation into NMN via NR kinase (NRK1/2)

(Bieganowski and Brenner 2004), prior to direct conversion of NMN into NAD+ via NMNAT

129

Chapter 4 Investigating microbial NAD+ metabolism

1-3 (Raffaelli et al. 2002; Schweiger et al. 2001; Zhang et al. 2003), a pathway which clearly

does not involve deamidated intermediates.

Another difference in NAD metabolism between bacteria and eukaryotes is that

amidation in eukaryotes is fuelled by glutamine (C5H10N2O3) via the enzyme NAD synthetase

(NADSYN1) (Hara et al. 2003; Preiss and Handler 1958a, 1958b), whereas the bacteria use

free ammonia as a course of nitrogen for amidation of NaAD into NAD+. This likely reflects

differences in the availability of these nitrogen sources.

The aim of this work was to examine how bacteria metabolise NMN, in particular the

potential conversion of NMN into deamidated products. First, we reviewed the literature to

build the bacterial NAD pathways to identify functional contributions that are distinguished

from mammalian counterparts. We then generated metabolomic data from an in vitro model,

in which NMN was supplemented into the broth of cultured E. coli bacteria. The reconstructed

pathways and the metabolite data were then integrated to resolve NAD pathways that are active

in E. coli. Most importantly, the detection of labelled intermediates in the culture supernatant

upon supplementing labelled NMN indicated a putative role of the commensal gut microbiome in host NAD+ homeostasis. Following the work described in this chapter, an in vivo mouse

model is described in the next chapter to identify a host-microbiome integrated model of NAD+

homeostasis.

130

Chapter 4 Investigating microbial NAD+ metabolism

4.2 Methods

4.2.1 Bacterial culture and NMN treatment

A stab culture of the E. coli strain OP50 was inoculated into sterile Luria-Bertani (LB) broth

(consisting of 10 g/L tryptone, 5 g/L yeast extract and 10 g/L sodium chloride in deionized water) under aseptic conditions and incubated overnight at 37℃ on a shaking platform set at

200 rpm. This strain of E. coli (OP50) is a standard food source for growing the model nematode organism Caenorhabditis elegans (C. elegans), commonly used as a robust genetic

model for lifespan studies.

To measure the growth rate of E. coli, the overnight culture was sub-cultured (1:200)

into sterile LB broth in a new flask and the optical density was measured at 600 nm (OD600)

every 20 minutes (approximate doubling time) (Figure 4.1 A). This growth curve was used to

ensure samples were collected during the early-mid exponential growth phase (OD600 < 0.70),

as bacterial enzymes are more active during exponential growth phase than stationary phase

(Rahman et al. 2006). For samples, the overnight culture was sub-cultured (1:200) and

aliquoted into smaller volumes. The cultures were then supplemented with either vehicle

(water) or unlabelled NMN (0.5 mM) and collected at the following time points; time zero

(before NMN), time zero (immediately after NMN), 120, 160 and 180 minutes. The supernatant

of cells was separated from the cells via centrifugation (5000g for 10 minutes at 4℃) and stored immediately at -30℃. Meanwhile, the cell pellet was resuspended in cold (4°C) saline solution

(0.9% NaCl) and centrifuged as above, to rinse away residual media before storage at -30℃.

The OD600 (Figure 4.1 B) was measured for each sample and used to normalise metabolite

levels after LC-MS/MS analysis. In a separate experiment, the growth rate of E. coli (Figure

4.2 A) and OD600 from samples supplemented with M+6 labelled NMN1 (Figure 4.2 B) was

also measured at time zero (before NMN), time zero (after NMN), and 140, 160 and 180

minutes after supplementation with NMN1 at a final concentration of 0.1 mM. The reason for

131

Chapter 4 Investigating microbial NAD+ metabolism using isotope labelled NMN at a concentration of 0.1 mM, compared to initial experiments with unlabelled NMN at 0.5 mM, was the limited availability of isotope labelled material.

Figure 4.2 E. coli growth curve and sample OD600 values. (A) E. coli (OP50) bacteria incubated overnight was sub-cultured (1:200) in sterile LB broth. The OD600 was measured every 20 minutes to generate a growth curve. (B) E. coli culture broth was supplemented with 0.5 mM NMN and collected at time points 0 (before NMN), 0 (after NMN), 120, 160 and 180 minutes. OD600 was measured for samples at each collection time point. Data represents mean ± s.d. (n=3 technical replicates for control, 5 technical replicates for NMN, each sample OD600 read in triplicate).

132

Chapter 4 Investigating microbial NAD+ metabolism

Figure 4.3 E. coli growth curve and sample OD600 values. (A) E. coli (OP50) bacteria incubated overnight was diluted 1:200 with fresh LB broth. The OD600 was measured every 20 minutes to generate a growth curve. (B) E. coli culture broth was supplemented with 0.1 mM NMN1 and collected at time points 0 (before NMN1), 0 (after NMN1), 140, 160 and 180 minutes. OD600 was measured for samples at each collection time point. Data represents mean ± s.d. (n=1 technical replicates for control, 3 technical replicates for NMN1, each sample OD600 read in triplicate).

133

Chapter 4 Investigating microbial NAD+ metabolism

4.2.2 Extracting NAD+ metabolites from E. coli cell lysates and culture supernatants.

To separate the supernatant from cells, samples were immediately centrifuged at 5000 g for 10 minutes at 4℃. Samples were collected at 0, 120, 160 and 180 minutes following supplementation with unlabelled NMN. These time points represent the early-mid exponential phase of growth, determined by the first, second and third doubling time points measured by

OD600. When treating with labelled NMN1, samples were collected at time points 0 (before

and after the addition of NMN1), 140, 160 and 180 minutes. Following centrifugation, the

supernatant was collected and stored at -30℃ until ready for further processing. The remaining

cell pellet (cell lysate) was rinsed once with a cold saline (0.9% NaCl) solution to remove

residual media by centrifugation at 5000 g for 10 minutes at 4℃. The supernatant was

discarded, and the final pellet was snap frozen in liquid nitrogen and stored at -30℃ until ready

for further processing.

To extract NAD+ metabolites from cell lysates, pellets were resuspended in an extraction buffer (500 µL of a 2:2:1 mixture of acetonitrile, methanol and water at -30℃) by

repeated pipetting. These resuspended cells were centrifuged at 16,000 g for 10 minutes at 4℃

and supernatant, containing the extracted NAD+ metabolites, was transferred into a new tube.

Using a centrifugal vacuum system (Savant SpeedVac SPD140DDA Vacuum Concentrator,

Thermo Fisher Scientific), in the solution was evaporated to yield a dried pellet, which was

resuspended in LC-MS-grade water (50 µL, Fisher Healthcare, cat.no. PI51140) before

centrifugation at 16,000g for 10 minutes at 4℃. The supernatant was transferred into HPLC

vials and promptly analysed by LC-MS/MS (see Chapter 2 Section 2.3 LC-MS/MS). To extract

NAD+ metabolites from the supernatant, 30 µL of media was combined with 120 µL of

extraction buffer, consisting of a 1:1 ratio of acetonitrile and ethanol (HPLC grade) before

centrifugation at 16,000g for 10 minutes at 4℃. The supernatant was transferred to a new tube,

snap frozen in liquid nitrogen and stored at -30℃ before further processing. The supernatant

134

Chapter 4 Investigating microbial NAD+ metabolism

was dried down as above using the speed vac system and the dried pellet was resuspended in

30 µL of LC-MS-grade water, centrifuged as above and transferred into HPLC vials for LC-

MS/MS analysis. The internal standards, CSA, MES and thymine-d4 (see Chapter 2, Section

2.2.1 Preparation of extraction buffer) were added to each respective extraction buffer prior to extraction. The internal standard thymine-d4 was used for normalisation of peak intensities before normalising samples to the OD600 value.

Table 4.1 (below) details mass spectrometry (MS) parameters including DP, CE, CXP

and RT, and MRM transitions used to select and identify NAD+ metabolites. As this experiment

was repeated and analysed as two separate batch experiments (Run 1 and Run 2), the MS

parameters are shown for each LC-MS analysis. The first batch (Run 1) included one control

(water) and three NMN (0.5 mM) treated samples whereas the second batch (Run 2) included

two control and two NMN treated samples. Overall, the data represent three control and five

NMN treated samples. Similarly, Table 4.2 and Table 4.3 (below) shows the MS parameters

and MRM transitions used to select and identify NAD+ isotopologues from amidated (Table

4.2) and deamidated (Table 4.3) metabolites which have incorporated isotopes from labelled

ribose and nicotinamide moieties following supplementation with NMN1 (0.1 mM) and

includes one control and three NMN1 treated samples. The MS parameters, DP, CE and CXP

remained the same throughout all experiments and the slight changes in retention times are

likely due to differences in laboratory conditions and LC-column status.

135

Chapter 4 Microbial NAD+ metabolism

Table 4.1 MRM transitions and mass spectrometry (MS) parameters to detect NAD+ metabolites in E. coli (supernatant and lysates) following supplementation with unlabelled NMN using LC-MS/MS. Run 1 included one control and three NMN treated samples whereas Run 2 included two control and two NMN treated samples.

Run 1 MRM MS parameters Supernatant Lysate Metabolite Q1 Q3 DP CE CXP RT RT Isotopologue Q1_Q3 label (m/z) (m/z) (V) (V) (V) (mins) (mins)

NAM_0_0 NAM (M+0) 122.8 79.8 80.0 30.0 25.0 2.0 2.0 NMN_0_0 NMN (M+0) 335.2 123.1 47.7 24.0 11.0 18.1 18.1 NR_0_0 NR (M+0) 255.0 123.0 64.3 29.6 12.6 11.2 11.2 NAD+_0_0 NAD+ (M+0) 664.0 428.1 33.0 36.0 31.0 16.9 16.9 NA_0_0 NA (M+0) 123.8 77.8 70.0 25.0 10.0 4.2 4.4 NaR_0_0 NaR (M+0) 256.0 124.0 46.0 18.3 13.3 10.1 10.1 NaMN_0_0 NaMN (M+0) 336.1 123.8 66.0 27.7 10.7 17.8 17.8 NaAD_0_0 NaAD (M+0) 665.0 428.1 139.0 35.0 38.0 17.2 17.2 Run 2 MRM MS parameters Supernatant Lysate Metabolite Q1 Q3 DP CE CXP RT RT Isotopologue Q1_Q3 label (m/z) (m/z) (V) (V) (V) (mins) (mins) NAM_0_0 NAM (M+0) 122.8 79.8 80.0 30.0 25.0 2.0 2.0 NMN_0_0 NMN (M+0) 335.2 123.1 47.7 24.0 11.0 18.1 18.4 NR_0_0 NR (M+0) 255.0 123.0 64.3 29.6 12.6 11.2 11.2 NAD+_0_0 NAD+ (M+0) 664.0 428.1 33.0 36.0 31.0 16.9 16.9 NA_0_0 NA (M+0) 123.8 77.8 70.0 25.0 10.0 4.6 4.4 NaR_0_0 NaR (M+0) 256.0 124.0 46.0 18.3 13.3 10.1 10.1 NaMN_0_0 NaMN (M+0) 336.1 123.8 66.0 27.7 10.7 17.8 17.6 NaAD_0_0 NaAD (M+0) 665.0 428.1 139.0 35.0 38.0 17.2 17.2

136

Chapter 4 Microbial NAD+ metabolism

Table 4.2 MRM transitions and MS parameters to detect NAD+ isotopologues of amidated metabolites in E. coli (supernatant and lysates) following supplementation with NMN1 using LC-MS/MS.

137

Chapter 4 Investigating microbial NAD+ metabolism

Table 4.3 MRM transitions and MS parameters to detect NAD+ isotopologues of deamidated metabolites in E. coli (supernatant and lysates) following supplementation with NMN1 using LC-MS/MS.

138

Chapter 4 Investigating microbial NAD+ metabolism

4.3 Results

4.3.1 Supplementation of NMN in E. coli – changes to NAD+ metabolites.

To investigate the incorporation of NMN into the NAD+ metabolome in bacteria, E. coli were cultured in the presence of labelled and unlabelled NMN. Exogenous supplementation of NMN in E. coli led to changes in both amidated (NMN, NR, NAM and NAD+) and deamidated (NA,

NaR, NaMN and NaAD) metabolites, as expected. This was further supported by detecting the incorporation of labelled ribose (13C) and nicotinamide (15N) moieties into both the cell lysate and supernatant following supplementation with M+6 labelled NMN1 (Figure 4.5 and Figure

4.7). A breakdown of these findings will be discussed in detail in the following paragraphs.

NMN in the supernatant was detected in abundance immediately following its addition

(Figure 4.4 A), but rapidly diminished at subsequent time points indicating almost instant assimilation (Figure 4.4 B) and subsequent conversion into downstream metabolites during the exponential growth phase. This was confirmed by detection of M+6 labelled NMN in both the supernatant (Figure 4.5 A) and cell lysate (Figure 4.5 B), with a similar pattern of detection across the time points following supplementation with NMN1.

NMN can also enter the periplasmic space through the OmpP2 porin (channel-forming protein) (Andersen et al. 2003), where it undergoes dephosphorylation via the periplasmic

5’nucleotidase UshA (Burns and Beacham 1986; Wang et al. 2014b) to form NR. Consistent with this, NR was detected in the supernatant at all time points (Figure 4.4 C) which was confirmed through labelling at both the ribose and nicotinamide moieties (Figure 4.5 C). As

UshA is a periplasmic enzyme, this may be the result of contamination of periplasmic metabolites such as NR during the separation of the supernatant from cell lysate via centrifugation. NR may then betransported across the inner cytoplasmic membrane via PnuC

(Jaehme, Guskov, and Slotboom 2014), where it is re-phosphorylated by NadR in the cytoplasm to generate NMN. Interestingly, very little NR could be detected in the cell lysate

139

Chapter 4 Investigating microbial NAD+ metabolism

(Figure 4.4 D), including both labelled and un-labelled versions (Figure 4.5 D). This could be attributed to the high catalytic efficiency of NadR reported in previous studies (Kurnasov et al.

2002), preventing NR efflux back through PnuC and trapping NMN inside the cell to encourage

NAD+ biosynthesis (Grose, Bergthorsson, and Roth 2005).

In addition to detecting NR in the supernatant, an abundance of NAM was also observed

in the supernatant (Figure 4.4 E), but very little was detected in the cell lysate (Figure 4.4 F).

This was confirmed again through M+1 label at the pyridine ring base of NAM in Figure 4.5

E (supernatant) and Figure 4.5 F (cell lysate). NAM may enter the cytoplasm through niacin

transporters NiaX (Johnson et al. 2015) and NAM can be generated from the degradation of

NMN by NMN glycohydrolase embedded in the inner membrane. Once in the cytosol, NAM

can serve as the primary precursor for NA through its deamidation via PncA.

Consistent with this, treatment with unlabelled NMN increased NA in cell lysates

(Figure 4.6 B), and this was confirmed by the incorporation of labelled NMN into M+1

labelled NA (Figure 4.7 B). Interestingly, a small amount of NA was also detected in the

supernatant (Figure 4.6 A and Figure 4.7 A for labelled NA) almost immediately after

supplementation with NMN. This could suggest that some NA in the cell lysate permeates out

of the inner and outer membrane and into the supernatant, where in the context of an integrated

host-microbiome model of NAD homeostasis, it could be available as a precursor for cellular

uptake in mammals.

NaMN in the cell lysate was detected following supplementation with unlabelled NMN

(Figure 4.6 F) and confirmed through M+6 labelled NaMN following supplementation with

labelled NMN1 (Figure 4.7 F). NaMN is a substrate for NaAD synthesis via NadD, however

no NaAD was detected in the cell lysate (Figure 4.6 H) following supplementation with NMN,

even following supplementation with labelled NMN1 (Figure 4.7 H). One possibility for this

140

Chapter 4 Investigating microbial NAD+ metabolism

is that NaAD is rapidly assimilated into NAD+ during the exponential growth phase of bacteria, circumventing its detection. Nonetheless, due to the loss of 15N label by deamidation, the incorporation of NMN into NAD via NaAD can be inferred based on the detection of M+6 labelled NAD+ (Figure 4.5 H) in the cell lysate following NMN1 supplementation.

Both NAD+ (Figure 4.4 G) and NaAD (Figure 4.6 G) were largely absent from the

supernatant, which was expected due to the absence of dedicated transporters and the large- highly charged structures that are impermeable to membrane diffusion. This was mirrored in cells treated with labelled NMN1, as M+6 labelled NAD+ in the supernatant (Figure 4.5 G)

and NaAD (Figure 4.7 G) was not detected. In contrast, NaR, a molecule which was not

expected to be present in the extracellular space, was detected in the supernatant (Figure 4.6

C) and largely absent in the cell lysate (Figure 4.6 D). In human cells, while NaR may be

generated from the dephosphorylation of NaMN via cytosolic nucleotidases II (CN-II) and III

(CN-III) (Kulikova et al. 2015; Nikiforov et al. 2011), NaR was detected exclusively in the

supernatant, not in the cell lysate in this study, for reasons that are puzzling. Even if there were

bacterial homologues for the CNII and CNIII found in humans, bacterial 5’-nucleotidases

found in the cytosol seem to be conditionally lethal (Innes, Beacham, and Burns 2001). Further,

whether there is a specific mechanism for NaR to be transported from the cytosol into the

extracellular compartment remains unclear. As a nucleoside, NaR may be transported across

the plasma membrane through the equilibrative nucleoside transporters (ENTs) SLC29A1 and

SLC29A2 (Baldwin et al. 2004; Kulikova et al. 2015), however transport of NaR across the

plasma membrane through ENTs is reportedly less efficient than the transport of NR, the

amidated counterpart of NaR (Belenky et al. 2009). To test whether the presence of NaR in

the supernatant is due to increased transport of this metabolite across the plasma membrane

through ENTs, the pharmacological inhibitors of nucleoside transporters, S-(4-nitrobenzyl)-6-

141

Chapter 4 Investigating microbial NAD+ metabolism thioinosine (NBTI) and dipyridamole (Dip) may be used to further elucidate on these mechanisms in future studies (Kulikova et al. 2019; Nikiforov et al. 2011).

NaMN was also detected in the supernatant of cells (Figure 4.6 E), however enzymes that generate NaMN including the NMN deamidase PncC, the NA phosphoribosyltransferase

PncB, and NaR kinase are all located in the cytosol. Further, NaMN is not among the metabolites known to freely permeate the plasma membrane, nor has a specific transporter been identified for NaMN-specific efflux from the cell in any species. Further investigation is required to determine how NMN treatment can lead to the formation of NaMN in the supernatant in bacteria. One possibility is that they are released into the supernatant following their synthesis to facilitate NAD+ biosynthesis in neighbouring cells, a mechanism which has been previously reported in HEK293 (human embryonic kidney) and HepG2 (hepatocellular carcinoma) cell lines (Kulikova et al. 2015). In the context of a putative microbiome-host interaction, this may support the growth of bacterial populations in the GIT and/or serve as alternative precursors for uptake and utilisation by host cells.

142

Chapter 4 Investigating microbial NAD+ metabolism

Figure 4.4 Detection of amidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN. E. coli bacteria grown in suspension were supplemented with unlabelled NMN (0.5 mM). Whole cultures (supernatant + cells) were collected at time point, 0 (before NMN), 0 (after NMN), 120, 160 and 180 minutes after NMN supplementation. Following the separation of the supernatant from the cell lysate, NAD+ metabolites were extracted and detected using LC-MS/MS. Data represent mean ± s.d (n=3-5). (A) NMN, (C) NR, (E) NAM and (G) NAD+ in the supernatant. (B) NMN (D) NR, (F) NAM and (H) NAD+ in the cell lysate.

143

Chapter 4 Investigating microbial NAD+ metabolism

Figure 4.5 Detection of amidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN1. E. coli bacteria grown in suspension were supplemented with M+6 labelled NMN1 (0.1 mM). Whole cultures (supernatant and cells) were collected at time points, 0 (before NMN1), 0 (after NMN1), 140, 160 and 180 minutes after NMN1 supplementation. Following separation of the supernatant from the cell lysate, NAD+ metabolites were extracted and detected using LC-MS/MS. Data represent mean ± s.d (n=3-5). (A) NMN (M+6), (C) NR (M+6), (E) NAM (M+1) and (G) NAD+ (M+6) in the supernatant. (B) NMN (M+6) (D) NR (M+6), (F) NAM (M+1) and (H) NAD+ (M+6) in the cell lysate.

144

Chapter 4 Investigating microbial NAD+ metabolism

Figure 4.6 Detection of deamidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN. E. coli bacteria grown in suspension were supplemented with unlabelled NMN (0.5 mM). Whole cultures (supernatant and cells) were collected at time points, 0 (before NMN), 0 (after NMN), 120, 160 and 180 minutes after NMN. Following separation of the supernatant from the cell lysate, NAD+ metabolites were extracted and detected using LC-MS/MS. Data represent mean ± s.d (n=3-5). (A) NA, (C) NaR, (E) NaMN and (G) NaAD in the supernatant. (B) NA, (D) NaR, (F) NaMN and (H) NaAD in the cell lysate.

145

Chapter 4 Investigating microbial NAD+ metabolism

Figure 4.7 Detection of deamidated NAD+ metabolites in the supernatant and cell lysate from E. coli following supplementation with NMN1. E. coli bacteria grown in suspension were supplemented with M+6 labelled NMN1 (0.1 mM). Whole cultures (supernatant and cells) were collected at time points, 0 (before NMN1), 0 (after NMN1), 140, 160 and 180 minutes after NMN1. Following separation of the supernatant from the cell lysate, NAD+ metabolites were extracted and detected using LC- MS/MS. Data represent mean ± s.d (n=3-5). (A) NA (M+1), (C) NaR (M+6), (E) NaMN (M+6) and (G) NaAD (M+6) in the supernatant. (B) NA (M+1), (D) NaR (M+6), (F) NaMN (M+6) and (H) NaAD (M+6) in the cell lysate.

146

Chapter 4 Investigating microbial NAD+ metabolism

4.4 Discussion

NAD+ plays a critical role as a redox cofactor in metabolic reactions that are conserved across

bacteria and higher eukaryotes (Gazzaniga et al. 2009). As such, NAD+ precursors

administered through the oral route could benefit both the microbiome and the mammalian

host. This study therefore sought to shed light on pathways that may be active in the

microbiome, and could contribute to an integrated model of NAD+ homeostasis. Trammell et

al. (2016) reported the detection of NaMN and NaAD in mammals following the administration

of NR, despite the absence of a canonical pathway for NR deamidation in mammals. Our

results suggest that contributions from the gut microbiome in deamidating NMN could

contribute to this striking phenomenon. Based on our results, we proposed that NR is

phosphorylated into NMN and then deamidated into NaMN (Galeazzi et al. 2011; Sorci et al.

2014), which is converted into NaAD and then NAD+ (Preiss and Handler 1958a, 1958b).

We detected NaMN in the cell lysate following treatment with NMN, though could not

detect NaAD. This indicated that NMN deamidation was active, presumably by the bacterial

NMN deamidase PncC. Despite the inability to detect NaAD as an intermediate, the production

of M+6 labelled NAD+ at later timepoints confirmed that NMN did undergo incorporation via

deamidation. Our inference draws upon previous work that in bacteria, NAD+ must be synthesised from NaAD; further work would be required to validate these findings. The direct utilisation of NMN into NAD+ via NadR is unlikely, although may explain the absence of

NaAD in the cell lysate. NadR has both NR kinase as well as NMN adenylyltransferase

(NMNAT) activity, however previous studies show that the latter activity is weak and physiologically irrelevant (Grose, Bergthorsson, and Roth 2005). The fact that NMN utilisation

in bacteria has been reported to occur mostly through its deamidation into NaMN, rather than

through the direct synthesis of NAD+, supports this (Kurnasov et al. 2002). Another possible

explanation for the absence of NaAD in the cell lysate could be of its rapid assimilation into

147

Chapter 4 Investigating microbial NAD+ metabolism

NAD+ by the enzyme NadE, given abundant availability of ammonia as a nitrogen donor for

NaAD amidation into NAD (Willison and Tissot 1994).

The labelling of the amidated metabolites NMN, NR, and NAM as well as the deamidated metabolites, NA, NaR and NaMN contributed to resolving how NMN is taken up and utilised in E. coli. (Figure 4.1). The detection of a substantial pool of NR in the supernatant strongly suggests NMN was rapidly dephosphorylated to NR by the 5’-nucelotidase UshA

(Burns and Beacham 1986) in the periplasmic space, following which it can then enter the cytoplasm through the nucleoside transporter PnuC (Jaehme, Guskov, and Slotboom 2014).

Labelled NMN and NR in the cell lysate were detected immediately after supplementation with

NMN1, and both rapidly diminished at later time points (Figure 4.4 A and C, Figure 4.5 A and C). This suggests they were rapidly assimilated into the NAD metabolome.

Our results then suggest a potential bifurcation at NMN. The first is a direct intracellular route involving rapid conversion of NR-derived NMN to NaMN; this is consistent with the high catalytic efficiency (Kcat/Km) of NadR and PncC (Galeazzi et al. 2011; Kurnasov et al.

2002). The second extracellular route involves NMN hydrolysed into NAM by NMN glycohydrolase, an enzyme embedded in the inner membrane facing the periplasmic side

(Andreoli et al. 1972), followed by deamidation of NAM into NA by PncA (Frothingham et al.

1996). This was inferred based on the detection of labelled NAM and NA in the supernatant.

NAM diffuses across the inner membrane, and NA is actively transported via NiaP (Jaehme and Slotboom 2015; Jeanguenin et al. 2012), before both finally converging at NaMN via PncB

(Wubbolts et al. 1990).

The detection of NaR and NaMN in the supernatant was unexpected. It will be important to follow these results up to determine whether this is an experimental artefact from necrotic and lysed cells or if it represents the true transport of these metabolites from bacteria.

148

Chapter 4 Investigating microbial NAD+ metabolism

The NMN deamidase PncC is located in the cytoplasm, and there is no identified transporter

for NaMN. NaR in the supernatant could be generated from the dephosphorylation of NaMN

by 5’-nucleotidases in the cytosol, which have been identified in yeast as Isn1 and Std1 (Bogan et al. 2009) and humans cells as CNII and CNIII (Kulikova et al. 2015), though homologues

have not been identified in bacteria. It is not known whether the 5’-nucleotidase UshA,

responsible for the dephosphorylation of NMN to NR, can also dephosphorylate NaMN to

NaR, or whether any other 5’-nucleotidase is responsible for dephosphorylating NaMN in

bacteria, which may be located in different cell compartments (Bogan and Brenner 2010).

Importantly, the absence of NaR in cell lysates is consistent with the fact that 5’-nucleotidase activity in the cytoplasm is lethal (Innes, Beacham, and Burns 2001) and that NaR is also poorly imported (Belenky et al. 2009).

In summary, data from this study largely support canonical routes of NMN uptake and utilisation in bacteria through both dephosphorylation into NR and hydrolysis to release NAM.

The generation of the deamidated metabolite NaMN was also observed, supporting the hypothesis that bacterial enzymes in the GIT may be contributing to the metabolism of orally administered NAD+ precursors such as NMN. Although the deamidated metabolite NaAD was not detected in bacteria, as previously observed in mammalian blood following the administration of NR by Trammell and colleagues (Trammell et al. 2016a), other deamidated

NAD+ precursors such as NA, NaR and NaMN were also detected in the supernatant following

supplementation with NMN in bacteria, suggesting they may also serve as extracellular

precursors to neighbouring or host cells in the GIT.

Study Limitations

The findings from this study relied on the detection of NAD+ metabolites to infer enzymatic

routes of NMN uptake and utilisation in E. coli bacteria, specifically via dephosphorylation to

149

Chapter 4 Investigating microbial NAD+ metabolism

NR as well as hydrolysis to NAM. Measuring the enzymatic activity and/or expression of these

proteins would provide important insights into the interpretation of these metabolomics data,

and future work should aim to incorporate this (Burns, Mendz, and Hazell 1998). Detection of

metabolites in this study was mainly supported and confirmed by the incorporation of labelled

ribose and nicotinamide moieties from labelled NMN isotope, NMN1, which is labelled at five

carbons of the ribose (13C) and at the base nitrogen of the pyridine ring of the nicotinamide

moiety (15C). This is an important tool which has not been used to study the bacterial

metabolism of exogenously NMN, which was an advantage of this study.

Other than the possibility that periplasmic metabolites contaminated the supernatant

during sample processing, detection of NR in the supernatant may also suggest that dephosphorylation could be occurring outside the cell. The only known 5’-nucleotidase in

bacteria, UshA, however, is located in the periplasmic compartment, and not on the outer

membrane of E. coli. An alternative explanation for these data could be that NMN undergoes modification as a result of constituents present in growth broth, rather than through the action of bacterial enzymes. Future studies should include measurements for the stability of labelled

NMN in cell-free broth to exclude this possibility. There is some precedence for the concept that culture media could degrade or alter NMN. A recent study (Kulikova et al. 2019) measured

the impact of culture conditions on the chemical and enzymatic stability of NAD precursors,

showing that the presence of 10% fetal bovine serum (FBS) in mammalian cell culture media

resulted in nucleotide pyrophosphatase and 5’-nucleotidase activity, which degraded several

NAD+ metabolites, including the dephosphorylation of NMN, however the presence of FBS in

mammalian tissue culture is a very different scenario to the bacterial culture described here.

While not investigated in this study, it is a possibility, that NMN is deamidated and/or

dephosphorylated through non-enzymatic means including by chemical degradation from

compounds present in the growth media. The culture medium, Luria-Bertani (LB) broth used

150

Chapter 4 Investigating microbial NAD+ metabolism

to grow E. coli cells in this study was prepared with standard ingredients; 10 g/L tryptone, 5 g/L yeast extract and 10 g/L sodium chloride in deionized water. While it is possible that enzymes present in yeast extract could metabolise NMN, this broth undergoes autoclaving prior to use, and this heating would be expected to degrade any NAD consuming enzymes.

Regardless, future studies should incorporate a cell-free control group supplemented with

NMN to confirm this.

Another limitation of this study is the use of a single strain of a single bacterial species, in this case the OP50 strain of E. coli. This strain was chosen due to its immediate availability and ease of use. This species encodes enzymes involved in the de novo (NadD and NadE), salvage (NadR) and deamidation (PncB and PncC) pathways of NAD+ biosynthesis , and in

this study was considered to be a generic model organism for bacterial NAD metabolism. In

contrast, in the mammalian gut microbiome there are over 1000 species of bacteria and E. coli has been reported to account for only a small proportion (0.1%-2.1%) of the microbial population in the GIT, which is mainly dominated by Bacteroidetes and Firmicutes, which

together account for over 50-60% of the gut microbiome (D’Argenio and Salvatore 2015;

Huttenhower et al. 2012; Lynch and Pedersen 2016; Segata et al. 2012). Exploring the

differences in NMN uptake and utilisation pathways between different bacterial strains may

elucidate further interactions between cells and between species, which will be important to

understand in the context of stark changes in bacterial diversity based on various health and

disease conditions, including ageing (Biagi et al. 2010; D’Argenio and Salvatore 2015; Le

Chatelier et al. 2013; Ley et al. 2006; Mariat et al. 2009; Qin et al. 2012; Tilg and Kaser 2011;

Yatsunenko et al. 2012). These changes could in turn influence the bioavailability of NAD+

precursors to the host.

151

Chapter 5 Host-microbiome interactions in NMN metabolism

Host-microbiome interaction in NMN metabolism

5.1 Introduction

Nicotinamide adenine dinucleotide (NAD+) is an essential redox cofactor central to processes

involved in energy metabolism such as glycolysis, the TCA cycle and fatty acid oxidation

(Braidy et al. 2018; Kulkarni and Brookes 2019). It is also a key substrate consumed by NAD+-

dependent enzymes such as the sirtuins (Imai et al. 2000b) and poly(ADP-ribose) polymerases

(PARPs) (Chambon, Weill, and Mandel 1963) which are important epigenetic regulators of

gene expression and genome stability (Haigis and Sinclair 2010) and DNA repair (de Murcia

and de Murcia 1994). Given the essential role of this metabolite to cell survival, the decline in

NAD+ that occurs during biological ageing (Braidy et al. 2011a; Camacho-Pereira et al. 2016;

Clement et al. 2019; Gomes et al. 2013; Massudi et al. 2012b) and in various disease states

(Frederick et al. 2016; Zhou et al. 2016) is considered to be a potential cause of declining health. Boosting levels of NAD+ through pharmacological supplementation with NAD+

152

Chapter 5 Host-microbiome interactions in NMN metabolism precursors has gained attention as a promising strategy for therapeutic intervention (Aman et

al. 2018; Connell, Houtkooper, and Schrauwen 2019; Madeo et al. 2019; Mouchiroud,

Houtkooper, and Auwerx 2013; Rajman, Chwalek, and Sinclair 2018; Sultani et al. 2017;

Yoshino, Baur, and Imai 2018).

Boosting NAD+ levels through supplementation with NAD+ precursors such as nicotinamide mononucleotide (NMN) and nicotinamide riboside (NR) are emerging as promising strategies to combat various pathological conditions (Belenky et al. 2007; Cantó et al. 2012; Caton et al. 2011; Chi and Sauve 2013; Das et al. 2018; de Picciotto et al. 2016;

Diguet et al. 2018; Gong et al. 2013; Johnson, Wozniak, and Imai 2018; Lu et al. 2014a; Mills et al. 2016; Park et al. 2016; Pham et al. 2019; Tarantini et al. 2019; Trammell et al. 2016c;

Uddin et al. 2017; Wang et al. 2016b; Wei et al. 2017; Yamamoto et al. 2014b; Zhang et al.

2017). In comparison to other well-known NAD+ precursors such as nicotinic acid (NA) and

nicotinamide (NAM), collectively termed niacin, the use of NMN and NR has several

advantages. Unlike NA, the use of NMN and NR does not lead to uncomfortable flushing in

patients which is caused by the activation of the G-protein-coupled receptor, GPR109A (Benyó et al. 2005; Tunaru et al. 2003). At high doses, NAM can inhibit the sirtuins, a family of NAD+-

dependent histone and protein deacetylase enzymes involved in the regulation of gene

expression through silencing (Avalos, Bever, and Wolberger 2005; Bitterman et al. 2002), though NMN and NR do not display this effect.

Historically, niacin was used to treat pellagra, a disease prevalent in malnourished populations, caused by a chronic deficiency in dietary vitamin B3 and/or tryptophan, which are precursors for NAD+ synthesis (Alport, Ghalioungui, and Hanna 1938; Fouts et al. 1937;

Goldsmith et al. 1952; Spies, Cooper, and Blankenhorn 1938). NA has also been used to treat individuals with dyslipidaemia and hypercholesterolemia (Achor et al. 1958; Altschul, Hoffer, and Stephen 1955) but high doses lead to painful flushing side effects, which in some cases

153

Chapter 5 Host-microbiome interactions in NMN metabolism manifest as a deterioration in glycaemic and plasma uric acid control, leading to hyperglycaemia and hyperuricaemia, further limiting its therapeutic application (Benyó et al.

2005; Garg and Grundy 1990). The enzymatic synthesis of NAD+ from NAM involves the addition of ribose-5-phosphate from phosphoribose pyrophosphatase by the enzyme nicotinamide phosphoribosyltransferase (NAMPT) to form NMN, in a reaction which is considered the rate-limiting step in the salvage pathway (Revollo, Grimm, and Imai 2004). The enzyme nicotinamide mononucleotide adenylyltransferase (NMNAT1-3) converts NMN into

NAD+ in a reaction that uses ATP as a donor for AMP, yielding free pyrophosphate (Berger et al. 2005; Garavaglia et al. 2002; Schweiger et al. 2001; Zhang et al. 2003), while NAD+ synthesis from NR involves its phosphorylation into NMN via nicotinamide riboside kinase

(NRK1/2) (Tempel et al. 2007) prior to acting as a subsrate for NMNAT1-3. As such, NMN and NR are ideal candidates for raising NAD+ levels in a manner which is, to our knowledge, relatively safe and absent of any harmful and or/undesirable side effects, bypassing the rate- limiting step of NAD+ biosynthesis in the salvage pathway (Airhart et al. 2017b; Conze,

Brenner, and Kruger 2019; Dellinger et al. 2017; Irie et al. 2019; Martens et al. 2018; Mills et al. 2016).

When considering the translatability of NAD+ precursors in a clinical setting, it is important to understand of mechanisms of uptake and utilisation in the cell. The route of administration significantly impacts these mechanisms and influences the efficacy of treatment. A recent study by Liu and colleagues compared the metabolic fate of NMN when delivered intravenously versus orally, and found only intravenously delivered NMN remained intact in tissues, while the oral route resulted in breakdown into NAM in the liver (Liu et al.

2018). For orally delivered compounds to have an effective systemic effect, they must survival the gastrointestinal environment, where bacterial populations thrive and overcome the ‘first pass effect’, which refers to the reduction in drug concentration at the target site due to initial

154

Chapter 5 Host-microbiome interactions in NMN metabolism metabolism at a specific location in the body (Herman and Santos 2019). The first pass effect is often associated with the liver, a major site of drug metabolising enzymes such as cytochrome

P450 (CYP450) (Anzenbacher and Anzenbacherova 2001), but the microbiome in the gastrointestinal environment is also a major contributor to xenobiotic metabolism (Clarke et al.

2019; Collins and Patterson 2019; Spanogiannopoulos et al. 2016; Wilson and Nicholson

2017). Whether the microbiome plays a role in the metabolism of NAD+ precursors is not well understood, and may be important for future therapeutic applications.

The requirement for NAD+ as a co-factor is conserved across evolution, however the steps to NAD biosynthesis vary between kingdoms. In bacteria, NAD+ synthesis includes enzymatic steps that are, to our current knowledge, absent in mammalian NAD+ metabolism.

Bacteria use the NMN deamidase PncC to catalyse the conversion of NMN to its acidic form, nicotinic acid mononucleotide (NaMN) (Galeazzi et al. 2011; Sorci et al. 2014), a step which is not present in mammals. Bacteria then convert NaMN to nicotinic acid adenine dinucleotide

(NaAD) through the NaMN adenylyltransferase NadD (Mehl, Kinsland, and Begley 2000;

Preiss and Handler 1958b), which is then amidated via the NAD synthetase, NadE (Spencer and Preiss 1967; Willison and Tissot 1994), utilising ammonia as a nitrogen source for the final step in NAD+ synthesis, instead of glutamine as in mammals. Interestingly, a recent study by

Trammell and colleagues revealed the striking appearance of NaAD following oral administration of NR, the direct precursor to NMN, in mice and in a human volunteer

(Trammell et al. 2016a). Given that canonical pathways of NAD+ biosynthesis in mammals do not have a step that would allow NR to feed into the synthesis of NaAD, this finding is surprising. One option would be the existence of a mammalian NMN deamidase, however this has been controversial (Magni et al. 2008; Rongvaux et al. 2003; Sarma, Rajalakshmi, and

Sarma 1961). An alternative explanation is that the appearance of NaAD from NR could potentially occur through engaging bacterial deamidase enzymes such as PncC in the gut. NR

155

Chapter 5 Host-microbiome interactions in NMN metabolism can be phosphorylated to NMN via NR kinase enzymes (NRK1/2) (Tempel et al. 2007), and

one hypothesis is that NMN could then undergo deamidation to NaMN by the bacterial enzyme

PncC through interactions with the gut microbiome. These bacterial enzymes could facilitate

host NAD+ metabolism, or conversely, limit the availability of NAD+ precursors to the host through direct consumption. Given the highly conserved requirement for NAD+ in metabolism across evolution (Gazzaniga et al. 2009), it is conceivable that microbial species in the gut may consume and/or metabolise NAD+ precursors obtained from dietary sources, or when provided by exogenous supplements. Another alternative explanation could be that NAD+ undergoes

deamidation to NaAD, however the amidation of NaAD into NAD+ by NAD synthetase is, to

our knowledge, an irreversible reaction and for this to proceed in the opposite direction would

be thermodynamically unfavourable. We therefore propose the deamidation of NMN as a

reasonable hypothesis to explain the initial observation by Trammell and colleagues and

further, examine the role of the microbiome in NMN metabolism.

In addition to the role of the microbiome in NAD+ precursor metabolism, another key

question in the field is the mechanism of NMN transport into cells. There are two proposed

mechanisms, which will be referred to in this study as ‘direct’ or ‘indirect’ mechanisms of

NMN transport (Figure 5.12). The indirect mechanism involves the extracellular

dephosphorylation of NMN to NR by the ecto-5’-nucleotidase, cluster of differentiation 73

(CD73) (Grozio et al. 2013). Following dephosphorylation of NMN, NR enters the cell via

SLC29A1 and SLC29A2, which are the primary members of a family of equilibrative

nucleoside transporters (ENT1-3) selective for purine and pyrimidine nucleosides, some

nucleobases and adenosine (Baldwin et al. 2004; Young et al. 2008). Once transported into the

cell, NR kinases (NRK1/2) phosphorylate NR into NMN (Bieganowski and Brenner 2004;

Ratajczak et al. 2016), which is used by NMNAT enzymes as a substrate for NAD+ synthesis

(Berger et al. 2005; Garavaglia et al. 2002; Schweiger et al. 2001; Zhang et al. 2002). In

156

Chapter 5 Host-microbiome interactions in NMN metabolism contrast, a competing theory suggests that NMN undergoes direct, intact transport via a

dedicated NMN transporter which is highly expressed in the gut epithelium (Grozio et al.

2019a) where NAD+ is synthesised in one enzymatic step by the enzymes NMNAT1-3. In addition to addressing our hypothesis of NMN deamidation via bacterial enzymes in the gut, this study allowed us to also gain insights into which of these two mechanisms is more dominant, particularly in intestinal tissue where the putative NMN transporter is highly expressed (Grozio et al. 2019a). To address this, we paired the oral administration of strategically labelled NMN isotopes to ablation of the gut microbiome through antibiotic treatment to investigate the role of the microbiome in NMN metabolism. By tracing the incorporation of labelled nicotinamide and ribose moieties of NMN, we were able to investigate our hypotheses surrounding the contribution of the gut microbiome to the metabolism of orally administered NMN compounds and the mechanism of transport of NMN into cells. In these investigations, our emphasis was on the intestinal tissue, as our questions pertained to the contributions of the microbiome and the role of NMN transporters in the gut.

5.1.1 Rationale for NMN isotope design

In order to assess whether the gut microbiome contributes to the metabolism of orally delivered

NMN, isotope tracing was used to examine its routes of assimilation and in particular, to differentiate exogenously supplemented material from endogenous species of NAD+

metabolites that were already present in each sample. A key aspect was the strategic design of

NMN isotopes that would answer specific questions regarding the deamidation and

assimilation of NMN into the cell. To investigate this, two separate NMN isotopes were

designed that would provide complementary evidence for our hypotheses.

The first of these isotopes, designated as NMN1, was labelled with 13C-carbon at all

five carbon positions of the ribose, as well as with 15N-labelled at the pyridinyl ring of the

157

Chapter 5 Host-microbiome interactions in NMN metabolism nicotinamide moiety, for an overall mass shift of M+6 (Figure 5.1 A). This isotope was co-

administered with 15N-amide-labelled glutamine (Figure 5.1 B).

Figure 5.1 Labelled isotope compounds 13 15 (A) M+6 labelled NMN1; labelled at ribose ( C5) and base ( N) of pyridine ring position. (B) M+1 labelled glutamine; labelled at amide (15N) position. 13 15 15 (C) M+7 labelled NMN2; labelled at ribose ( C5), base ( N) and amide ( N) position.

The rationale for this experiment was that if NMN undergoes deamidation prior to its

incorporation into NAD+, it would be metabolised via the de novo pathway, where the last step

involves the amidation of NaAD into NAD+ by the enzyme NAD synthetase (NADSYN). In

mammals, NADSYN catalyses the transfer of an amine group (NH2) from glutamine to NaAD,

+ replacing the hydroxyl group (R-OH) with an amide (R-NH2), to form NAD (Figure 5.2). By

generating an isotope labelled NMN with an M+6 mass shift and administering in the presence

of 15N-amide-labelled glutamine, the detection of NAD+ with an overall mass shift of M+7, at

quantities that exceed the expected background for naturally occurring 13C, would provide

evidence for the metabolism of NMN via the de novo route. To determine whether microbial

metabolism would contribute to this proposed step, the isotope was delivered to mice treated

with antibiotics to deplete their intestinal microbiome. If this proposed deamidation step was

158

Chapter 5 Host-microbiome interactions in NMN metabolism mediated by the microbiome, it could be expected that there would be a reduction in the

appearance of M+7 labelled NAD+, while if this step was mediated by an unknown mammalian

host enzyme, there would be no reduction in M+7 labelling of NAD+.

The second of these isotopes, designated as NMN2, was labelled at all five carbons of the ribose moiety with 13C with a mass shift of M+5, and was also labelled at the pyridine ring

and amide with 15N on the nicotinamide moiety for an additional M+2 mass shift, giving it a final mass shift of M+7 (Figure 5.1 C). To answer our hypothesis, if NMN underwent deamidation prior to its incorporation into NAD+, we would anticipate the loss of one mass

unit, as the 15N label at the amide group of the nicotinyl moiety would be displaced by a

hydroxyl (-OH) group forming M+6 labelled NaMN as in bacteria, and NAD+ would carry an overall mass shift of M+6, rather than M+7 (Figure 5.3). NMN2 was also used to explore other aspects of NMN metabolism, such as NMN transport through direct and/or indirect mechanisms, by tracing its appearance into several metabolic fates as illustrated in Figure 5.4.

Given that one mechanism for NMN transport involves its extracellular dephosphorylation by

CD73 prior to uptake, measuring the appearance of M+7 labelled NR would quantify the relative contribution of this mechanism to NMN uptake and NR availability.

As illustrated in Figure 5.4, we proposed that NMN2 would result in the formation of

M+2 labelled free nicotinamide (NAM), due to the cleavage of M+7 labelled NAD+ by NAD+

consuming enzymes such as the PARPs and sirtuins, however, we do not exclude the possibility

that M+2 labelled NAM could represent cleavage of NMN at its labile glycosidic bond. This

M+2 labelled NAM could then be re-incorporated into NAD+ via the recycling pathway and

the actions of the enzymes NAMPT and NMNAT1-3, resulting in the appearance of M+2

labelled NAD+. NMN2 further allowed us to explore the hypothetical involvement of bacterial

deamidases, PncA and PncC, in the NAD+ metabolome. PncA converts nicotinamide (NAM)

to nicotinic acid (NA) (Frothingham et al. 1996) while PncC uses NMN as a substrate to form

159

Chapter 5 Host-microbiome interactions in NMN metabolism NaMN (Galeazzi et al. 2011). Deamidation of NMN2 via PncC would form M+6 labelled

NaMN and in the presence of NMNAT1-3 and glutamine-dependent NADSYN, result in M+6

labelled NaAD and NAD+, respectively. Cleavage of M+6 labelled NAD+ by NAD+-

consuming enzymes would result in M+1 labelled NAM, which has two different fates. The

first involves normal recycling through the salvage pathway, where M+1 labelled NMN and

NAD+ are formed via NAMPT and NMNAT1-3 enzymes. The second involves deamidation

via PncA, forming M+1 labelled NA which may be used to synthesise M+1 labelled NaMN,

NaAD and NAD+ as part of the Preiss-Handler pathway (Figure 5.4). If bacterial enzymes meaningfully contribute to the NAD+ metabolome, we expect antibiotic treatment to reduce

M+6 labelling of NaMN as well as reduce M+1 labelling of NA, following the loss of 15N- labelled amide from NMN2. If this were the case, given that NA is a precursor to NaMN in the

Preiss-Handler pathway, M+1 labelled NA could result in M+1 labelling of NaMN, NaAD and

NAD+.

The NMN2 isotope was better suited to addressing these metabolic fates than the

NMN1 isotope, as an M+1 mass shift in free nicotinamide from the NMN1 isotope could be

difficult to distinguish from background 13C isotopomers of nicotinamide at low levels of

incorporation.

As one of the main aims of this study was to investigate whether the microbiome was

contributing to the metabolism of NMN, findings from these experiments are shown

predominantly in the intestinal tissue. The intestinal tissue samples were derived from the entire

length of the gastrointestinal tract including the small and large intestine as well as the proximal

and distal colonic segments (excluding cecum).

160

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.2 Schematic of the NMN deamidation and re-amidation hypothesis using NMN1 and 15N-glutamine. Incorporation of isotope labels into M+6 labelled NaMN, NaAD and M+7 labelled NAD+, following administration with NMN1 (50 mg/kg, oral gavage) and 15N-glutamine (5.0 mmoles/kg, approximately 735.7 mg/kg, intraperitoneal injection).

161

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.3 Schematic of NMN deamidation hypothesis using NMN2. Loss of 15N-amide and incorporation of isotope labels into M+6 labelled NaMN, NaAD and NAD+, following administration with NMN2 (50 mg/kg, gavage).

162

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.4 Schematic for the potential biosynthesis pathways and NAD+ metabolites involved in the utilisation of NMN2. NMN2 (box on left) was labelled with 13C at all 5 carbon positions of the ribose moiety (highlighted in red) and with 15N at the base (highlighted in blue) and amide (highlighted in orange) of the nicotinamide moiety for a total mass shift of M+7. The expected mass shift for each species depending on the potential route of utilisation is shown, with steps catalysed by mammalian enzymes represented by solid black lines, and steps that are only known to be carried out by bacterial enzymes represented by the dashed lines.

163

Chapter 5 Host-microbiome interactions in NMN metabolism 5.2 Methods

5.2.1 Animal housing

All animal experiments were performed according to procedures and guidelines approved by

UNSW Animal Care and Ethics Committee (ACEC) under the ethics protocol 18/133A. The

UNSW ACEC operates under the Animal Ethics Guidelines from the National Health and

Medical Research Council (NHMRC) of Australia. All mice were housed in individually ventilated cages of no more than five mice per cage and fed a standard chow diet and drinking water ad libitum. The cages were maintained under a 12-hr light/12-hr dark cycle in a temperature-controlled room (22 ± 1℃) at 80% humidity. All cages were changed twice per week and monitored daily.

5.2.2 Antibiotic and NMN treatment regimen

Four-week-old female C57BL/6J mice were acclimatised for one week prior to treatment. At five weeks, all mice were body-weight-matched and randomly assigned into one of the following treatment groups.

Antibiotic treatment

For antibiotic treatment, mice were administered a cocktail of antibiotics consisting of vancomycin (0.5 g/L; Sigma SBR00001), neomycin (1 g/L; Sigma N6386), ampicillin (1 g/L;

Sigma A9393) and metronidazole (1 g/L; Sigma M3761) (herein, referred to as VNAM). This antibiotic cocktail was administered in the drinking water of mice for four days with the addition of sucrose (3 g/L; Bundaberg Sugar) in all treatment groups including controls to increase palatability and prevent aversion to the drinking water during antibiotic treatment

(Jimeno, Brailey, and Barral 2018). The body weight of mice was recorded during the antibiotic treatment period however, there was a reduction in water consumption in the VNAM cocktail group (Figure 5.5 A) which can increase their risk of illness or death due to dehydration

164

Chapter 5 Host-microbiome interactions in NMN metabolism (Reikvam et al. 2011). As a result, VNAM was replaced by ampicillin treatment only, with

sucrose, for an additional week which was previously shown to effectively reduce gut bacterial

density by 103-fold (Jimeno, Brailey, and Barral 2018; Ubeda et al. 2010).

NMN2 cohort

After the antibiotic treatment period, mice were orally administered an equivalent bolus of

water or NMN2 by gavage according to the following treatment groups: 1) Vehicle control

(water) (n=3), 2) Antibiotics only (Abx) (n=4), 3) NMN2 only (n=4) and 4) NMN2+Abx (n=4).

NMN1+15N-amide-glutamine cohort

After the antibiotic treatment period, mice were orally administered an equivalent bolus of

water or NMN1 by gavage and/or an intraperitoneal injection of water or 15N-amide-glutamine

according to the following treatment groups: 1) Vehicle control (water) (n=4), 2) Antibiotics

only (Abx) (n=4), 3) 15N-amide-glutamine only (n=3), 4) 15N-amide-glutamine+Abx (n=4), 5)

NMN1+15N-amide-glutamine (n=4) and 6) NMN1+15N-amide-glutamine+Abx (n=4).

The synthesis of labelled compounds, NMN1, NMN2 and 15N-amide-glutamine is detailed in

Chapter 2, Section 2.2.1. The final concentration of NMN isotopes and 15N-amide-glutamine were 50 mg/kg and 5.0 mmoles/kg (approximately 735.7 mg/kg), respectively, based on previous studies (Liu et al. 2018; Wang et al. 2019). All blood and tissue samples were collected four hours after gavage. The oral gavage was staggered by five mins between each mouse with the treatment groups alternating to avoid experimental bias during the procedure.

5.2.3 Plasma collection and metabolite extraction

For blood collection, all mice were placed under general anaesthesia with isoflurane gas maintained at 1.5-2% v/v and O2 flow set at 1-1.5 litres per minute. Blood was collected by

cardiac puncture via insertion of a 25-gauge needle through the diaphragm with at least 600 µL

165

Chapter 5 Host-microbiome interactions in NMN metabolism of blood drawn from each mouse. Caution was taken to draw the blood slowly into a 1mL

syringe to prevent the collapse of heart chambers due to pressure changes. The volume of blood

obtained was immediately expelled into a 1.5 mL tube pre-filled with 10 µL of 0.5 M

ethylenediaminetetraacetic acid (EDTA) solution which was thoroughly mixed up and down

with a pipette to prevent the blood from clotting. Plasma was separated from whole blood

contents via centrifugation at 2000 g for 15 minutes at 4℃ before being transferred to a clean

pre-labelled tube and snap frozen immediately in liquid nitrogen. Samples were stored at -80℃

until further processing. On the day of sample acquisition, the following protocol was used for

extraction of NAD+ metabolites from plasma samples; 20 µL of plasma was combined with 80

µL of cold (-30℃) extraction buffer consisting of a 1:1 ratio of LC-MS-grade acetonitrile and

methanol (ACN:MeOH) containing a mixture of internal standards, methionine sulfonate

(MES), camphorsulfonic acid (CSA) and thymine-d4, as in Chapter 2, Section 2.2.1. Samples

were vortexed and centrifuged at 16,000 g for 10 minutes at 4℃ after which the supernatant

was transferred to a clean pre-labelled tube and dried under a Savant SpeedVac™ system

(Thermo Scientific) under vacuum for approximately 2 hours, as in Chapter 2, Section 2.2.3.

The resulting dry pellet was resuspended in 30 µL of LC-MS-grade water and centrifuged again as described above. The supernatant was then transferred to pre-labelled mass spectrometry vials and analysed promptly by LC-MS, as described in Chapter 2, Section 2.3. Data was extracted using in-house MATLAB scripts as described in Chapter 2, Section 2.4. For data analysis, samples were normalised by dividing the raw peak area of the sample by the raw peak area of the internal standard, thymine-d4, for each metabolite. The standard curve for each metabolite was then used to calculate the molar amount (µM) of metabolite in each sample. All graphs were generated and statistically analysed using GraphPad Prism software as described in Chapter 2, Section 2.5.

166

Chapter 5 Host-microbiome interactions in NMN metabolism 5.2.4 Tissue collection, metabolite extraction and data analysis

For collection and processing of mouse tissue samples, the following protocols were used.

Livers were resected and weighed immediately before rinsing in ice-cold 1X phosphate

buffered saline (PBS) to remove any residual blood from the tissue. Intestinal tissue was

resected from the small intestine to the colon (excluding cecum) and the inside was flushed

with ice-cold 1X PBS to remove any faecal contents remaining in the intestinal tract. Both

tissues were snap frozen immediately in liquid nitrogen and stored at -80℃ until further

processing. Faecal pellets were collected from the colonic region of the intestinal tract,

immediately snap frozen in liquid nitrogen and stored at -80℃ until ready for further

processing. One day prior to sample acquisition, the liver and intestinal tissues were crushed

with a mortar and pestle on liquid nitrogen to pulverise the frozen tissues. Approximately 50

mg of liver and intestinal tissue was weighed into pre-labelled tubes containing 1.4 mm ceramic

beads (Precellys® CK14, Bertin Technologies). NAD+ metabolites were extracted from

samples as described in Chapter 2, Section 2.2.2. Briefly, 500 µL of cold (-30℃) extraction buffer (HPLC-grade acetonitrile-methanol-water mixture, 2:2:1) was added to each sample with the internal standard mixture (Chapter 2, Section 2.2.1). The samples were then homogenised using a mechanical homogeniser (Precellys® 24, Bertin Technologies) at 5500 rpm for 15 seconds at room temperature. All samples were centrifuged at 16,000 g for 10 minutes at 4℃ and the supernatant was transferred to a clean pre-labelled tube before snap freezing in liquid nitrogen and storage at -80℃. All sample extracts were dried using the speed vac system (Savant SpeedVac SPD140DDA Vacuum Concentrator, Thermo Fisher Scientific) and the dried pellet was resuspended in 50 µL of LC-MS-grade water and centrifuged once more, as described above. The supernatant was transferred to pre-labelled mass spectrometry vials and analysed promptly by LC-MS/MS as described in Chapter 2, Section 2.3. The data collected from the LC-MS/MS acquisitions were extracted and analysed as described in

167

Chapter 5 Host-microbiome interactions in NMN metabolism Chapter 2, Section 2.4. For data analysis, samples were normalised by dividing the raw peak area of the sample by the raw peak area of the internal standard, thymine-d4, for each metabolite. The standard curve for each metabolite was then used to calculate the molar amount

(pmol) of metabolite in each sample and normalised again to the frozen tissue weight (mg).

Final measurements are presented as pmol/mg tissue. The data were statistically analysed using

GraphPad Prism software as described in Chapter 2, Section 2.5.

5.2.5 DNA extraction from faecal pellets

Solid faecal pellets taken from the colonic and rectal region of the gastrointestinal tract were stored in -80℃ until further processing. DNA was extracted from frozen faecal pellets using the QIAamp® PowerFecal® DNA kit (Qiagen, Cat. No. 12830-50) according to the manufacturer’s protocol. DNA concentration was determined using the NanoDrop™

(DeNovix®, DS-11 FX) and the purity of double-stranded DNA (dsDNA) was also determined by measuring the 260/280 ratio. All DNA extracts were stored at -80℃ until further processing.

5.2.6 Nanopore 16S Sequencing

Full length 16S rRNA genes were amplified by PCR using the Oxford Nanopore 16S

Barcoding Kit (SQK-RAB204; Oxford Nanopore Technologies, Oxford, UK). Briefly, 10 ng genomic DNA, 1 µL 16S Barcode (10 µM) and 25 µL LongAmp Taq 2X Master Mix (New

England Biolabs, Ipswich, MA, USA) were combined in a 50 µL reaction for PCR on a Bio-

Rad T100TM Thermal Cycler (Bio-Rad Laboratories Pty Ltd, Hercules, CA, USA). PCR cycling condition were as follows; initial denaturation at 95 ℃ for 1 minute, 25 cycles of denaturation at 95 ℃ for 20 seconds, annealing at 55 ℃ for 30 seconds and extension at 65 ℃ for 2 minutes before a final extension at 65 ℃ for 5 minutes.

PCR products were purified as per Oxford Nanopore Technologies (ONT) protocol using AMPure XP magnetic beads (Beckman Coulter, Indianapolis, IN) and DNA quantified

168

Chapter 5 Host-microbiome interactions in NMN metabolism using the NanoDrop™ (DeNovix®, DS-11 FX). Barcodes were pooled to a total of 100 fmol in 10 μL of 10 mM Tris-HCl, pH 8.0 with 50 mM NaCl for library loading. Sequencing was performed using R9.4.1 ONT Flow Cells on the MinIONTM sequencing platform and data acquired using MinKNOW software version 19.10.1 (Oxford Nanopore Technologies).

5.2.7 Data Analysis for 16S sequencing

Data extraction and analysis for full length 16S sequences obtained from nanopore sequencing was kindly carried out by Timothy Chalmers. Sequencing reads acquired from the MinION runs (i.e. FAST5 data) were basecalled to fastq files using Guppy software version 3.4.4

(Oxford Nanopore Technologies). Fastq files were demultiplexed using Porechop

(https://github.com/rrwick/Porechop) and trimmed to 1400bp with Trimmomatic version 0.39

(Bolger, Lohse, and Usadel 2014). Reads were imported to QIIME2 for dereplication and chimeric reads screened and filtered from the dataset. Operational taxonomic unit clustering was completed within QIIME2 version 2019.7.0 (Bolyen et al. 2018) at 85% similarity to account for typical sequencing errors obtained from long-read sequencing. Taxonomy was assigned to reads using a pre-trained classifier on the SILVA 132 16S rRNA representative sequences. Data was imported into R version 3.6.1 with qiime2R version 0.99.13

(https://github.com/jbisanz/qiime2R) for visualisation and alpha diversity analysis using raw and rarefied data with the phyloseq version 1.30.0 (McMurdie and Holmes 2013) package. The

R package DESeq2 version 1.28.1 (Love, Huber, and Anders 2014) was used to normalise and model-fit the read counts for differential expression testing between treatment groups.

169

Chapter 5 Host-microbiome interactions in NMN metabolism 5.3 Results

5.3.1 Antibiotic ablation of the gut microbiome

To interrogate the contribution of the gut microbiome to the metabolism of orally delivered

NMN, C57BL/6J female mice were exposed to a course of antibiotics for eleven days through

delivery in drinking water (Figure 5.5 A). Water intake (Figure 5.5 A) and body weight

(Figure 5.5 B) was measured throughout the treatment period in order to monitor antibiotic

consumption and animal welfare. The antibiotic cocktail including vancomycin (0.5 g/L),

neomycin (1 g/L), ampicillin (1 g/L) and metronidazole (1 g/L) (VNAM) was administered in the drinking water of mice, but after four days was associated with a severe decline in water consumption (Figure 5.5 A), though no signs of body weight loss or dehydration were observed (Figure 5.5 B). Due to the potential risks of dehydration and illness from reduced water intake for a prolonged period, the VNAM cocktail was replaced with ampicillin treatment alone, which rapidly increased water consumption within two days.

Antibiotic treatment also led to differences in the internal organs of mice including an enlargement of the cecum (Figure 5.5 D) and a reduction in the weight of spleens (Figure 5.5

E) and livers (Figure 5.5 F), with no significant reduction in body weight compared to the

controls (Figure 5.5 G). These observations are consistent with previous studies using

antibiotic treatment (Cresci, Nagy, and Ganapathy 2013; Jimeno, Brailey, and Barral 2018;

Puhl et al. 2012; Reikvam et al. 2011; Xu et al. 2017) and are also observed in germ-free mice

(Al-Asmakh and Zadjali 2015; Niimi et al. 2019; Niimi and Takahashi 2019; Smith, McCoy,

and Macpherson 2007) indicating these effects are likely due to the under-representation of

commensal bacteria in the gut, rather than due to an adverse side effect of antibiotic treatment.

No significant difference was observed when antibiotic treated mice were administered NMN,

which is expected due to the acute treatment period of four hours. Antibiotic treatment also

170

Chapter 5 Host-microbiome interactions in NMN metabolism dramatically reduced the DNA concentration of mouse stool samples compared to controls

(Figure 5.5 C), inferring an overall reduction in the gut microbiome from antibiotic treatment.

To test the effects of antibiotic treatment on the make-up of the remaining species in

the gut microbiome, gut contents were subjected to microbiome sequencing. Using generic

primers, full-length 16S rRNA was amplified from samples that were normalised to the amount

of starting DNA, and subjected to long-read sequencing using an Oxford Nanopore instrument

(Benítez-Páez, Portune, and Sanz 2016; Kilianski et al. 2015; Shin et al. 2016). This yielded a

significant reduction in bacterial species diversity in stools of mice treated with antibiotics

compared to controls, with the only surviving species in antibiotic-treated mice being from the

Burkholderia genus (Figure 5.6 A). This change is highlighted by alpha diversity analysis

using the Shannon diversity index (Allen, Kon, and Bar-Yam 2009) which showed

significantly reduced diversity in antibiotic treated mice, represented here at the genus level

(Figure 5.6 B). There was no change in microbial diversity when antibiotic-treated mice were

co-administered NMN, which was expected due to the acute treatment period of 4 hours

following a single oral gavage. A similar effect was observed in antibiotic-treated mice co-

administered NMN1 and 15N-glutamine (Figure 5.7) where microbial diversity was

significantly reduced, with the Burkholderia genus remaining. Differential expression analysis

showed antibiotic treated mice had significant downregulation of genes compared to vehicle

control, though there were very little significant changes observed in NMN treated mice

compared to vehicle control (Appendix Figure A.1 and Figure A.2).

The Burkholderia genus is made up of gram-negative bacteria and are common inhabitants of soil that thrive in acidic environments (Sermswan et al. 2015; Stopnisek et al.

2014). Members of Burkholderia are well known for developing resistance to antimicrobials, largely owing to low outer membrane permeability and unique lipopolysaccharide (LPS) structure which reduces the net negative charge of the cell envelope and in turn, reduces

171

Chapter 5 Host-microbiome interactions in NMN metabolism permeation of cationic antimicrobials such as polymyxins (Rhodes and Schweizer 2016). In animals and humans, there are several Burkholderia species that have emerged as opportunistic pathogens which cause severe infections in cystic fibrosis and immunocompromised patients

(Carlier et al. 2014; Mann et al. 2010; Zlosnik et al. 2015). Recent evidence, however, identified new Burkholderia species with beneficial traits that promote plant growth and stress resistance (Pinedo et al. 2015; Sheibani-Tezerji et al. 2015; Su et al. 2015) with potential use in agriculture and industry. Comparative genomics studies to identify common virulence factors in Burkholderia species reveal universal virulence factors in multiple organisms and disease models is largely host-specific and remains a challenge (Angus et al. 2014; Schwager et al. 2013; Uehlinger et al. 2009). Improved methods to better assess pathogenicity of

Burkholderia strains are warranted to identify beneficial from harmful strains which in turn may be helpful in the diagnosis and treatment of diseases affected by infection by Burkholderia species. Regardless, these data suggest that antibiotic treatment was overwhelmingly effective in reducing bacterial content, with the exception of this genus.

172

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.5 The effect of antibiotic treatment in mice. The (A) water intake and (B) body weight during antibiotic treatment was recorded. Antibiotic treatment significant decreased (C) DNA stool concentration and caused (D) enlargement of the cecum, decreased (E) spleen and (F) liver weight but had no effect in (G) body weight after one week of antibiotic treatment compared to controls. For statistical analysis a two-way ANOVA with Sidak’s multiple comparisons test was performed. Data represents mean ± s.d. (n=3-5) with a p-value less than 0.05 to be considered statistically significant. *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

173

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.6 Reduced diversity of the murine faecal microbiome upon antibiotic treatment in NMN2 cohort. (A) Stacked bar plots represent the total reads and relative abundance of bacterial taxa at the genus level for mice treated with vehicle control, antibiotics (Abx), NMN2, or NMN2+Abx, measured by full length 16S rRNA Nanopore sequencing. Taxa with < 0.1% abundance grouped together. (B) Shannon diversity measures of murine gut microbiome by Wilcoxon rank sum test. Data presented as mean ± s.d. (n=3-4). *p < 0.05

174

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.7 Reduced diversity of the murine faecal microbiome upon antibiotic treatment in NMN1 cohort. (A) Stacked bar plots represent the total reads and relative abundance of bacterial taxa at the genus level for mice treated with vehicle control, antibiotics (Abx), 15N-glutamine, 15N- glutamine+Abx, NMN1+15N-glutamine, and NMN1+15N-glutamine+Abx, measured by full length 16S rRNA Nanopore sequencing. Taxa with < 0.1% abundance grouped together. (B) Shannon diversity measures of murine gut microbiome by Wilcoxon rank sum test. Data presented as mean ± s.d. (n=3-4). *p < 0.05

175

Chapter 5 Host-microbiome interactions in NMN metabolism 5.3.2 Antibiotic treatment increases NAD+ metabolite and NMN availability in the

intestinal tissue of mice.

Due to its greater mass shift and therefore ability to discriminate between species, we initially

used the NMN2 isotope for the first part of this investigation. Following antibiotic treatment,

mice were orally administered by gavage a bolus of either water or NMN2 (50 mg/kg). Four

hours later, animals were euthanased and tissues rapidly collected and snap frozen. NAD+

metabolites were detected using the optimised LC-MS/MS method developed in Chapter 3.

In addition to NMN2, NMN1 was also administered in mice combined with 15N-amide-

glutamine (Figure 5.1 B), a necessary cofactor in the last step of the Preiss-Handler pathway

where NaAD is converted into NAD+ via the enzyme NAD synthetase (NADSYN).

Incorporation of all labels from NMN1 (M+6) and 15N-amide-glutamine (M+1) into NAD+,

can be detected by LC-MS/MS as M+7 labelled NAD+ and may provide evidence to suggest

NMN deamidation into NaMN and its re-amidation through the Preiss-Handler pathway. If this

occurs through bacterial deamidases in the gut, mice treated with antibiotics would show a

dampening of this effect.

In the intestinal tissue, an increase in the abundance of unlabelled NAD+ metabolites,

NR, NAD+, NAM and NMN, was observed in mice treated with antibiotics alone (Figure 5.7

A-D), even without the exogenous supplementation with NMN. This was a surprising observation as our original hypothesis anticipated a reduction in metabolites with antibiotic treatment, rather than an increase, if bacterial enzymes in the gut facilitated NAD+ biosynthesis.

In contrast to our hypothesis, these results suggest that bacteria in the gut may be competing

with the host for baseline uptake of trace nutrients from dietary sources (Chi and Sauve 2013;

Mielgo-Ayuso et al. 2018; Mills et al. 2016; Yao et al. 2011), implicating the involvement of

the gut microbiome in host NAD+ availability.

176

Chapter 5 Host-microbiome interactions in NMN metabolism In addition to differences in the levels of NAD+ metabolites derived from dietary

sources, this “competition” relationship between the microbiome and the host was tested

through treatment with an exogenous bolus of NMN, effectively overriding the host-

microbiome competition for relatively scarce dietary NAD+ precursors. The labelled species that may be detected for each of these metabolites can be categorised into two distinct groups, which are referred to here as ‘complete’ (Figure 5.7 E-H) or ‘partial’ (Figure 5.7 I-L) labelling of metabolites species. Complete labelling of metabolites represents the incorporation of both labelled ribose and nicotinamide moieties from the NMN2 isotope, while partial labelling of metabolites represents the incorporation of labelled nicotinamide species formed from the

NAD+ degradation from NAD+-consuming activities. As modelled in Figure 5.4, the

contribution of the microbiome in the gut the specific tracing of metabolites allow the

contribution of the gut microbiome to the metabolism of exogenously supplemented NMN in

the host to be determined. The incorporation of M+7 label into the NAD+ metabolome occurred

almost exclusively in antibiotic treated animals as denoted by the red bars in Figure 5.7 E-H.

These data suggest that even at this small bolus dose, the gut microbiome is still in competition

with the host for NAD+ metabolites, limiting the availability of supplemented NMN, and/or

impeding its intestinal absorption in the host. These effects were extended beyond the

gastrointestinal tract. The simultaneous increased abundance of partially labelled M+2 NAD+

(Figure 5.7 J) and NMN (Figure 5.7 L) in intestinal tissue, M+7 NAD+ (Figure 5.8 F) and

M+2 NAM (Figure 5.8 G) in the liver and M+2 NAM in the plasma (Figure 5.9 B) add weight

to the availability of NMN to the host.

The effect of antibiotic treatment on NAD+ metabolites detected in the intestinal and

liver homogenates from the NMN2 cohort was also reflected in the cohort of mice administered

NMN1, which has an overall mass shift of M+6. Similar to observations in the NMN2 cohort,

antibiotic treatment significantly increased the endogenous unlabelled pool of NAM in

177

Chapter 5 Host-microbiome interactions in NMN metabolism intestinal tissues with a similar, but non-significant trend observed in NR, NAD+ and NMN

(Figure 5.10 A-D). In the liver, unlabelled NAD+ and NAM significantly increased with

antibiotic treatment with a similar, but non-significant, trend observed in unlabelled NR and

NMN (Figure 5.11 A-D). A significant increase in the incorporation of M+6 label into NR,

NAD+ and NMN was detected in intestinal homogenates following the administration of

NMN1 to antibiotic treated mice and denoted by the red bars in Figure 5.11 E, F and H. The

turnover of M+6 labelled NAD+ in the intestine was also affected by antibiotic treatment,

forming M+1 labelled NAM at the pyridine base, which significantly increased exclusively in

antibiotic treated mice following a bolus dose of NMN1 (Figure 5.11 G). The partially labelled

metabolites, M+5 NR (Figure 5.11 I), M+1 NAM labelled at the amide from 15N-glutamine

(Figure 5.11 K) and M+1 NMN (Figure 5.11 L), also significantly increased following

antibiotic treatment, highlighting the effect of the microbiome on NMN utilisation as well as

on metabolic processes involved in NAD+ degradation and biosynthesis. Unlike the intestinal

tissue, the complete and partial incorporation of labels into NAD+ metabolites detected in the

liver following administration with NMN2 (Figure 5.8) or NMN1 (Figure 5.11), were not as

striking. This could be due to the majority of metabolites being degraded and/or consumed by

bacterial enzymes in the intestinal environment prior to reaching the liver. Alternatively,

antibiotic treatment may have induced damage to the liver, as well as the spleen (Figure 5.5

E) and possibly the integrity of the intestinal epithelium (though was not investigated here), as there was a reduction in liver weight in antibiotic treated mice compared to controls (Figure

5.5 F, above). Collectively, the results from both cohorts highlight the significant increase in the availability of orally delivered NMN and NAD+ metabolites to host tissues with antibiotic

treatment, likely mediated by changes to the microbiome. It is possible that the microbiome

acts in competition with the host for NAD+ metabolites for its own growth and proliferation,

178

Chapter 5 Host-microbiome interactions in NMN metabolism and/or impeding intestinal absorption of NMN in the host, however more investigations need

to be conducted in order to further support these claims.

Metabolites detected in the blood represent those capable of circulating between tissues

such as the intestines and the liver, acting as an extracellular source of NAD+ precursors to ensure neighbouring cells and tissues can maintain intracellular NAD+ levels. The main NAD+

metabolite detected in the plasma of NMN administered mice was nicotinamide (NAM) which

can be derived from NAD+-consuming activities or from breaking the glycosidic bond between

the ribose and nicotinamide moieties of NMN2, yielding M+2 NAM (Figure 5.9 B), or NMN1,

yielding M+1 NAM (Figure 5.12 B). Treatment with antibiotics had little impact on these

labelled NAM isotopologues (red bars) in the plasma, whereas in the intestines and liver a

significant increase in M+2 NAM (Figure 5.7 G for intestines and Figure 5.8 G for liver) and

M+1 NAM (Figure 5.10 G for intestines) was observed following an exogenous bolus of

NMN2 or NMN1, respectively, indicating NAM metabolism in these tissues is significantly

impacted by ablation of the microbiome through antibiotic treatment.

179

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.8 An increase in amidated NAD+ metabolites detected with antibiotic treatment in the intestinal tissue after NMN2 administration. Five-week old C57BL/6 mice were treated with or without antibiotics (Abx) before receiving a single oral gavage of vehicle (water) or NMN2 (50 mg/kg). The abundance of NAD+ metabolites in the intestinal tissue, shown above, was analysed using LC-MS/MS. (A-D) represents the abundance of unlabelled species. (E-H) represents complete labelling while (I-L) represents partial labelling of NR, NAD+, NAM and NMN. Data were analysed by two-way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. (n=3-4 mice per group). **p<0.01, ***p<0.001, ****p<0.0001.

180

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.9 Detection of amidated NAD+ metabolites with antibiotic treatment in the liver after NMN2 administration. Five-week old C57BL/6 mice were treated with or without antibiotics (Abx) before receiving a single oral gavage of vehicle (water) or NMN2 (50 mg/kg). The abundance of NAD+ metabolites in the liver, shown above, was analysed using LC-MS/MS. (A-D) represents the abundance of unlabelled species. (E-H) represents complete labelling while (I-L) represents partial labelling of NR, NAD+, NAM and NMN. Data were analysed by two-way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. (n=3-4 mice per group). *p<0.05, **p<0.01, ***p<0.001.

181

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.10 Detection of nicotinamide (NAM) with antibiotic treatment in the plasma after NMN2 administration. Five-week old C57BL/6 mice were treated with or without antibiotics (Abx) before receiving a single oral gavage of vehicle (water) or NMN2 (50 mg/kg). The abundance of NAM (nicotinamide) in the plasma, shown above, was analysed using LC-MS/MS. (A) M+0 represents unlabelled NAM which is endogenously present in the plasma, while (B) M+2 labelled NAM represents NAM labelled with 15N at the pyridine ring and amide functional group. It can be formed from the degradation of M+7 labelled NAD+, involving cleavage at the glycosidic bond between the nicotinamide and ribose moieties from NAD+-consuming activities. Data were analysed by two-way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. (n=3-4 mice per group). *p<0.05, **p<0.01, ****p<0.0001.

182

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.11 An increase in amidated NAD+ metabolites detected with antibiotic treatment in the intestinal tissue after NMN1 administration. Five-week old C57BL/6 mice were treated with or without antibiotics (Abx) before receiving a single oral gavage of vehicle (water) or NMN2 (50 mg/kg). The abundance of NAD+ metabolites in the intestinal tissue, shown above, was analysed using LC-MS/MS. (A-D) represents the abundance of unlabelled species. (E-H) represents complete labelling while (I-L) represents partial labelling of NR, NAD+, NAM and NMN. Data were analysed by two-way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. (n=3-4 mice per group). *p<0.05, **p<0.01, ***p<0.001.

183

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.12 Detection of amidated NAD+ metabolites with antibiotic treatment in the liver after NMN1 administration. Five-week old C57BL/6 mice were treated with or without antibiotics (Abx) before receiving a single oral gavage of vehicle (water) or NMN2 (50 mg/kg). The abundance of NAD+ metabolites in the liver, shown above, was analysed using LC-MS/MS. (A-D) represents the abundance of unlabelled species. (E-H) represents complete labelling while (I-L) represents partial labelling of NR, NAD+, NAM and NMN. Data were analysed by two-way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. (n=3-4 mice per group). *p<0.05.

184

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.13 Detection of nicotinamide (NAM) with antibiotic treatment in the plasma after NMN1 administration. Five-week old C57BL/6 mice were treated with or without antibiotics (Abx) before receiving a single oral gavage of vehicle (water) or NMN2 (50 mg/kg). The abundance of NAM in the plasma, shown above, was analysed using LC-MS/MS. (A) Unlabelled NAM increased with NMN1 administration which may be due to a frameshift increase in NAD+ metabolites following an exogenous bolus of NMN. (B) No significant change was observed in antibiotic treated mice. (C) Labelling of NAM at the amide comes from 15N-amide-labelled glutamine (15N-gln), however, this could be due to a frameshift increase in NMN rather than from 15N- gln as no change was observed between mice injected with 15N-gln (in green) to controls (in grey). Data were analysed by two-way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. (n=3-4 mice per group). *p<0.05.

185

Chapter 5 Host-microbiome interactions in NMN metabolism 5.3.3 Evidence for NMN uptake facilitated by dephosphorylation into NR

The design of our isotope labelled NMN allowed us to obtain insights into the proposed

mechanisms of NMN transport by intestinal cells in the gut. The two proposed mechanisms for

NMN uptake are either directly via a dedicated transporter (Grozio et al. 2019a) or indirectly,

via dephosphorylation into NR prior to uptake where NR is re-phosphorylated into NMN by

NR kinases (NRK1/2) (Ratajczak et al. 2016). These competing mechanisms are illustrated in

Figure 5.13. Both the NMN transporter, SLC12A8, and the ecto-5’-nucleotidase, CD73, which

dephosphorylates NMN into NR, sit on the apical side of the intestinal epithelium, and for this

reason the intestinal tissue was a key focus of this investigation. NR can enter cells via an

equilibrative nucleoside transporter (ENT), SLC29A1 or SLC29A2 (Baldwin et al. 2004;

Young et al. 2008), which is embedded in the plasma membrane. It is possible that these

mechanisms run parallel to each other, rather than being mutually exclusive events. The

question, however, is the degree to which each mechanism contributes to the availability of

intracellular NMN, or whether these are condition- or tissue-specific events. If the direct route via SLC12A8 prevailed, we would expect to see significant intact (M+7) NMN incorporation relative to the endogenous (M+0) pool, and similarly for downstream products that may include

NR.

Ratios between the ‘complete’ and unlabelled isotopologues are reported here as proxies for the relative presence and turnover of metabolites (Figure 5.14). These ratios capture

the dilution of the existing endogenous pool by the influx of supplemented NMN and NMN-

derived products. Strikingly, we observed strong labelling of the NR, but not NMN pools

(Figure 5.14 A). Isotope (M+7) labelling of the NR pool following NMN2 treatment was three-

fold greater than endogenous NR (M+0), this increased to a ten-fold enrichment in animals

treated with antibiotics. In stark contrast, only around 5% of the NMN pool was M+7 labelled,

representing a small proportion of labelled NMN detected intact. The inability of exogenous

186

Chapter 5 Host-microbiome interactions in NMN metabolism NMN to displace the endogenous NMN pool, combined with the surge of NR derived directly from supplemented NMN, provides strong evidence that the availability of intracellular NMN bypasses direct transport. These data would instead support the dephosphorylation of NMN into NR to facilitate its intestinal absorption, as described in Figure 5.13. If direct transport of

NMN does occur, we propose it is in competition with the microbiome, as the M+7 labelling that was detected only occurred in the intestinal tissue of antibiotic treated animals (Figure 5.7

H and Figure 5.10 H). NR also had, by far, the highest labelled-to-unlabelled ratio compared to all other metabolites (Figure 5.14 A-B). As such, these data support the uptake of NMN in the gastrointestinal tract via an NR intermediate. There are, however, important caveats to the interpretation of these data which will be discussed in detail in the discussion.

187

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.14 Schematic for the direct or indirect transport of NMN in intestinal cells. Direct transport of NMN into cells may occur via the membrane bound transporter, Solute Carrier Family 12 Member 8, SLC12A8 (Grozio et al. 2019a). Intracellular NMN is then able to synthesise NAD+ via NMN adenylyltransferases (NMNAT1-3) (Berger et al. 2005). The indirect transport of NMN requires dephosphorylation of NMN into nicotinamide riboside (NR) via the ecto-5’-nucleotidase, cluster of differentiation 73 (CD73) (Grozio et al. 2013), lying on the apical side of the membrane and faces the lumen. NR may enter the cell via equilibrative nucleoside transporters (ENT1-3), SLC29A1 or SLC29A2 (Baldwin et al. 2004; Young et al. 2008). NR is then re-phosphorylated into NMN via NR kinases (NRK1/2) in the NRK pathway (Ratajczak et al. 2016) before NAD+ synthesis via NMNAT1-3 (Berger et al. 2005). NAD+-consuming enzymes such as the sirtuins and poly(ADP-ribose)-polymerases (PARPs) degrade NAD+ into NAM which can be recycled through the salvage pathway to maintain intracellular NAD+ levels. NAM is an uncharged compound small enough to diffuse across the basolateral membrane and enter the blood. It is possible for NR to also enter the blood as the NR transporters, ENT1-3, are also embedded on the basolateral membrane of mammalian cells (Baldwin et al. 2004; Young et al. 2008).

188

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.15 The ratio of the labelled-to-unlabelled isotopologues of NAD+ metabolites (A) The ratio of complete labelled-to-unlabelled species in the intestinal homogenates of antibiotic treated-mice following oral administration with M+7 labelled NMN2 is shown above. The enrichment of the label into NR but not NMN is observed which increased in antibiotic treated mice. Similarly, the ratio of partially labelled-to-unlabelled species in the intestinal homogenates is shown in (B) showing enrichment of the label into NR, which is significantly greater compared to all other metabolites, including NMN. Data were analysed by two-way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. with the mean value for each treatment group annotated above each bar (n=3-4 mice per group). *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001, ns=not significant.

189

Chapter 5 Host-microbiome interactions in NMN metabolism 5.3.4 NMN deamidation is independent of the microbiome.

In the canonical pathways of NAD+ biosynthesis in mammals, the amidated metabolites NMN,

NR and NAM are not converted into their deamidated counterparts nicotinic acid

mononucleotide (NaMN), nicotinic acid riboside (NaR), and nicotinic acid (NA), respectively

(refer to schematic in Figure 5.4). These deamidated forms are confined to the Preiss-Handler pathway (Preiss and Handler 1958a), the final step of which involves the enzyme, NAD synthetase (NADSYN), amidating NaAD into NAD+ (Figure 5.4). Surprisingly, other research

groups have reported that treatment with the amidated precursor, NR, leads to the formation of

the deamidated metabolite, NaAD (Trammell et al. 2016a). This could potentially be explained

by a bottleneck at the enzyme NADSYN, caused by exogenous treatment with NMN or NR.

Alternatively, bacteria possess deamidase enzymes such as the NMN deamidase PncC

(Gazzaniga et al. 2009), absent in mammals, which may contribute to the formation of NaAD

observed by Trammell and colleagues, by deamidating NMN and forming NaMN, the

precursor to NaAD in the Preiss-Handler pathway (Figure 5.4).

Our hypothesis proposed that a bacterial enzyme-mediated NMN deamidation step may

be involved in the formation of the deamidated metabolite, NaAD, following oral

administration with the amidated NAD+ precursor, NR. To investigate this hypothesis, we

specifically designed the labelled isotopes of NMN so that 15N labelling at both the pyridine ring and amide group of the nicotinamide moiety of NMN was achieved. As described in

Section 5.3.1 and illustrated in Figure 5.4, NMN deamidation forming NaMN, followed by the reamidation of NaAD into NAD+, would result in the loss of a single mass unit from the 15N

amide being removed. This would result in the conversion of M+7 NMN into M+6 NaMN via

PncC, formation of M+6 NaAD via NMNAT 1-3 (Berger et al. 2005) and final synthesis of

M+6 NAD+ via NADSYN (Hara et al. 2003) (Figure 5.4). Further, the appearance of M+6

NaAD would provide evidence for NMN or NR deamidation, rather than a bottleneck in

190

Chapter 5 Host-microbiome interactions in NMN metabolism endogenous NAD+ synthesis at the enzyme NADSYN. This strategy would allow us to test our theory that the microbiome contributes to NMN deamidation, potentially via enzymes such as

PncC. Antibiotic treatment ablates bacterial species, and hence their deamidation enzymes, and subsequently we would expect a reduction in the abundance of M+6 labelled species detected in deamidated metabolites.

In this study, a small degree of M+6 labelled NaMN (Figure 5.16 B), NaAD (Figure

5.16 C) and NAD+ (Figure 5.16 D) was detected in the metabolite extracts from intestinal

homogenates following administration with NMN2. The formation of M+6 labelled NaMN

and NaAD with exogenous NMN treatment may suggest that the direct deamidation of NMN

through bacterial enzymes in the gut, and biosynthesis of NAD+ through the Preiss-Handler pathway metabolites, is a possibility. In contrast to our hypothesis, however, an increase in

M+6 labelled NaAD and NAD+ was observed in antibiotic treated mice following

administration with NMN, as denoted by the red bars in Figure 5.16 C and D, rather than an

expected decrease. These data contradict our hypothesis that NMN is deamidated through

interaction with bacterial deamidase enzymes in the gut. Instead, the presence of bacterial

species seems to be limiting the availability of NMN (Figure 5.16 A and E) and generation of

NaAD (Figure 5.16 C and G) and NAD+ (Figure 5.16 D and H) in the intestinal tissue.

Alternatively, these results indicate the generation of deamidated metabolites may not be

occurring through bacterial enzymes in the gut at all, as barely any M+6 labelled NaMN and

NaAD was detected in intestinal homogenates from mice treated with NMN1 (Figure 5.16 F

and G). Rather, these results put forward the possibility of a mammalian deamidase capable of

direct NMN or NR deamidation. While this poses an important question as to the identity of

this enzyme, whether it will meaningfully contribute to overall NAD+ metabolism in the host

is uncertain, as only a negligible proportion of deamidated metabolites, NaMN and NaAD

(<1pmol/mg tissue), were detected in this study (Figure 5.16 B and C).

191

Chapter 5 Host-microbiome interactions in NMN metabolism

Figure 5.16 Deamidation pathway in the intestinal tissue of antibiotic treated mice following administration with NMN2 or NMN1 and 15N-amide-glutamine (15N-gln). Five-week-old female C57BL/6 mice were supplemented antibiotics in their drinking water for 11 days before NMN2 administration (50 mg/kg) by gavage. The intestinal tissues were collected at the 4-hr time point, and labelled isotopologues were detected in metabolite extracts from tissue homogenates using LC-MS/MS in MRM mode. The hypothesis that bacterial enzymes in the gut play a role in NMN metabolism by generating deamidated metabolites such as NaMN and NaAD, was contradicted by observing an increase in these metabolites following antibiotic treatment as observed in (A-D) for NMN2 and (E-H) for NMN1, and NaMN was largely absent after (B) NMN2 or (F) NMN1 administration. This may suggest a mammalian NMN deamidase could exist, though the physiological relevance may be negligible due to the low picomolar amounts detected. Data were analysed by two- way ANOVA with Sidak’s multiple comparisons test and represent mean ± s.d. (n=3-4 mice per group). *p<0.05, **p<0.01, ***p<0.001.

192

Chapter 5 Host-microbiome interactions in NMN metabolism 5.4 Discussion

While intravenous delivery of NMN results in intact assimilation into peripheral tissues such

as the liver, kidney, brain and muscle, recent studies have revealed that the oral delivery of

NMN results in its cleavage at the glycosidic bond into free nicotinamide (Liu et al. 2018).

This was attributed to cleavage activity in the liver, however, orally administered NMN must also survive the gastrointestinal environment where bacteria have diverse enzymes to utilise

NAD+ precursors for their own metabolism (Gazzaniga et al. 2009), prior to reaching host

tissues.

In this chapter, we showed that the availability of orally delivered NMN in the intestinal tissue is tightly limited by competition with the gut microbiome. While this does satisfy the hypothesis that the microbiome contributes to the metabolism of orally administered NMN in vivo, the increase in NAD+ metabolites observed with antibiotic treatment was not anticipated

(Figure 5.8-Figure 5.13). This conclusion was drawn based on the specific conditions of this

study which involved a relatively modest dose of NMN (50 mg/kg) and its effect on levels of

NAD+ metabolites detected in the intestinal and liver tissue at an acute time point (4 hours). In

this study, the complete labelled versions of NMN were not detected in mice plasma from the

NMN2 or NMN1 cohort, which was consistent with the findings from a study conducted by

Liu and colleagues who also did not detect labelled NMN (or NR) in circulation at any time

point (0, 5, 15, 45 and 135 minutes) after orally administering the same bolus dose of NMN

(50 mg/kg) (Liu et al. 2018). In a study conducted by Mills and colleagues NMN was detected

in the plasma, with an increase detected as early as 2.5 minutes and returning back to baseline

by 15 minutes, however, the orally administered dose of NMN was much higher, at 300 mg/kg

body weight (Mills et al. 2016). As such, it is conceivable that the gut microbiome could

contribute to a threshold effect for dosing, thereby exogenously administered NMN or NR may

only reach peripheral tissues at a dose beyond the amount needed to saturate the microbiome

193

Chapter 5 Host-microbiome interactions in NMN metabolism (i.e. >50 mg/kg), and mass balance studies that incorporate the terminal excretion of NMN and

other NAD+ metabolites from the body will be crucial to establish this. Importantly, while these

results demonstrate a tight competition for the uptake of NMN into the intestinal epithelium,

they were in close alignment with the finding by Liu and colleagues that a substantial

proportion of orally delivered NMN is decomposed to free nicotinamide in the liver (Liu et al.

2018). This study adds to these findings, where in addition to detecting an abundance of M+2

(Figure 5.9 G) and M+1 labelled NAM (Figure 5.12 G) in the liver, M+2 and M+1 NAM

were also abundantly detected in the intestinal tissues of mice administered NMN2 and NMN1,

respectively (Figure 5.8 G and Figure 5.11 G). This suggests that the cleavage of NMN at its

glycosidic bond may occur before it reaches the liver, in the gastrointestinal tract. As the half-

life for NAD+ turnover in the small intestine (< 30 minutes) is faster than that in the liver (2

hours) (Liu et al. 2018), detecting these labelled NAM isotopologues in the intestinal tissue at

earlier time points and comparing them to their detection in the liver is crucial to confirm this.

The increase in M+2 NAD+ in the intestinal tissues (Figure 5.8 J) and livers (Figure

5.9 J) of NMN2 administered mice, at levels higher than M+7 NAD+ (Figure 5.8 F, Figure

5.9 F), suggests new NAD+ is synthesised from the salvage of M+2 NAM rather than directly

from M+7 NMN. These data are consistent with a previous report (Liu et al) suggesting that

orally delivered NMN and NR undergo rapid metabolism by the liver to release free NAM,

raising the question of whether it is necessary to use NMN, rather than NAM, to raise NAD

levels. This also raises the question as to whether NMN acts as a prodrug of NAM and whether

administering NAM directly may be more effective in increasing cellular NAD+ levels, though

these comparative studies have not been conducted but are urgently warranted. There are

however, limitations to the therapeutic use of NAM as it inhibits the sirtuin enzymes (Avalos,

Bever, and Wolberger 2005; Bitterman et al. 2002), whereas NMN administration at a

relatively high dose (300 mg/kg) in mice over a 12-month treatment period yielded no such

194

Chapter 5 Host-microbiome interactions in NMN metabolism side effects (Mills et al. 2016). A recent study revealed that NMN has longer retention in the

body compared to NAM (Kawamura, Mori, and Shibata 2016), with lower urinary excretion

of NAD+ catabolites, suggesting NMN has longer NAD+-raising potential over NAM. Future studies comparing the metabolic changes and therapeutic effects of NMN and NAM are needed to confirm this.

This study was initially undertaken to address questions of how the delivery of the amidated NAD+ precursor, NR, the precursor to NMN, could lead to a spike in the formation

of a deamidated metabolite, NaAD (Trammell et al. 2016a). We hypothesised that a

deamidation step could be mediated by bacterial enzymes in the gut microbiome, contributing

to an integrated model of host-microbiome NAD+ metabolism that has been described for the deamidation of NAM into NA by the nicotinamide deamidase, PncA (Frothingham et al. 1996),

in which case a reduction in the production of M+6 labelled NaAD following antibiotic

treatment was expected following NMN2 administration in mice. Contrary to this hypothesis,

the reverse occurred where an increase in M+6 NaAD labelling was observed (Figure 5.16 C

& G). Even if the involvement of bacterial deamidases to generate deamidated metabolites were true, based on the very low detection of M+6 labelled NaAD, whether it will meaningfully contribute to overall biosynthesis and metabolism of NAD+ inside the cell is uncertain.

Similarly, in the NMN1 cohort, the deamidation of M+6 labelled NMN1 isotope followed by

the re-amidation of M+6 labelled NaAD to M+7 labelled NAD in the presence of M+1 labelled

15N-glutamine was hypothesised (Figure 5.2), however, the opposite effect of targeting

bacterial enzymes using antibiotic treatment was observed (Figure 5.16 H). These data,

therefore, do not support the hypothesis that the appearance of deamidated metabolites

following oral delivery of amidated NAD+ precursors is mediated by bacterial enzymes in the

gut, and the question of why exogenous NR treatment leads to the acute formation of NaAD

observed by Trammell and colleagues, remains unanswered. Nevertheless, the appearance of

195

Chapter 5 Host-microbiome interactions in NMN metabolism NaAD levels following supplementation with NR is consistently observed (Remie et al. 2020)

and is emerging as a sensitive biomarker for increasing intracellular NAD+ in cells and tissues.

While one reason for the appearance of NaAD could be from the deamidation of excess NAD+

as a way for cells to maintain a narrow range of cellular NAD+ concentration to regulate NAD+

homeostasis (Hara et al. 2019), this is unlikely to be the case as the amidation of NaAD into

NAD by NADS is an ATP dependent process, and the reversibility of this reaction is

thermodynamically unfavourable. Another possibility is that NaAD may be formed from

dephosphorylation of NAADP, a secondary messenger that plays a role in intracellular calcium

mobilisation, though the enzymes responsible for this proposed step are also yet to be

identified. Whether the formation of deamidated metabolites, NaMN and NaAD, can occur

through non-enzymatic acid-catalysed hydrolysis in the stomach or by another non-specific

deamidase enzyme is unknown. The redox state of NAD dictates its pH sensitive hydrolyisis,

with the reduced form NADH susceptible to degradation under acidic conditions but resistant

to high pH, and the inverse for NAD+ (Lowry, Passonneau, and Rock 1961). The products of

this acid-mediated degradation are not known to include deamidated products, and it is unclear

whether the pH of the stomach could influence the deamidation of NMN. Comprehensive

bioinformatic analysis of the entire NMN deamidase family, however, has revealed that NMN

deamidases are mainly found in bacteria, with a few examples in fungi (Hughes and

Williamson 1953; Sánchez-Carrón et al. 2013) and studies concerning deamidase activity in

mammals has been associated with microorganisms found in the stomach (Shimoyama et al.

1971).

Given our clear evidence for the preferential utilisation of orally delivered NMN by the

microbiome prior to host uptake, future studies should aim to characterise changes to the

microbiome itself during dosing with NAD+ precursors. Given our growing understanding of

the role of the gut microbiome in health, it is conceivable that physiological benefits observed

196

Chapter 5 Host-microbiome interactions in NMN metabolism in preclinical models of ageing (Rajman, Chwalek, and Sinclair 2018) could, in part, be

mediated by changes to the microbiome. Overall, our findings for the role of the gut

microbiome to NMN metabolism will be important to the ongoing clinical development of

NAD+ precursors as drug candidates, with important implications for dosing and strategies

concerning their route of administration.

Study limitations

NAD+ metabolism in microbial organisms has been well-characterised and used as a guide to elucidate mammalian NAD+ metabolism (Gazzaniga et al. 2009). The idea that the gut

microbiome plays a role in the metabolism of NAD+ precursors is not unexpected, however most animals studies that use NAD+ precursors, such as NMN and NR, fail to take this into

consideration. The preliminary findings from this study provide valuable insight into the role

of the gut microbiome in the metabolism of NAD+ precursors such as NMN. In future, it will

be important to consider whether the route of administration and dose of NAD+ precursors in

preclinical and clinical studies is appropriate.

A higher dose may not necessarily translate to improved efficacy or superior therapeutic

outcomes, in fact, a single dose of NMN at 62.5 mg/kg had the strongest protective effect

against ischemic brain injury in mice compared to the higher doses administered at 125, 250

and 500 mg/kg in mice (Park et al. 2016). In another study, long term administration of NMN

at a lower dose (100 mg/kg/day) was better at improving functional parameters such as oxygen

consumption, energy expenditure and physical activity, whereas the higher dose (300

mg/kg/day) was associated with greater improvements in tear production, bone density and

myeloid-lymphoid composition (Mills et al. 2016), indicating that the optimal dose of NMN

may differ significantly depending on the physiological target. A small bolus dose of NMN at

50 mg/kg was administered to mice in this study which may have been directly consumed by

197

Chapter 5 Host-microbiome interactions in NMN metabolism microbial organisms in the gut, accounting for the low levels of NAD+ metabolites detected in

peripheral tissues. Future studies investigating NMN metabolism should aim to test a wider

range of doses to establish whether a higher dosing can overcome the potential threshold effects

of the microbiome on host availability of orally administered NAD+ precursors.

One major limitation of this study is that the small (duodenum, proximal jejunum,

middle jejunum, distal jejunum, and ileum) and large (cecum, ascending colon, and descending

colon) intestinal segments were combined, causing a loss of functional heterogeneity. Different

segments not only harbour different populations of microbes, but also identify with different

cell types such as enterocytes, dendritic cells, neuroendocrine cells, goblet cells, proliferating

stem cells, Paneth cells, immune cells, muscle cells, neurons and adipocytes (Kong, Zhang,

and Zhang 2018; Zhang et al. 2018a; Zhao et al. 2015). The unique functions and differences

in metabolism of these cell types along the length of the intestines could significantly vary and

performing metabolite analyses in separate intestinal segments will provide further insight into

this.

Another limitation is that the changes to the NAD+ metabolome in this study were

examined at a single time point, rather than over a time course. The 4-hour time point chosen

in this study may have been too long to examine the minute-order kinetics of NMN transport into the cell, as previously reported (Mills et al. 2016; Yoshino et al. 2011). Future studies should aim to sample at tighter and more frequent time points to account for the minute- and hourly-order kinetics of NMN transport and metabolism.

The high variability in the intestinal concentrations of metabolites between NMN2

(Figure 5.8) and NMN1 (Figure 5.11) cohorts may be attributed to the sample measurements being acquired in separate LC-MS batches. In addition to the natural biological variance between experimental mice, slight differences in HPLC column conditions over time and/or

198

Chapter 5 Host-microbiome interactions in NMN metabolism sample processing periods may change metabolite signals, contributing to the variability seen

between intestinal samples across separate cohorts. Further, the standard calibration curves

used to calculate the molar amounts of NAD metabolites were generated for each sample batch

and included internal standards however, were prepared in LC-MS-grade water rather than the sample matrix which consists of components that may cause ion suppression, a significant reduction in analyte signal due to competing or interfering matrix components with the metabolite of interest (Annesley 2003; Stokvis, Rosing, and Beijnen 2005). While standard calibration curves and samples were all normalised to the internal standard (thymine-d4), the difference between the internal standard values in standards and samples was on average approximately 7-fold higher in the NMN2 cohort and 10-fold higher in the NMN1 cohort, suggesting a potential matrix effect on ionisation efficiency. This limitation should be addressed in future studies by performing experiments which test the degree of ion suppression

(Annesley 2003), determine the conditions that influence ionisation efficiency (such as type of sample matrix, extraction method and buffers, or structure of the metabolite of interest) and develop ways to minimise these interferences to achieve greater accuracy between batches.

One of the main aims of this study was to explore the mechanism of NMN transport into the cell using labelled isotopes of NMN. This assumed the presence and function of transporters SLC12A8 and ENTs (SLC29A1 and SLC29A2) as well as enzymes CD73 and

NRK1/2, however this was not experimentally demonstrated. This should be addressed in future studies through genetic deletion of SLC12A8 (Grozio et al. 2019a), NRK1/2 (Ratajczak et al. 2016) or supplementation with small molecule inhibitors such as adenosine-5′ -(α ,β - methylene)-diphosphate (AOPCP) which inhibits CD73 activity (Bhattarai et al. 2015) and dipyridamole (DIPY) or S-(4-Nitrobenzyl)-6-thioinosine (NBTI) which inhibit plasma membrane nucleoside transporters such as ENTs (Jarvis 1986; Soriano-Garcia et al. 1984).

199

Chapter 5 Host-microbiome interactions in NMN metabolism Finally, to consolidate the hypothesis that the gut microbiome acts in competition with host intestinal tissues for the uptake of orally delivered NMN, the measurement of NAD+ metabolites in the faecal contents of mice will provide better insight into whether it is being properly absorbed in the gut and metabolised, rather than being excreted.

200

Chapter 6 General Discussion

General Discussion

It has been over 110 years since NAD+ was first discovered as an essential redox cofactor

required for yeast fermentation, and its biological significance has since grown to include other

roles including as a critical substrate for proteins regulating DNA repair, metabolism and

longevity (Katsyuba et al. 2020). Declining NAD+ levels with advanced age are implicated in

age-related diseases (Camacho-Pereira et al. 2016; Gomes et al. 2013; Massudi et al. 2012a),

compromising cellular redox reactions and the activity of enzymes such as the sirtuins, PARPs

and CD38/CD157, which consume NAD+ as a co-substrate (Braidy et al. 2011b; Camacho-

Pereira et al. 2016; Frederick et al. 2016; Pang et al. 2015; Schultz and Sinclair 2016; Wang et

al. 2013; Wilk et al. 2020; Yaku, Okabe, and Nakagawa 2018a). In a bid to halt the decline in

NAD+ levels, supplementation with NAD+ precursors such as NMN and NR has show potential to improve healthspan and lifespan (Bertoldo et al. 2020; Das et al. 2018; de Picciotto et al.

2016; Diguet et al. 2018; Mills et al. 2016; Tarantini et al. 2019; Trammell et al. 2016c;

201

Chapter 6 General Discussion

Yoshino et al. 2011; Zhang et al. 2016a) with clinical studies underway (Airhart et al. 2017a;

Dolopikou et al. 2019; Elhassan et al. 2019; Irie et al. 2020; Martens et al. 2018; Remie et al.

2020).

Despite the popularity of this strategy, key details of how these supplements raise

NAD+ levels in the body are missing, which may lead to more effective ways to increase their

therapeutic potential. Immediately prior to submission of this thesis, a new pathway for NAD+

biosynthesis was revealed. This involved the reduced form of NR (NRH) (Giroud-Gerbetant et

al. 2019; Yang et al. 2019) acting as a substrate for the enzyme adenosine kinase (AK) (Yang

et al. 2020). Critically, the action of this pathway allows for NRH treatment to increase NAD+

levels up to 10-fold in mammalian cell lines and mouse models (Giroud-Gerbetant et al. 2019;

Yang et al. 2019; Yang et al. 2020), compared to the 2-4 fold increases in NAD+ levels achieved by other precursors such as NMN and NR (Trammell et al. 2016b; Yoshino et al. 2011). The limited increase in NAD+ levels with NMN or NR treatment aligns with other arguments that

NAD homeostasis is tightly maintained, even when the supposed rate-limiting enzyme

NAMPT is over-expressed (Hara et al. 2019). Understanding how this new arm of NAD+

synthesis involving NRH and AK escapes this regulatory constraint, and importantly NAD

metabolism in general, will have important therapeutic implications.

Our work was motivated by the fact that in clinical trials, the effects of treatment with

NAD+ precursors have been mixed. While NR is well tolerated at high doses of up to 2 grams

per day, only mild benefits on blood pressure and exercise performance were demonstrated in

elderly individuals (Dolopikou et al. 2019; Martens et al. 2018). We speculate that this underwhelming results is due to the fact NAD+ precursors are commonly administered via the

oral route and must persist in the gastrointestinal tract to deliver their therapeutic payload

(Airhart et al. 2017a; Elhassan et al. 2019; Irie et al. 2020; Remie et al. 2020; Trammell et al.

2016b). Differences in the gut microbiome of test subjects may be a source of variation in

202

Chapter 6 General Discussion

results. . Currently, the contribution or effect of the microbiome has on the metabolism and

bioavailability of these compounds to the host is unknown. For example, whether the

microbiome plays a role in the appearance of deamidated metabolites such as NaAD and

NaMN following oral administration with the amidated precursor NR (Elhassan et al. 2019;

Trammell et al. 2016a) has neither been confirmed nor ruled out. The studies conducted in this

thesis sought to achieve a better understanding of the gut microbiome interactions with host

metabolism of the NAD+ precursor NMN. Observations of E. coli rapidly catabolising NMN

in the periplasmic space (Chapter 4) and antibiotic treatment increasing NMN uptake via NR

at the gut epithelium (Chapter 6) convinced us that the gut microbiome acts in competition with

the host for orally supplemented NMN. The conclusions drawn from these preclinical findings

demonstrate the importance of reconsidering the route of administration and dosing

requirements in the therapeutic use of NAD+ precursors.

6.1 Quantifying the NAD metabolome using LC-MS/MS—So close, yet [not] so far

At the start of this thesis program, LC-MS-based methods using multiple-reaction-monitoring

(MRM) to quantify the NAD+ metabolome were limited. There was no method on hand that

could simultaneously measure the different NAD metabolites and their isotopologues. The

latter is needed to trace how NMN is assimilated and to reveal the pathways by which NMN is

transported into the cell and utilised in vivo. A crucial first step in this thesis was to establish a

reliable LC-MS/MS method to quantify how isotope labelled material is propagated through

the NAD metabolome. A key challenge was achieving LC separation of the acidic metabolites

NaAD and NaMN from their amidated counterparts NAD+ and NMN, as the difference in mass

shift of only a single Dalton in mass can confound downstream analyses. Ultimately, we

developed and validated an LC-MS approach that can be used to quantify metabolism of NMN

that is isotopically labelled. This method can be applied to biological samples from in vitro and

in vivo models.

203

Chapter 6 General Discussion

The quantification of NAD metabolites has come a long way, but progress has been

most rapid in recent years. Nearly two decades ago, Evans and colleagues were the first to

report an LC-MS/MS method for the measurement of NAD+ in cellular extracts (Evans et al.

2002). They used four stable isotope labelled precursors tryptophan (Trp), quinolinic acid

(QA), nicotinic acid (NA) and nicotinamide (NAM) to measure the incorporation of labelled

moieties into NAD+ but no other NAD+ metabolites were quantified. Further, an ion trap mass

spectrometer was used which requires each selected reaction monitoring (SRM) transition to

be scanned individually, rather than a triple-quadrupole mass spectrometer which uses MRM

to measure multiple metabolites of interest in a single injection. Shortly after, Yamada and

colleagues improved NAD+ quantification using a triple-quadrupole mass spectrometry

method and were the first to directly measure NAD+ metabolites NMN and NaMN in any cell

type. They were however, limited by a subset of NAD+ metabolites where a majority of the metabolites (NADH, NADP+, NADPH, QA, NR, NaR, NA, NAM and NaAD) were below the

detection limit (Yamada et al. 2006). In 2010, Evans and colleagues developed an LC-MS/MS

based assay with hydrophilic interaction liquid chromatography (HILIC) using a Luna NH2

column which provided good retention and chromatographic resolution of a defined core set of

NAD+ metabolites (Evans et al. 2010). Following this, Trammell and colleagues developed a

highly reliable and robust LC-MS/MS method to quantify a total of 19 NAD+ metabolites

including all major metabolites involved in NAD+ biosynthesis pathways (NAM, NMN,

NAD+, NR, NA, NaR, NaMN and NaAD) (Trammell et al. 2016a). The only limitation was the

need for two separate extraction methods, acidic and basic, to accurately quantify NAD+

metabolites.

The LC-MS/MS method developed in this thesis (Chapter 3) was not adapted from the

aforementioned methods specialising in the detection of NAD metabolites, but instead from a

metabolomics protocol by Yuan and colleagues that targeted more than 250 compounds and

204

Chapter 6 General Discussion

covered all major metabolic pathways including glycolysis, the TCA cycle and the pentose

phosphate pathway (PPP) (Yuan et al. 2012). By using a type of HILIC-based chromatography,

i.e., amide phase, to retain extremely polar compounds, we achieved good resolution of the

eight core NAD+ metabolites (NAM, NMN, NAD+, NR, NA, NaR, NaMN and NaAD) in a

sensitive and reproducible manner. The strongest advantage of the method developed here was

the use of a triple quadrupole mass spectrometer in MRM mode to detect a total of 64 individual

isotopologues each with unique MRM (Q1>Q3) transitions, all in a single injection for each

sample. While there were challenges with the initial separation of the acidic metabolites NaAD

and NaMN to their amidated counterparts NAD+ and NMN respectively, we overcame this by

adjusting the pH of our mobile phase buffer from pH 9.4 to pH 5 using acetic acid and

developing deconvolution scripts to resolve the overlapping pairs of closely related metabolites using MATLAB software.

This method was then validated in primary hepatocytes isolated from mice and in

C2C12 and HEK293 mammalian cell lines to show the successful quantification of both unlabelled and labelled NAD+ metabolites can be detected in a biologically relevant in vitro model. This also served as a validation of our isotope labelling strategy, confirming that NMN is metabolised in mammalian cells via the canonical pathway, as expected. This involved the direct synthesis of NAD+ via NMNAT and recycling of NAM, the by-product of NAD+-

consuming activities through the salvage pathway, as observed by increases in labelled NAD+

and NAM isotopologues in cell lysates following supplementation with labelled NMN

isotopes.

In recent years, LC-MS-based metabolomics have become one of the most reliable and

sensitive techniques to quantify the NAD+ metabolome and further, to study NAD+ precursor

metabolism and transport into mammalian cells (Bustamante et al. 2018; Grozio et al. 2019a;

Liu et al. 2018; Ratajczak et al. 2016; Trammell et al. 2016a). Despite uncertainties around

205

Chapter 6 General Discussion

which biological mechanisms are most important for mediating the benefits of elevating NAD+

levels, the importance of rigorous measurements of NAD+ and its metabolites are increasingly

recognised. For example, the consistent appearance of elevated NaAD levels in peripheral

tissues following the administration of the NAD+ precursor NR under preclinical and clinical

conditions (Remie et al. 2020; Trammell et al. 2016a) suggest there are aspects of NAD+

metabolism which are not understood, which may have gone unnoticed without their

serendipitous observation using LC-MS-based techniques. Future studies will require further

evaluation of these pathways and additional isotope tracer studies that employ a diverse range

of biological systems are needed.

6.2 Mechanisms of NMN uptake in gut epithelial tissue — ¿Por qué no los dos?

Whether the gut epithelium takes up NMN intact has been a highly debated question in this

field. The dilemma was whether the direct or indirect route is the most efficacious absorption

route for NMN, with important implications for which NAD+ precursor has greater efficacy.

Ultimately, our results added weight to the idea that NMN is taken up indirectly as NR. Our

findings highlighted the role and physiological importance of NR in raising NAD+ levels in the cell.

Briefly recapping that ongoing debate, recent work argued that the fast pharmacokinetics of exogenously supplemented NMN is due to direct and intact transport of

NMN, where it is immediately utilised for NAD+ biosynthesis (Mills et al. 2016; Yoshino et

al. 2011). This led to the hypothesis that there is an effective NMN transporter, which remained

elusive for several years until a recent paper characterised Solute Carrier Family 12 Member 8

(SLC12A8) as a dedicated NMN transporter (Grozio et al. 2019a). The interpretation of this

work has been challenged, due to differences in the bioanalytical method used to detect these

metabolites (Schmidt and Brenner 2019), with counter-arguments (Grozio et al. 2019b) that

have fuelled this debate. A competing idea argues that NMN transport is indirect and requires

206

Chapter 6 General Discussion

dephosphorylation to NR prior to uptake, where it is re-phosphorylated by NR kinases (NRK1

or NRK2) to form NMN, as a rate-limiting step required for the utilisation of exogenous NMN

to raise intracellular NAD+ levels (Ratajczak et al. 2016).

Using isotope labelled NMN, the results presented in this thesis supported the indirect

mechanism for NMN transport into the cell involving the dephosphorylation of NMN to NR.

This was based on the significant detection of intact labelled NR following supplementation

with labelled NMN in extracts from primary hepatocytes, C2C12 and HEK293 cells (Chapter

3), supernatants of E. coli bacteria (Chapter 4) and intestinal and liver tissues of mice (Chapter

5). These findings are consistent with previous tracer studies in the human hepatocellular

carcinoma (HepG2) cell line where treatment with 18O-NMN labelled at the carboxamide

oxygen of the nicotinamide moiety resulted in the appearance of 18O-NR in the conditioned

media, parallel to the disappearance of 18O-NMN, concurrent with steady intracellular

enrichment of 18O-NR. These results support the mechanism by which exogenous NMN first

undergoes dephosphorylation to NR prior to entry into the cell (Ratajczak et al. 2016).

Our results did not entirely exclude the possibility that NMN may still be transported

intact and directly via a dedicated NMN transporter, as intact labelled NMN was detected at

earlier time points in both C2C12 and HEK293 cell lysates (Chapter 3). In future work, it may

be interesting to determine whether there are conditions under which utilisation of one

mechanism over another may be favoured in the context of treating pathological conditions or

age-related diseases. For example, a recent study reported that high expression levels of the

NMN transporter SLC12A8 and its genetic polymorphism leads to a better prognosis for

patients with pancreatic ductal adenocarcinoma (Feigin et al. 2017) and breast cancer (Kim et al. 2018) respectively. Furthermore, SLC12A8 expression is upregulated in aged ileum of mice and supplementation with NMN could maintain NAD+ in this tissue at levels comparable to

207

Chapter 6 General Discussion

those of young mice (Grozio et al. 2019a). These results highlight that NMN and its transporter

SLC12A8 could be used as potential new therapeutic targets in the treatment of certain cancers.

One argument to support the direct NMN transporter mechanism shown in previous studies is in NRK1-deficient mice, intraperitoneal injection of NMN still increased NAD+

levels in the liver, kidney, brown adipose tissue and skeletal muscle, though the statistical

significance of these results was not shown. An alternative explanation for these results could

be that expression of NRK2 is highly restricted to skeletal muscle and could compensate for

the loss of NRK1 to re-phosphorylate NR to NMN in this tissue (Ratajczak et al. 2016).

Another study treated primary myotubes from NRK1/NRK2 double knockout mice with

unlabelled NMN, with increased NAD+ levels compared to controls, though again, significance

was not reported (Fletcher et al. 2017). The strongest argument supporting the existence of

direct NMN transport is that the minute-order uptake of NMN reported in previous studies

(Grozio et al. 2019a; Mills et al. 2016; Yoshino et al. 2011) cannot be explained by the hourly

kinetics of NMN to NR conversion, which can occur over the course of 24 hours (Ratajczak et

al. 2016). Nonetheless, these nascent findings suggest the ability to raise NAD+ levels from

exogenous NMN may not be entirely dependent on its conversion to NR prior to entry, and its

assimilation into the cell via the NMN transporter may be of equal physiological importance.

6.3 Host-microbe interactions in NAD+ precursor metabolism—Friend or foe?

The results of this investigation were surprising. While we established that the gut microbiome

did play a major role in the metabolism of orally administered NMN, it was not in the direction of boosting NAD+ metabolism, as initially hypothesised. Our discovery will be an important contribution to determining which route may be most effective for delivering NAD+ precursors.

It was suggested that high level of expression of SLC12A8 in the duodenum, ileum and jejunum allows the gut to directly transport NMN (Grozio et al. 2019a). In this study, intact

208

Chapter 6 General Discussion

NMN was detected in the intestinal and liver extracts of antibiotic-treated mice only (Chapter

5). This providing evidence that the gut microbiome may limit NMN bioavailability following a bolus dose. Reducing the bacterial load in the gut translated to a greater abundance of most

NAD+ metabolites in host tissues, indicating that this impact is systemic. Remarkably, antibiotic-treated mice that did not receive a bolus of NMN still showed more abundant NAD+ metabolites. From this, we speculated that the competition between the host and microbiome is not only for NMN, but also for NA and NAM, which may be obtained from background dietary sources.

The above interpretation does not take into account the effect of antibiotic treatment on the integrity of the intestinal epithelium, as the gut microbiome is important to support the renewal of the epithelial lining, maintain optimal barrier permeability and protect against epithelial cell injury (Kang et al. 2013a; Mathewson et al. 2016; Stefka et al. 2014; Zhan et al.

2013). Methods to test gut permeability in vivo, for example by orally administering non- digestible tracer molecules and quantifying them in the urine or blood of antibiotic-treated mice, will be crucial to establish this (González-González et al. 2019).

We acknowledge that the lack of NMN detected in our studies may reflect the relatively late timing of sampling, where sampling at four hours would have missed minute-order transport of NMN into the cell as indicated in previous studies (Yoshino et al. 2011; Mills et al. 2016). With this in mind, caution is needed in the interpretation of these data. Time course experiments were not an option for us due to limited availability of our isotope labelled NMN

. The timing of sampling (4 hours) was based on previous studies that detected a peak in the deamidated metabolite NaAD in the peripheral tissues of NR-administered mice (Trammell et al. 2016b). Our data and those from others show that the NAD+ precursors NAM, NA and NR have unique pharmacokinetic profiles, where the same metabolite peaked at different times following oral administration (Trammell et al. 2016b), highlighting the importance of

209

Chapter 6 General Discussion analysing samples over a time course, rather than a single time point. Future studies should address these study limitations by sampling at tighter and more frequent time points to address the potential pharmacokinetic influences on NMN transport mechanisms.

Likewise, spatial issues could explain our inability to detect NMN. We analysed a bulk sample of the whole length of the small intestine, rather than separating and analysing different intestinal segments (duodenum, ileum, jejunum, cecum and colon). These subsections differ in the density and diversity of bacterial species, with likely differences in the metabolism of

NAD+ precursors via different routes (Gazzaniga et al. 2009). Further, expression of the putative NMN transporter SLC12A8 is not uniform across the entire intestine, and a homogenous tissue lysate may have missed these differences.

6.4 Preliminary evidence for host NMN deamidation—it’s not you, it’s me

Our investigations here were based on the hypothesis that the appearance of deamidated NAD+ intermediates NaMN and NaAD following oral administration with NR (Trammell et al.

2016b) is mediated by bacterial NMN deamidase enzymes in the gut, such as PncC. Consistent to our hypothesis, we showed that mammalian cells in culture lacked the ability to deamidate

NMN because the levels of labelled NaMN and NaAD detected in our in vitro studies were negligible (Chapter 3). In parallel, we detected labelled NaMN in E. coli culture media (Chapter

4), which indicated the conversion of NMN to NaMN by intracellular PncC and NaMN export by an unknown mechanism (Galeazzi et al. 2011). Based on these in vitro results, we concluded the conversion of NMN to NaMN is dependent on the action of bacteria. During preparation of this thesis, an elegant study by Shats and colleagues showed that orally delivered

NAM undergoes deamidation into NA by bacterial deamidase enzymes in the gut (Shats et al.

2020). This study used a combination of deuterated NAM, antibiotic treatment and the repopulation of germ-free wild-type mice with wild-type and pncA knockout E. coli to

210

Chapter 6 General Discussion

ultimately confirmed PncA-mediated conversion of NAM to NA is drives the appearance of

deamidated NAD+ metabolites in host tissues.

Interestingly, our in vivo experiments yielded different results. We found that labelled

NMN deamidated into NaMN in antibiotic treated mice, but not the control group (Chapter 5,

Figure 5.14). Validation is still required because the amount of NaMN and NaAD detected

were low compared to the amidated metabolites NR, NAM, NAD+ and NMN. A crucial difference between this study and that of Shats el al (2020) is the use of NMN rather than NAM, with differences in the route of uptake that could explain this discrepancy. In these results, when the gut microbiome was ablated, host tissues were exposed to a greater concentration of

NMN, and it is quite plausible that NMN deamidation pathways were engaged under this circumstance. This means that although NAM deamidation is definitely bacterial dependent, whether mammalian cells are incapable of NMN deamidation remains to be addressed.

Importantly, this work may contribute to understanding the changes that occur in microbial and host NAD+ metabolism—separately and as a whole—following supplementation with NAD+

precursors.

6.5 Beyond NAD metabolism—blue-sky thinking

Beyond their potential role in the metabolism of NAD+ precursors, the microbiome is well-

known to contribute to the metabolism of xenobiotics including commonly used compounds

such as metformin, gemcitabine, oxaliplatin and levodopa, which are important for the

treatment of type II diabetes, cancer and Parkinsons’s disease, respectively (Clarke et al. 2019;

Kang et al. 2013b; Pryor et al. 2020). This can have important clinical impacts.

A recent work by Rekdal and colleagues demonstrated the detrimental impact of the microbiome on xenobiotic compounds. This group uncovered fundamental pathways for the microbial metabolism of levodopa (L-dopa), an orally administered drug in the treatment of

211

Chapter 6 General Discussion

Parkinson’s disease (PD) (Rekdal et al. 2019). Microbial metabolism of L-dopa was associated

with reduced efficacy of the drug, high interpatient variability and development of adverse side

effects. Thus understanding these mechanisms and host-microbe interactions has the potential

to enhance the development of L-dopa in the treatment of PD, and it is conceivable that a

similar phenomenon may be at play in the case of therapeutic administration of NAD

precursors.

On the opposite end of the spectrum, we also see beneficial impacts for the the

microbiome in mediating the benefits of drug treatments. A recent paper suggested the

therapeutic effect of the anti-diabetic drug metformin may be due to changes in gut microbial

diversity and abundance (Pryor et al. 2020), with improvements The in microbiota-induced

maintenance of intestinal barrier integrity, and regulation of the secretion of gut hormones

involved in appetite control.

Given the biological significance of NAD+ in microbial systems, with more pathways

and enzymes involved in its biosynthesis than in mammals (Gazzaniga et al. 2009), it is

conceivable that the benefits of NAD+ precursors are in part mediated by complex host-microbe

interactions. Whether these interactions benefit or limit the efficacy of orally administered

NAD+ precursors remain to be seen, and may have important clinical implications for the use of NAD+ precursors in the treatment of age-related disease.

212

APPENDIX

Appendix

Figure A.1 Differential expression analysis of combined bacterial taxonomic levels in NMN2 cohort. Panels show MA-plots for differential testing between treatment groups. Y- axis shows the log2-fold change of the OTU. X-axis shows the mean of normalised counts of the OTU. Differential OTUs from treatments were tested using the Wald test in the R package, DESeq2, with red coloured points significant at an adjusted p.value < 0.05 using the Benjamini- Hochberg correction for multiple testing.

213

APPENDIX

Figure A.2 Differential expression analysis of combined bacterial taxonomic levels in NMN1 cohort. Panels show MA-plots for differential testing between treatment groups. Y- axis shows the log2-fold change of the OTU. X-axis shows the mean of normalised counts of the OTU. Differential OTUs from treatments were tested using the Wald test in the R package, DESeq2, with red coloured points significant at an adjusted p.value < 0.05 using the Benjamini- Hochberg correction for multiple testing.

214

APPENDIX

Nicotinamide mononucleotide (NMN) deamidation and indirect regulation of the NAD metabolome

Lynn-Jee Kim1, Timothy J. Chalmers1, Greg C. Smith1, Abhirup Das1, Eric Wing Keung Poon2, Jun Wang2,3, 4 1,5 6* 1* Simon P. Tucker , David A. Sinclair , Lake-Ee Quek , Lindsay E. Wu

1School of Medical Sciences, UNSW Sydney, NSW 2052, Australia

2GeneHarbor (Hong Kong) Biotechnologies Limited, Hong Kong Science Park, Shatin, Hong Kong SAR, China

3School of Life Sciences, The Chinese University of Hong Kong, Hong Kong SAR, China

4Jumpstart Fertility Pty Ltd, Melbourne, Victoria, Australia

5Harvard Medical School, Boston, MA, United States of America

6School of Mathematics and Statistics, The University of Sydney, NSW 2007, Australia

*These authors contributed equally to this work. Correspondence: [email protected] +61 2 9385 1621

ABSTRACT

Treatment with nicotinamide mononucleotide (NMN) is a prominent strategy to address the

age-related decline in nicotinamide adenine dinucleotide (NAD+) levels for maintaining

aspects of late-life health. It is assumed that exogenous NMN is directly incorporated into the

NAD+ metabolome in mammals via the canonical recycling pathway. Here, we show that NMN

can undergo direct deamidation and incorporation via the de novo pathway, which is in part

mediated by the gut microbiome. Surprisingly, isotope labelling studies revealed that

exogenous NMN treatment potently increased the endogenous production of unlabelled NAD

metabolites, suggesting that exogenous NMN impacts the NAD metabolome through indirect

means, rather than through its direct incorporation. This included a striking increase in

endogenous production of the metabolites nicotinic acid riboside (NaR) and nicotinamide

riboside (NR) which was amplified in antibiotics treated animals, suggesting the production of

endogenous NaR/NR through altered metabolic flux, enzyme kinetics and/or an as-yet unidentified pathway that interacts with the gut microbiome.

215 APPENDIX

Nicotinamide adenine dinucleotide (NAD+) is an essential redox cofactor central to metabolic

processes such as glycolysis, the tricarboxylic (TCA) cycle and fatty acid oxidation1,2. NAD+

is also consumed by enzymes such as the sirtuins3 and poly(ADP-ribose) polymerase (PARP) enzymes4 which are mediators of genome stability5 and DNA repair6. Given the essential role

of this metabolite, the decline in NAD+ that occurs during biological ageing7-12 and disease

states13-15 has gained attention as a target for therapeutic intervention16. Strategies to boost

NAD+ levels through supplementation with NAD precursors such as nicotinamide

mononucleotide (NMN) and nicotinamide riboside (NR) are emerging as promising

therapeutics12,16-23. Historically, dietary supplementation with the NAD precursors nicotinic acid (Na) or nicotinamide (Nam) was used to prevent chronic NAD deficiency, which causes pellagra. When these micronutrients are replete, the step converting Nam into NMN by the enzyme nicotinamide phosphoribosyltransferase (NAMPT) is rate limiting in NAD synthesis24,

and the use of NAD precursors that occur after this step, namely NMN and NR, have gained

prominence as a strategy to raise NAD+.

One surprising aspect of this strategy is the striking appearance of the deamidated metabolite nicotinic acid adenine dinucleotide (NaAD) following oral delivery with the amidated metabolite NR25. NR is phosphorylated into NMN by NR kinases (NRK1/2)26,27, and then

adenylated into NAD+ by NMNAT enzymes (NMNAT1-3)28-33, effectively bypassing NaAD,

which is an intermediate of the Preiss-Handler or de novo pathway34,35. In contrast, bacteria

have a well-characterised NMN deamidase enzyme, PncC36 that prevents the accumulation of

NMN, which inhibits the bacterial DNA ligase37-39. One theory to explain the increase in NaAD

with NR treatment25 could be that NMN and NR assimilation follows a non-canonical route

that combines steps of both microbial and mammalian processes, whereby NMN is deamidated

into NaMN or NaR prior to its uptake into mammalian tissue, and then assimilated into NAD+

via the intermediate step of NaAD. This could explain the appearance of NaAD following NR

216 APPENDIX

supplementation25, however an unexplained aspect is that the delivery of labelled NR results

in the formation of unlabelled NaAD25.

Here, we use targeted metabolomics to trace the in vitro and in vivo metabolism of strategically designed NMN isotopologues to answer these questions. We show that NMN can be incorporated following its deamidation and metabolism via the de novo route, which is in part mediated by the microbiome. We further show that ablation of the microbiome by antibiotic treatment increases the uptake and conversion of orally delivered NMN into the NAD metabolome, and that isotope labelled NMN overwhelmingly presents in intestinal tissue in the form of NaR and NR. Contrary to the assumption that exogenous NMN treatment raises NAD+

levels solely through its direct incorporation into the NAD metabolome, we show that treatment

with isotope labelled NMN increases the levels of endogenous, unlabelled NAD metabolites.

Overall, our results provide unique insights into the assimilation of orally delivered, exogenous

NMN into gastrointestinal tissue, and raise questions around how exogenous precursors alter

the NAD metabolome.

RESULTS

NMN treatment alters the de novo arm of NAD+ synthesis

According to canonical models of mammalian NAD homeostasis, the metabolism of NMN, an

amidated intermediate in the recycling pathway, does not intersect with the de novo pathway,

which utilises deamidated intermediates. Unlike mammals, bacteria present in the gut

microbiome do encode deamidase enzymes such as PncC, which deamidates NMN into

nicotinic acid mononucleotide (NaMN) for metabolism via the de novo pathway36. To test whether the gut microbiome alters the in vivo metabolism of orally administered NMN, we

used mice that were exposed to a course of antibiotics to ablate the gut microbiome (Supp. Fig.

1). These animals received a bolus of NMN (500 mg/kg) by oral gavage, and four hours later,

217 APPENDIX animals were sacrificed and tissues rapidly preserved for targeted metabolomic analysis (Fig.

1). We focused our analyses on the gastrointestinal tract (GIT) and the liver, as these two tissues have high levels of NAD synthetase (NADS) activity40, and are the primary sites of uptake and metabolism for orally delivered compounds. In agreement with previous work25, NMN treatment increased the abundance of the de-amidated metabolites NaR and NaMN in both the gastrointestinal tract (GIT) (Fig. 1a, b) and liver (Fig. 1d, e), while NaAD was increased in the liver (Fig. 1f), matching previous findings for NR25. Interestingly, this was completely abolished in antibiotic treated animals, where NMN treatment instead led to a spike in the amidated metabolites NR (Fig. 1g, j) and NMN (Fig. 1h, k), and abolished the increase in liver

NaAD (Fig. 1f). To highlight the inverse relationship between amidated and de-amidated metabolites during antibiotics treatment, the abundance of each deamidated metabolite was expressed as a ratio of its amidated counterpart (Fig. 1m-r), highlighting a profound role for the microbiome in dictating the roles of the de-amidated and amidated arms of NAD metabolism.

Strategic isotope tracing of NMN metabolism

We next sought to carefully test whether exogenous NMN was indeed undergoing direct deamidation prior to its incorporation into the NAD metabolome using isotope tracing studies.

We designed two separate isotopologues of NMN that were strategically labelled at positions that would answer our hypothesis of NMN deamidation. The first of these, designated as

NMN1, was 13C labelled at all five carbon positions of the ribose moiety for an M+5 mass shift, and 15N labelled at the pyridine ring for an overall M+6 mass shift (Fig. 2a, Supp. Fig.

2). In the last step of the de novo pathway, the enzyme NAD synthetase (NADS) amidates the carboxylic acid of NaAD using an ammonia intermediate derived from the amide group of glutamine, yielding glutamate (Fig. 2). By delivering the NMN1 (M+6) isotope in the presence of 15N-amide labelled glutamine (M+1), the presence of M+7 labelled NAD+ with an additional

218 APPENDIX

mass shift from the nicotinyl amide would indicate that the original amide N atom had been

lost during deamidation and replaced by the 15N amide from 15N-Gln, indicating incorporation

of NMN into NAD+ via prior deamidation and the de novo pathway (Fig. 2a). To complement

this experiment, we designed a second isotope, designated as NMN2, where all five carbons of

the ribose moiety were 13C labelled, and both the pyridine ring and primary amide positions

were 15N labelled, for an overall M+7 mass shift (Fig. 2b, Supp. Fig. 2). When delivered in a

separate experiment, if NMN2 (M+7) underwent deamidation prior to its incorporation, the

15N amide would be lost and replaced by an unlabelled amide from the endogenous glutamine

pool, resulting in M+6 labelled NAD+. By comparing the ratios of M+7 and M+6 labelled

NAD+ in each experiment, we could quantify the proportion of NMN that had been

incorporated into NAD+ following deamidation and assimilation by the de novo pathway. This

would be supported by comparing the ratios of M+1 and M+2 labelled Nam, which is released

by NAD+ consuming enzymes, however this interpretation would be complicated by the

recently described role of the bacterial nicotinamide (Nam) deamidase PncA in systemic

mammalian NAD+ homeostasis41. By using triple-quad mass spectrometry and multiple

reaction monitoring (MRM) for targeted metabolomics, we could further refine these data to

determine where mass shifts occurred, including whether Nam was labelled at the pyridine ring

or amide positions, and whether M+6 or M+7 labelling of NAD was from the NMN rather than

the adenosine phosphate moiety.

15N-Gln labelling of NAD+ biosynthesis

To test whether this scheme would lead to labelling of the NAD pool as anticipated, we first

used primary rat hepatocytes grown in vitro, to avoid contributions from the microbiome.

Hepatocytes were treated for 24 hr with 15N-glutamine (M+1) in the presence or absence of

NMN1 (M+6), or with NMN2 (M+7) (Fig. 3). Cell lysates were subject to targeted

metabolomic analysis to assess the degree of isotope incorporation into each metabolite (Fig.

219 APPENDIX

3a-e). Delivery of each of these isotopes yielded the expected M+6 and M+7 mass shifts of

NMN (Fig. 3b) as well as its de-phosphorylated counterpart NR (Fig. 3a), which is consistent

with the indirect transport of NMN26, though these data do not exclude the direct transport of

NMN via the putative transporter SLC12A842 – for this reason, the data in this investigation

could be interpreted as evidence for deamidation of NMN and/or NR, rather than NMN alone.

To test the strategy of using 15N glutamine to label NAD+ synthesis, we compared the ratio of

M+1 (nicotinamide labelled) to M+0 (endogenous) NAD+ (Fig. 3f). As expected, 15N-Gln

+ + treatment increased M+1 labelling of endogenous NAD , with M+1 NAD labelling in

untreated samples due to baseline levels of naturally occurring isotopes. High levels of M+1

NAD+ labelling (Fig. 3f) were observed in samples treated with NMN1, likely due to recycling

of the M+1 labelled Nam moiety (Fig. 3g) following the breakdown of NAD+ (Fig. 2), or by

cleavage of the NMN glycosidic bond between the ribose and nicotinamide groups. As

expected, treatment with NMN1 (M+6) and NMN2 (M+7) led to M+6 and M+7 labelling of

NAD+ (Fig. 3c). While we had hypothesised that in vivo treatment with NMN1 (M+6) and 15N-

Gln (M+1) would lead to M+7 labelled NAD+ due to the deamidation of NMN by the gut

microbiome, in these primary hepatocytes we observed that 15N-Gln co-treatment with NMN1

(M+6) increased the formation of M+7 labelled NAD+, when compared to NMN1 (M+6) alone

(Fig. 3h). In line with the expected recycling of labelled Nam from NAD+ (Fig. 2a), this

increased formation of M+7 labelled NAD+ during NMN1 (M+6) and 15N-Gln co-treatment

was matched by an identical increase in M+2 labelling of free Nam (Fig. 3i), which was re-

incorporated into the nicotinyl moiety of NAD+ (Fig. 3j). 15N-Gln treatment increased M+1

labelling at the amide position of Nam (Fig. 3i), but not the base N atom of the pyridine ring

(Nambase, Fig. 3k), which does not undergo substitution by NADS, with NMN1 (Fig. 2a)

treatment serving as a positive control for labelling at this position. Overall, these data verified

our system of labelling, and demonstrated the specificity of our targeted analytical approach,

220 APPENDIX

based on triple quadrupole mass spectrometry and MRM targeted metabolomics. As mammals

do not encode a known NMN, NR or Nam deamidase enzyme, we next sought to measure the

incorporation of labelled NMN into the NAD+ metabolome of bacteria, which can deamidate

NAD precursors including NMN36 and Nam43.

NMN deamidation by bacteria

Unlike mammals, bacteria rely on an NAD+ dependent DNA ligase that is inhibited by NMN,

the product of its own reaction37-39, resulting in the accumulation of intracellular NMN during

exponential growth44. This NMN is salvaged through the bacterial NMN deamidase PncC, yielding NaMN as a substrate for NAD+ synthesis by the Preiss-Handler pathway36. To model

whether extracellular NMN would undergo deamidation by bacteria, growth phase E. coli cultures were supplemented with NMN1 (M+6) (Fig. 2a) and subjected to targeted metabolomics of both cell lysates and extracellular culture media (Fig. 4). Consistent with the role of PncC in NMN metabolism in bacteria, treatment with labelled NMN resulted in the rapid incorporation of isotope labels into NaMN, with vastly increased labelling of NaMN compared to NMN (Fig. 4). Similarly, a role for the Nam deamidase PncA is strikingly reflected in the abundance of nicotinic acid (Na) compared to nicotinamide (Nam) in the cell pellet compared to the culture supernatant, where the ratio of Nam to Na in growth media was completely reversed. Overall, the avid uptake of NMN, followed by its rapid shunting into deamidated metabolites such as NaMN (Fig. 4) supported our hypothesis that the gut microbiome could contribute to the metabolism of orally administered NAD precursors such as NMN.

Antibiotic treatment alters NMN deamidation in vivo

To directly trace whether the increase in deamidated metabolites following NMN

administration (Fig. 1) was indeed due to the direct deamidation and incorporation of these

221 APPENDIX

metabolites, we next delivered our strategically designed isotopes into animals that had

similarly been treated with antibiotics to deplete the gut microbiome, as confirmed by

reductions in faecal DNA concentration (Supp. Fig. 1a), full-length 16S rRNA sequencing

(Supp. Fig. 1c) and reduced alpha diversity (Supp. Fig. 1e-r). Following antibiotic treatment,

animals received a single oral gavage (50 mg/kg) of the NMN1 (M+6) isotope (Fig. 2a), in

parallel with an i.p. bolus of 15N-Gln (M+1). Four hours later, animals were sacrificed and

tissues rapidly preserved for targeted metabolomic analysis (Fig. 5, Supp. Fig. 3-4). In a separate experiment, a different cohort of antibiotic treated animals (Supp. Fig. 1b, d, f-r)

received a bolus of the NMN2 (M+7) isotope (Fig. 2b) alone, following which tissues were

similarly collected 4 hr later for targeted metabolomic analysis (Fig. 6, Supp. Fig. 5, 6).

In tissues from animals treated with NMN1 (M+6), the deamidation of NMN could be

quantified by comparing the ratio of M+6 NAD+, which would assume incorporation following

the canonical route, to M+7 NAD+, which had incorporated an extra mass shift from co- treatment with 15N-Gln (M+1) (Fig. 2a, 7a). In this experiment, an increased ratio of M+7 to

M+6 labelled NAD+ would indicate the deamidation of NMN. The reason for using ratios,

rather than the overall amounts of each isotope (Fig. 5, Supp. Fig. 3, 4), is that they internally

control for differences in bioavailability within each animal. From the intact labelling of NAD+

in the GIT from NMN1 treatment, around 13% was M+7 labelled (Fig. 7b, c). Importantly,

these data likely underestimates incorporation via the deamidated route, as this scheme relied

on the availability of exogenous 15N-Gln relative to the endogenous pool of unlabelled Gln,

which composed only 9-13% of the total plasma Gln pool at the 4 hr timepoint (Fig. 7k).

Consistent with our hypothesis, the ratio of M+7 to M+6 labelling in the GIT was reduced in

antibiotic treated animals (Fig. 7b), suggesting reduced deamidation of orally administered

NMN when contributions from the microbiome were reduced. This was reflected by a

reduction in the ratio of M+2 to M+1base labelled Nam in the GIT, liver and plasma (Fig. 7d-f),

222 APPENDIX

however this change also likely reflected reduced contributions from the bacterial nicotinamide

de-amidase PncA43 following antibiotic treatment41. This change in Nam labelling carried into

the M+2 to M+1 ratio of NMN (Fig. 7g, h) and NAD+ (Fig. 7i, j), with reduced labelling ratios

of these recycled isotopes during antibiotic treatment likely reflecting a combination of

possible NMN/NR deamidation (Fig. 7a, b), and contributions from the bacterial Nam

deamidase PncA41.

To complement this approach, in the NMN2 (M+7) experiment (Fig. 2b), we would anticipate

that deamidation by the microbiome would result in loss of the 15N amide label, resulting in

the formation of M+6 NAD+ at the expense of M+7 NAD+ (Fig. 2b, 7l). In contrast to the

previous NMN1 experiment, the ratio of M+7 to M+6 NAD+ would instead decrease as the

rate of deamidation increased. Further, interpretation of deamidation in this NMN2 experiment

was not limited by the availability of exogenous 15N-Gln relative to a large, endogenous pool

of Gln, as was the case with the NMN1 experiment (Fig. 7a, k). In this experiment, the ratio of

M+7 to M+6 NAD+ was around 3:1 (Fig. 7m, n), suggesting that around 25% of orally

administered NMN undergoes deamidation prior to its intact incorporation into NAD+. In

agreement with the previous experiment, the ratio of M+7 to M+6 labelled NAD+ was increased in the GIT and liver of antibiotic treated animals (Fig. 7m, n). This was similarly matched by an increased ratio of M+2 to M+1base labelled Nam in the GIT (Fig. 7o), liver (Fig. 7p) and plasma (Supp. Fig. 7q) of antibiotic treated animals, reflecting decreased incorporation following deamidation, though this could instead be due the deamidation of Nam rather than

NMN. As in the previous experiment, these labels were recycled into M+2 labelled NMN (Fig.

7r, s) and NAD+ (Fig. 7t, u). In addition to differences in the isotope labelling of NAD+ (Fig.

7), these experiments replicated the inverse relationship between NaMN and NMN levels

following antibiotic treatment (Fig. 5b, e, Fig. 6b, e) observed in our earlier experiment with

unlabelled NMN (Fig. 1b, h, n). Overall, these data from two complementary isotope labelling

223 APPENDIX

approaches support the concept that orally delivered NMN or NR can undergo deamidation

prior to incorporation, and a role for the microbiome in mediating this. While these data could

in part explain the spike in the de-amidated metabolites NaMN and NaAD following treatment

with the amidated precursors NR25 or NMN (Fig. 1), it is important to note that when measured

as a proportion of the overall NAD+ pool, the contribution of both M+7 and M+6 intact labelled

NAD+ was small. Partially labelled NAD+ (M+2) was around 10-fold more abundant than intact

labelled NAD+ (M+7) (Fig. 5d, 6d, Supp. Fig. 3-6), indicating either cleavage of the labile glycosidic bond of NMN, or rapid recycling of NAD+45. Following cleavage of the glycosidic

bond to release free Nam, its deamidation in the GIT46,47 by the bacterial enzyme PncA41 also

likely contributes to these changes.

Exogenous NMN boosts the endogenous NAD+ metabolome

The abundance of partially labelled NAD+ with labelling at the Nam position only is consistent

with previous findings45 that orally delivered NMN and NR undergo cleavage at the glycosidic bond to release free Nam, with only a small proportion of orally delivered material being incorporated into tissues intact. Given that NMN and NR are overwhelmingly incorporated in the form of free Nam45, why do the downstream biological effects of NR or NMN differ from

the delivery of Nam25,48-50, a widely available nutrient present in dietary sources? One basic

assumption is that exogenous NAD precursors increase levels of NAD+ and other metabolites

due to their direct incorporation, as expected by classic mass-balance models. Following the

delivery of near 100% isotope labelled material, we found that the increase in metabolites such

as NR includes the increased production of endogenous, unlabelled metabolites. For example,

treatment with NMN1 or NMN2 in antibiotics treated animals increased levels of unlabelled

NaR (Fig. 5f, 6f, Supp. Fig. 3f, 5f) in the GIT, and increased unlabelled NR (Fig. 5b, 5p, 6b,

6p Supp. Fig. 3b, 5b) and NaAD (Fig. 5n, 6n, Supp. Fig. 4g, 6g) in the liver. Notably, treatment

with labelled NMN in antibiotics treated animals increased the production of unlabelled NR in

224 APPENDIX

the GIT by over 3-fold, where this single metabolite accounted for the vast majority of the

entire NAD metabolome in the gut, as summarised in Figure 5p and 6p.

Host-microbe interactions in the bioavailability of orally delivered NAD+ precursors

Another unexpected aspect of these data was the overall increase in levels of these metabolites as a result of antibiotics treatment alone, which more than doubled the labelling of the metabolites NMN, NR, NAD+ and Nam (Fig. 1g-i, 5a-d, 6a-d, Supp. Fig. 3h-k, 4h-k, 5h-k, 6h- k). This increase even occurred in unlabelled metabolites in animals that did not receive exogenous NMN (Supp. Fig. 3a-d, 5a-d). When NMN1 (M+6) was delivered, the incorporation of exogenous labels into NAD+ metabolites was vastly increased in antibiotic treated animals,

in the case of NR in the gut, by an order of magnitude (Fig. 5c, 5p, Supp. Fig. 3i), a trend that

was recapitulated in a separate cohort of animals receiving the NMN2 (M+7) isotope (Fig. 6b,

6p, Supp. Fig. 5i) and in animals that received unlabelled NMN (Fig. 1g). The overwhelming

abundance of NR as the dominant NAD metabolite in the GIT, especially following NMN

delivery in antibiotics treated animals is worthy of later investigation, as the abundance of this

single metabolite was greater than all other NAD+ metabolites combined, including NAD+

itself (summarised in Fig. 5p, 6p). Together, the striking increase in the uptake and overall

abundance of both labelled and unlabelled NAD metabolites in antibiotics treated animals

suggests that the microbiome could be in competition with mammalian tissue for the uptake of

orally administered, exogenous NAD precursors, and the uptake of NAD precursors from

dietary sources. Future studies should measure isotope labelling of NAD+ metabolites in faecal

contents of mice to confirm whether these compounds are being utilised by the microbiome,

rather than being excreted via other mechanisms, and should use animals in which the

microbiome has been reconstituted to control for the effects of antibiotics treatment.

225 APPENDIX

Evidence for NMN uptake following dephosphorylation into NR

NMN uptake can occur following the dephosphorylation of NMN into NR by the cell surface

enzyme CD73, prior to uptake by ENT nucleoside transporters and re-phosphorylation into

NMN inside the cell by NRK1/2 (Fig. 8)26,27. Alternatively, the solute carrier protein SLC12A8

has been described as a dedicated NMN transporter42. As with CD73 and ENT, SLC12A8 is

located on the apical side of the intestinal tissue. As both mechanisms could co-exist, the

question is the degree to which each mechanism contributes to the uptake of NMN51. If the direct route via SLC12A8 prevailed, we would expect to see high levels of labelled (M+7 or

M+6) NMN, with lesser uptake of labelled NR. In contrast, if the indirect transport of NMN following its dephosphorylation into NR was dominant, there would be a higher levels of NR labelling. In primary hepatocytes (Fig. 3), NMN1 (M+6) treatment resulted in near complete labelling of the NR pool (Fig. 2b), with slightly lesser labelling of the NMN pool (Fig. 2a).

Strikingly, in vivo treatment showed strong labelling of the NR, but not NMN pools (Fig. 5b, c; Fig. 6b, c; Supp. Fig. 3a-b, h-i; Supp. Fig. 5a-b, h-i; Supp. Fig. 7). Intact (M+6 or M+7) labelled NR levels (Supp. Fig. 3i, 5i) were five-fold higher than endogenous NR (M+0) (Fig.

5c, 6c, Supp. Fig. 3h, 5h; Fig. 8), which increased to a ten-fold greater enrichment with

antibiotics treatment (Supp. Fig. 8). In stark contrast, only around 5% of the NMN pool was

M+7 labelled (Fig. 5b, Fig. 6b; Supp. Fig. 3a, h; Supp. Fig. 5a, h, Fig. 8). The inability of

exogenous NMN to displace the endogenous NMN pool, combined with the surge of labelled

NR, suggests that NMN uptake bypasses direct transport, and would instead support the

dephosphorylation of NMN into NR to facilitate its intestinal absorption (Fig. 8). If direct

transport of NMN does occur, it (along with NR) is in competition with the microbiome, as

even when M+6 or M+7 labelling of NMN was observed at low levels, this only occurred in

antibiotic treated animals (Fig. 5b, 6b). An important caveat of this interpretation is that limited

availability of isotope labelled material meant that this study used a single time point, rather

226 APPENDIX

than a time course which also encompassed very early timepoints, possibly missing the minute-

order kinetics of direct NMN transport that were previously reported20,52.

Chronic NMN treatment does not alter microbial diversity

Given the potential role for host-microbiome interactions in the metabolism of orally delivered

NMN, it might be expected that NMN treatment would alter the composition of the

microbiome. To test this, we treated aged, 97-week old animals with chronic NMN dosing via

addition to drinking water (~400 mg/kg/day) for 8-10 weeks prior to cull7,9,11,53. 16S rRNA

long-read sequencing revealed no overall change in the alpha diversity of the gut microbiome

(Fig. 9), however there were a number of changes among individual species, available as a

Supplementary File.

DISCUSSION

Together, this work provides evidence for the partial incorporation of exogenous NMN into the NAD metabolome via the deamidated route, and for contributions of the gut microbiome to the metabolism of exogenous NMN. This is in line with recent findings around the role of

Nam deamidation by bacteria41, however a role for the microbiome in the uptake of exogenous

NAD precursors has not been described. It will be interesting to determine whether this relationship persists for other precursors, or is unique to NMN. Rather than being evidence for a “competition” relationship, differences in the uptake of exogenous NMN could reflect its role as an inhibitor of bacterial DNA ligase37-39, and bacterial mechanisms to prevent its accumulation. In addition to NMN deamidase enzymes, this could include a role for bacterial

NAD glycohydrolase enzymes, and/or bacterial SARM-like enzymes54.

While intravenous delivery of NR or NMN results in a small degree of intact assimilation into peripheral tissues such as the liver, kidney and muscle, oral delivery of NMN results in hepatic cleavage at the glycosidic bond yielding free nicotinamide due to the action of the liver 45. Our

227 APPENDIX results were in close alignment with those findings45, where the ratio of intact M+7 or M+6 to

M+0 unlabelled NAD+ was around 2%, whereas the ratio of M+2 labelled to M+0 unlabelled

NAD+, presumably as a result of incorporation of free Nam, was over 10% (Fig. 5d, k; 6d, k).

Given this evidence for the decomposition of NMN into free Nam prior to its uptake, a key question is why downstream precursors in NAD+ synthesis such as NMN and NR lead to different outcomes compared to Nam alone48-50. A surprising aspect of these results was that treatment labelled NMN led to an increase in unlabelled NAD metabolites. In the case of both

NR and NaR, treatment with NMN1 (M+6) or NMN2 (M+7) led to a stark increase in endogenous (M+0) levels, particularly in antibiotics treated animals (Fig. 5c, f, 6c, f, Supp. Fig.

3b, f, 5b, f). We also observed that exogenous NMN increased liver NaAD levels (Fig. 1f, 5g, n, 6g, n), as was previously reported for exogenous NR treatment25, however the majority of this increase was from unlabelled NaAD (Supp. Fig. 4g, 6g). These results were similar to those of Trammel et al25, where isotope tracing of double-labelled NR showed that while total NaAD levels increased by over 40-fold following NR treatment, only around 45% of this NaAD was isotope labelled – with the endogenous origin of the remaining 55% remaining unexplained.

We argue that these findings run against the assumed model that exogenous NAD+ precursors raise NAD+ levels through their direct incorporation into the NAD metabolome, and instead could suggest that treatment with exogenous precursors could indirectly trigger endogenous

NAD+ biosynthesis. The mechanism for this is not yet clear, though given the profound effect of antibiotic treatment, in particular for the overwhelming abundance of NR in the gut (Fig. 5c, p, 6c, p, Supp. Fig. 3b, 5b), it is likely to involve interplay with the gut microbiome. One possibility for the changes in endogenous NAD+ metabolites following exogenous NMN/NR treatment could be that exogenous NMN or NR treatment triggers unknown signalling pathways that indirectly alter endogenous NAD metabolism, rather than the direct incorporation of exogenous material.

228 APPENDIX

Another explanation is that increased substrate levels alter the in vivo kinetics of NAD

biosynthetic enzymes, increasing the utilisation of endogenous substrates. An important

question regarding NR in particular is how its endogenous production is increased by

exogenous NMN. NR is available from dietary sources27, and is an intermediate in the uptake

of extracellular NMN55,56. NMN accumulation in neurons can trigger cell death through the

NADase SARM157, and disposal of NMN through its adenylation into NAD+ can protect

against neuronal death58. It is possible that exogenous NMN triggers pathways that degrade

endogenous NMN into NR, which could act as a reservoir for NAD precursors, however CD73,

the NMN ectonucleotidase that carries this out, sits on the extracellular face of the plasma

membrane55,56 rather than the cytosol. In addition, the sheer molar quantity of unlabelled NR that we observed in the gut (Fig. 1g, 5c, 6c) relative to other metabolites (Fig. 5p, 6p) challenges this idea. NAD homeostasis is tightly maintained within a defined range59, and the activity of

NMNAT enzymes that carry out the last step of NAD biosynthesis is reversible60. It is possible

that exogenous NAD precursors push the equilibrium of this step in the opposite direction,

increasing endogenous NMN production from NAD+, though how intracellular NMN could be

dephosphorylated to an NaR/NR reservoir in mammals is unknown. This concept of increased

NAD breakdown during treatment with exogenous NMN in young animals is also supported

by the increased formation of unlabelled Nam in plasma (Fig. 5o, 6o). Further, while labelled

NMN treatment in vitro (Fig. 3) results in the formation of intact labelled NAD+, this occurs at

the cost of unlabelled NAD+, for a net zero change in total NAD levels (Fig. 3c), suggesting

that when NAD+ is replete, the utilisation of exogenous NMN into newly synthesised NAD+

results in a commensurate breakdown of existing material – potentially explaining the

formation of unlabelled metabolites such as NR during treatment with exogenous, labelled

NMN.

229 APPENDIX

Rather than degrading or recycling existing metabolites into NR, another possibility is that

exogenous NMN or NR could trigger a currently unknown step in mammals that leads to

endogenous NR production. While NR can be produced by the reversible phosphorolysis of

Nam and ribose-5-phosphate by a purine nucleophosphorylase (PNP) in E. coli61, this step is

irreversible in mammals56,62,63, and other potential steps involved in endogenous NR

production in mammals are unknown. Further work is needed to understand how this occurs,

for example, whether it is due to the acute up-regulation of NAD+ biosynthetic enzymes, the

time-scale by which this increase occurs, and a direct comparison of different isotope labelled

NAD+ precursors to identify which metabolites trigger the production of endogenous

metabolites under normal circumstances and during depletion of the microbiome. Regardless,

the ability to trigger the production of endogenous NAD metabolites could explain why

exogenous NR and NMN treatment lead to differences in pharmacokinetics, metabolite

production and therapeutic effects when compared to Nam alone48-50, despite their rapid

metabolism into free Nam by the liver45 (Fig. 4, 5).

Another speculative idea is the existence of a signalling pathway in the GIT that is sensitive to

both exogenous NAD precursors and to microbial metabolites, which can mediate endogenous

NAD metabolism. Metabolite sensing members of the G-protein coupled receptor (GPCR) family are putative candidates for this role as there are already GPCRs known to respond to extracellular nicotinic acid and to NAD+ itself64. One possible candidate is GPR109a, which

acts as a receptor both for nicotinic acid65 and for butyrate, released from the microbial

fermentation of dietary fibre66. This could link the observations including the deamidation of

orally derived NAD precursors into their acid equivalents, a role for microbiome depletion in

triggering the production of endogenous NAD metabolites, and evidence for the poor

incorporation of intact NR or NMN into the NAD metabolome. Identifying cell surface

230 APPENDIX receptors and downstream signalling pathways in the gut that are sensitive to both microbial metabolites and exogenous NAD+ precursors will be a key goal of testing our hypothesis around the role of exogenous metabolites in endogenous NAD+ metabolism. Together, these findings regarding the deamidation of NMN and its effects on the endogenous NAD metabolome have profound importance for the therapeutic development of NAD precursors.

231 APPENDIX

Methods

Methods are available in Supplementary Material, with raw data available on our Mendeley data site.

Acknowledgements

Funding was from the National Health and Medical Research (NHMRC) of Australia as a

Career Development Fellowship APP1122484 to LEW, and sponsored research from Jumpstart

Fertility. We wish to thank anonymous donors for philanthropic support.

Author contributions

LJK conducted experiments, analysed data, prepared figures, wrote manuscript. TJC conducted microbiome analyses. EWKP, TTC, JW prepared isotope labelled NMN. SPT and DAS provided critical feedback and interpretation. LEQ conducted experiments, extracted and analysed data, wrote manuscript. LEW conceived of and designed study, obtained funding, supervised experiments, analysed data, prepared figures, wrote manuscript.

Declaration of interests

EWKP and JW are employees and shareholders of GeneHarbor Biotechnologies. SPT is the

CEO of Jumpstart Fertility, which is developing NAD+ raising compounds for therapeutic use.

LEW and DAS are co-founders, shareholders, directors and advisors of Jumpstart Fertility and the Life Biosciences group which includes Jumpstart Fertility, Continuum Biosciences,

Senolytic Therapeutics, Selphagy, and Animal Biosciences. LEW and DAS are also advisors to and shareholders in the EdenRoc group of companies, which includes Metro Biotech NSW and Metro International Biotech, Arc-Bio, Dovetail Genomics, Claret, Revere Biosciences, and

Liberty Biosecurity. LEW is an advisor and shareholder in Intravital Pty Ltd. DAS is an

232 APPENDIX inventor on a patent application that has been licensed to Elysium Health. Updated affiliation are at https://genetics.med.harvard.edu/sinclair-test/people/sinclair-other.php.

233 APPENDIX

Figure 1

Figure 1. NMN treatment leads to the microbiome-dependent formation of deamidated

NAD+ metabolites in vivo. Mice treated with antibiotics (Abx) to ablate the gut microbiome

were administered a single dose of unlabelled nicotinamide mononucleotide (NMN) (500

mg/kg, oral gavage). Gastrointestinal tissue (GIT) (a-c) and liver tissue (d-f) were subject to targeted mass spectrometry to quantify the deamidated metabolites (a-f) nicotinic acid riboside

(NaR) (a, d), nicotinic acid mononucleotide (NaMN) (b, e) and nicotinic acid adenine

dinucleotide (NAAD) (c, f), as well as their amidated counterparts (g-l) nicotinamide riboside

234 APPENDIX

(NR) (g, j), NMN (h, k) and nicotinamide adenine dinucleotide (NAD+) (i, l). These data were

then expressed as ratios between de-amidated and amidated counterparts in GIT (m-o) and liver

(p-r). Data analysed by 2-way ANOVA with Sidak’s post-hoc test, exact p-values and F values in supplementary files. N=4-5 animals per group, *p<0.05, **p<0.01, ***p<0.001,

****p<0.0001.

235 APPENDIX

Figure 2

Figure 2. Scheme for assimilation of isotope labelled NMN into the NAD+ metabolome.

NMN (box on left) was labelled with 13C at all 5 carbon positions of the ribose moiety

(highlighted in red) and with 15N at the base (highlighted in blue) and amine (highlighted in orange) of the nicotinamide moiety for a total mass shift of M+7. The expected mass shift for

236 APPENDIX each species is shown, with steps catalyzed by mammalian enzymes in solid black lines, and steps that are only known to be carried out by bacterial enzymes shown with dashed lines.

237 APPENDIX

Figure 3

Figure 3. 15N-glutamine labelling of NAD+ synthesis. Primary hepatocytes were treated with

NMN1 (M+6) or NMN2 (M+7) isotopes (200 µM) in the presence of unlabelled or amide labelled 15N-glutamine (M+1) (4 mM) for 24 hr, to measure the degree of NAD synthesis and

the incorporation of exogenous NMN via the de novo pathway, which incorporates the amide

238 APPENDIX

label of 15N-Gln through the enzyme NAD synthetase (NADS). Exogenous NMN1 and NMN2

isotopes in the presence of 15N-Gln led to the expected isotopic labelling of a) NR, b) NMN, c)

+ NAD d) Nam and e) Gln. Ratios of f) M+1amide labelled to unlabelled NAD, g) M+7 labelled

15 NAD+, h) M+2 labelled NAD+ and i) M+1amide Nam were consistent with labelling by N-

Gln, with no change in j) M+1base labelled Nam acting as a negative control, as no change in

labelling at the ring position is expected. This resulted in l) M+2 labelled Nam during NMN1

and 15N-Gln treatment. Data analysed by 2-way ANOVA with Sidak’s post-hoc test, n=3 biological replicates.

239 APPENDIX

Figure 4

Figure 4. NMN deamidation in bacteria. Liquid cultures of E. coli OP50 bacteria were supplemented with M+6 labelled NMN1 (0.1 mM) at inoculation of a fresh culture. Samples were taken at time 0 (after NMN), 140, 160 and 180 minutes after NMN supplementation.

240 APPENDIX

Following separation of the culture supernatant (top) from the cell lysate (bottom), metabolites

were extracted and subjected to targeted LC-MS/MS mass spectrometry to detect the incorporation of the M+6 isotope label into NMN, NR, NaMN and NAD+ as well as M+1

labelling of nicotinamide (Nam) and nicotinic acid (Na) in both the culture supernatant (top)

and cell lysates (bottom). Data represents mean ± s.d. (n=3-5 samples per time point).

241 APPENDIX

Figure 5

Figure 5. Incorporation of NMN1 (M+6) isotope into the NAD metabolome in vivo.

Animals were treated with antibiotics (Abx) to deplete the microbiome, followed by an oral gavage (50 mg/kg) of NMN1 (M+6) with adjacent i.p. administration of 15N-Gln (735 mg/kg,

10ml/kg body weight). Four hr later, GIT (a-g), liver (h-n) and plasma (o) were rapidly

preserved for targeted metabolomics analysis to identify labelling of (a, h, o) Nam, (b, i) NMN,

(c, j) NR, (d, k) NAD+, (e, l) NaMN, (f, m) NaR and (g, n) NaAD. Data presented as stacked

bars of each isotopologue, with raw data points for each isotopologue overlaid on bar charts.

242 APPENDIX

(p, q) Relative molar abundance of all NAD metabolites in (p) GIT and (q) liver, including

endogenous (M+0), intact labelled and partially labelled (“part”) isotopologues. n=3-4 animals per group, n.d. = not detected.

243 APPENDIX

Figure 6

Figure 6. Incorporation of NMN2 (M+7) into the NAD metabolome in vivo. Animals were treated with antibiotics (Abx) to deplete the microbiome, followed by an oral gavage (50 mg/kg) of NMN2 (M+7). Four hr later, GIT (a-g), liver (h-n) and plasma (o) were rapidly preserved for targeted metabolomics analysis to identify labelling of (a, h, o) Nam, (b, i) NMN,

(c, j) NR, (d, k) NAD+, (e, l) NaMN, (f, m) NaR and (g, n) NaAD. Data presented as stacked bars of each isotopologue, with raw data points for each isotopologue overlaid on bar charts.

(p, q) Relative molar abundance of all NAD metabolites in (p) GIT and (q) liver, including

244 APPENDIX endogenous (M+0), intact labelled and partially labelled (“part”) isotopologues. n=3-4 animals per group n.d. = not detected.

245 APPENDIX

Figure 7

Figure 7. Contribution of the microbiome to NMN deamidation. Antibiotic treated animals were orally administered with the a) NMN1 (M+6) isotope in the presence or absence of 15N- glutamine (M+1), with the formation of M+7 labelled NAD+ or M+2 labelled Nam reflective

246 APPENDIX

of incorporation following deamidation and reamidation by the enzyme NADS. Antibiotic

treatment reduced (b, c) M+7 labelling of NAD+, (d-f) M+2 labelling of nicotinamide (Nam),

(g, h) M+2 labelling of recycled NMN, and (i-j) M+2 labelling of recycled NAD+, which

reflects the incorporation of recycled Nam. Data are expressed as ratios to M+6 or M+1 NAD+

and M+1base Nam, which are the expected isotope products of NMN or NR assimilation via the canonical (amidated) route. Incorporation of this extra label was limited by the availability of

15N-glutamine (M+1) as k) a proportion of the endogenous glutamine pool in plasma. In a separate cohort, animals were treated with l) NMN2 (M+7), where loss of the amide 15N label

+ to form M+6 labelled NAD or M+1base labelled Nam would reflect deamidation. Antibiotic treatment protected M+7 NAD+ (m, n), M+2 Nam (o-q), and M+2 NAD+ (n, o) against loss of

the NMN2 15N amide compared to untreated animals, which carried into M+2 labelling of recycled NMN, and (i-j) M+2 labelling of recycled NAD+. Tissues shown in the left column

(b, d, g, I, m, o, r, t) are gastrointestinal tract (GIT), middle column (c, e, h, j, n, p, s, u) are liver, right column (f, k, q) are plasma. NMN1 and NMN2 experiments were run in separate cohorts of animals, each measurement represents tissue from a separate animal. Comparisons of isotope treated groups with or without antibiotic treatment were analysed by Mann-Whitney

U-test, *p=0.0286, n=4 animals per group.

247 APPENDIX

Supplementary Figure 8

Supplementary Figure 8. Isotope labelled NMN treatment results in greater labelling of the NR than NMN pool, suggesting indirect uptake. (a) The two proposed mechanisms for

NMN uptake are either directly through the putative NMN transporter SLC12A8, or indirectly by dephosphorylation into NR via the ecto-5’-nucleotidase CD73 which is present on the apical side of intestinal cells. To compare the contributions of either direct or indirect transport mechanisms, the contribution of isotope labelled NMN to the overall pool of each metabolite is shown for the intestinal tissue of the NMN2 cohort (data from Fig. 6). Error bars are s.d., each data point represents tissue from a separate animal.

248 APPENDIX

Figure 9

Figure 9. Effects of chronic NMN treatment on the aged gut microbiome. Aged (97-week old) male and female mice were treated with NMN through addition to drinking water (~400 mg/kg/day) for 8-10 weeks prior to cull, and faecal microbiome samples subject to long-read

16S rRNA sequencing. (a) Stacked bar plots represent the total reads and relative abundance of bacterial taxa at the genus level for untreated or NMN treated animals, also shown as differential expression by the (b) genus level and (c) by operational taxonomic unit (OTU) level. There was no change in microbiome diversity, shown by the (d) Shannon alpha diversity index.

249 APPENDIX

SUPPLEMENTARY FIGURES

250 APPENDIX

Supplementary Figure 1

Supplementary Figure 1. Antibiotics treatment ablates the gut microbiome. Following

antibiotic treatment in the NMN1 and NMN2 mouse cohorts, (a-b) DNA was extracted from

faeces to measure changes in DNA concentration. Uniform amounts of DNA were then

subject to (c-d) full-length 16S rRNA Nanopore sequencing, with species abundance shown

here at the genus level. Sequencing revealed a reduction in (e-f) operational taxonomic units

(OTUs), the (g-h) Chao1 and (i-j) ACE species richness indices, and the (k-l) Shannon, (m-n)

Simpson, (o-p) Inverse Simpson and (q-r) Fisher diversity indices. Data shown are non-

251 APPENDIX rarefied; rarefication showed identical results (data not shown). Each data point represents samples from a separate animal

252 APPENDIX

Supplementary Figure 2

Supplementary Figure 2. Chromatogram of isotope labelled NMN and glutamine (Gln) using MRM LC-MS/MS. The above chromatograms represent individual peaks (ion count) for a) 100µM unlabelled NMN in combined NAD metabolite standard curve mixture, b) M+6 labelled NMN1 and c) M+7 labelled NMN2, as well as d) unlabelled and 15N-amide labelled glutamine.

253 APPENDIX

Supplementary Figure 3

254 APPENDIX

Supplementary Figure 3. Incorporation of NMN1 (M+6) into the GIT (raw data for Fig.

5). As in Figure 4 of main text, animals were treated with antibiotics (Abx) and M+6 isotope

labelled NMN1 followed by metabolomics analysis of intestinal tissue as described in Figure

1 of main text. Animals were also intraperitoneally injected with a concurrent bolus of 15N- amide labelled glutamine (735 mg/kg, 10ml/kg body weight). The left column (a-g) represents the abundance of unlabelled, endogenous species, middle column (h-n) represents intact label incorporation, right column (o-u) represents partial labelled species from the recycling of metabolites, as predicted in Fig. 2a. Data analysed by 2-way ANOVA with Sidak’s post-hoc test. n=3-4 animals per group, each data point represents a separate animal, error bars are SD.

255 APPENDIX

Supplementary Figure 4

256 APPENDIX

Supplementary Figure 4. Incorporation of NMN1 (M+6) into the liver (raw data for Fig.

5). As in Figure 4 of main text, animals were treated with antibiotics (Abx) and M+6 isotope

labelled NMN1 followed by metabolomics analysis of intestinal tissue as described in Figure

1 of main text. Animals were also intraperitoneally injected with a concurrent bolus of 15N-

amide labelled glutamine (735 mg/kg, 10ml/kg body weight). The left column (a-g)

represents the abundance of unlabelled, endogenous species, middle column (h-n) represents

intact label incorporation, right column (o-u) represents partial labelled species from the

recycling of metabolites, as predicted in Fig. 2a. Data analysed by 2-way ANOVA with

Sidak’s post-hoc test. n=3-4 animals per group, each data point represents a separate animal, error bars are SD.

257 APPENDIX

Supplementary Figure 5

258 APPENDIX

Supplementary Figure 5. Incorporation of NMN2 (M+7) into the GIT (raw data for Fig.

6). Animals were treated with antibiotics (Abx) and M+7 isotope labelled NMN2 followed by

metabolomics analysis of GIT tissue as described in Figure 6 of main text. Each data point

represents measurements from a different animal. The left column (a-d) represents the

abundance of unlabelled, endogenous species, middle column (e-h) represents intact label incorporation, right column (i-l) represents partial labelled species from the recycling of metabolites, as predicted in Fig. 1. Metabolites assayed are nicotinamide riboside (NR), nicotinamide adenine dinucleotide (NAD+), nicotinamide (Nam) and nicotinamide

mononucleotide (NMN). Data are analysed by two-way ANOVA with Sidak’s multiple

comparisons test. Data are mean ± s.d. (n=3-5 mice per group). *p<0.05, **p<0.01,

***p<0.001, ****p<0.0001, ns=not significant.

259 APPENDIX

Supplementary Figure 6

260 APPENDIX

Supplementary Figure 6. Incorporation of NMN2 (M+7) into the liver (raw data for Fig.

6). Animals were treated with antibiotics (Abx) and M+7 isotope labelled NMN2 followed by

metabolomics analysis of liver tissue as described in Figure 6 of main text. Each data point

represents measurements from a different animal. The left column (a-d) represents the

mononucleotide (NMN). Data are analysed by two-way ANOVA with Sidak’s multiple comparisons test. Data are mean ± s.d. (n=3-5 mice per group). *p<0.05, **p<0.01,

***p<0.001, ****p<0.0001, ns=not significant.

261 APPENDIX

SUPPLEMENTARY METHODS

262 APPENDIX

Synthesis of isotope labelled NMN

The isotopes used here were generated through a two-step process starting with the custom

synthesis of nicotinamide labelled with 15N at the nitrogen base and amide positions. This

custom isotope labelled version of nicotinamide was then used with [U5]-13C- ribose which

was 13C labelled at all five carbon positions (Cambridge Isotope Laboratories, cat. no. CLM-

3652) and ATP in an enzyme-based protocol using recombinant phosphoribosyl synthetase

(PRS) and recombinant nicotinamide phosphoribosyl transferase (NAMPT) to synthesise

NMN. The two enzymes were added into the reaction buffer that contains 1 mM ribose, 1 mM

nicotinamide, 3 mM ATP, 1 mM dithiothreitol, 10 mM MgCl2 and 50 mM Tris-HCl (pH 7.5)

and incubated at 37℃ for 30 min. The reaction was terminated with the addition of 0.01%

Trichloroacetic acid (TCA). The purification was proceeded with size-exclusion columns and

ion exchange columns. Isotope labelled NMN samples of >95% purity were concentrated by

lyophilization, and labelling confirmed by mass spectrometry (Supp. Fig. 2).

Animal experiments

All experiments were performed according to procedures approved by UNSW Animal Care

and Ethics Committee (ACEC) under ethics protocol 18/134A. The UNSW ACEC operates

under the animal ethics guidelines from the National health and Medical Research Council

(NHMRC) of Australia. Mice were fed standard chow ad libitum and housed under a 12-hr

light/12-hr dark cycle in a temperature-controlled room (22 ± 1 °C) at 80% humidity in

individually ventilated cages. Four-week old female C57BL/6J mice were acclimatised for one

week prior to treatment and body weight matched before random assignment into groups. For

antibiotic treatment, mice were administered a cocktail of antibiotics consisting of vancomycin

(0.5 g/L; Sigma SBR00001), neomycin (1 g/L; Sigma N6386), ampicillin (1 g/L; Sigma

A9393) and metronidazole (1 g/L; Sigma, M3761) (VNAM) with addition of sucrose (3 g/L;

263 APPENDIX

Bundaberg Sugar) to increase palatability for 4 days, and switched to ampicillin (1 g/L) with sucrose (3 g/L) for an additional week, which can reduce gut bacterial density by 1000-fold 67.

During treatment with the VNAM combination there was a reduction in water consumption

(below), which was the reason for the subsequent switch to ampicillin alone. Sucrose treatment

(3 g/L) was maintained as a vehicle control in animals that did not receive antibiotic treatment.

Above: Water intake and body weight of mice during antibiotics treatment.

To maintain consistency between the three cohorts presented here (unlabelled NMN – Fig. 1,

NMN1 – Fig. 5, NMN2 – Fig. 6), this antibiotic treatment protocol was used for all in vivo

experiments (Figs. 1, 5, 6). For NMN treatment, mice received a single oral gavage of NMN1

(Fig. 5) or NMN2 (Fig. 6) isotopes at 50 mg/kg, or for unlabelled NMN (Fig. 1) at 500 mg/kg,

with water vehicle used as a control. Four hours later, animals were placed under anaesthesia,

and blood was obtained by cardiac puncture, followed by euthanasia by cervical dislocation,

rapid dissection and snap freezing of tissues. Gavages were staggered between mice in

alternating treatment groups to avoid any experimental bias. On the day of cull mice were all

5-6 weeks old. Differences in NMN dosing between unlabelled and isotope labelled NMN were

due to limited availability of isotope labelled NMN.

264 APPENDIX

Blood plasma collection and preparation for mass spectrometry

Approximately 1 mL of blood was collected via cardiac puncture in anaesthetised mice (1-2%

isoflurane) into 1.5 mL eppendorf tubes prefilled with 10 µL of EDTA (0.5 M) and mixed

thoroughly with a pipette to prevent clotting. Blood samples were then spun at 2000 g for 10

min and the top layer was transferred to a new tube and snap frozen immediately in liquid

nitrogen. All samples were stored in -80°C until further processing. On the day of sample

acquisition plasma samples were thawed on ice and 20 µL of plasma was added to 80 µL of

extraction buffer (acetonitrile:methanol) with an internal standard mixture containing MES,

CSA and thymine-d4. Samples were vortexed and centrifuged at 16,000g for 10 mins at 4°C

and the supernatant was transferred to a new eppendorf tube and dried down completely using

a speed vacuum concentrator (Savant SpeedVac ® SPD140DDA, Thermo Scientific). The

resulting pellet was then resuspended in 30 µL of LC-MS-grade water and centrifuged as above and the supernatant analysed promptly by LC-MS.

Gastrointestinal and liver tissue collection and preparation for mass spectrometry

Intestinal contents (small intestine and colon without cecum) were resected and flushed with

ice cold 1 x phosphate buffered saline (PBS) to clear faecal contents before snap freezing

immediately with liquid nitrogen. Livers were resected and weighed before being rinsed in ice

cold 1x PBS and snap frozen in liquid nitrogen. All tissue samples were stored in -80 °C until

further processing. Frozen tissue samples were crushed using a mortar and pestle on liquid

nitrogen and approximately 50 mg was weighed into tubes containing ceramic beads (Precellys,

Bertin Technologies, France) to which 500 µl of cold (-30 °C) extraction buffer

(acetonitrile:methanol:water, 2:2:1) with internal standard mixture as above was added. All samples were homogenised using an automated tissue homogeniser (Precellys24, Bertin

Technologies, France) at 5,000-6,000 rpm for 15 seconds and immediately centrifuged at

265 APPENDIX

16,000g for 10 mins at 4 °C. The supernatant was transferred to a new tube and dried down completely using a speed vacuum concentrator (Savant SpeedVac ® SPD140DDA, Thermo

Scientific). All samples were resuspended in 50 µL of LC-MS-grade water, centrifuged as above and the supernatant analysed promptly by LC-MS.

Bacterial culture and NMN treatment A stab culture of the E. coli strain OP50 was inoculated into sterile Luria-Bertani (LB) broth

(10 g/L tryptone, 5 g/L yeast and 10 g/L sodium chloride in deionized water) under aseptic conditions and incubated overnight at 37℃ on a shaking platform set at 200 rpm. To measure the growth rate of E. coli, the overnight culture was sub-cultured (1:200) into sterile LB broth in a new flask and the optical density was measured at 600 nm (OD600) every 20 minutes

(approximate doubling time) and samples were collected during the early-mid exponential growth phase (OD600 < 0.70), as bacterial enzymes are more active during exponential growth phase than stationary phase 68. For samples, the overnight culture was sub-cultured (1:200) and aliquoted into smaller volumes. The cultures were then supplemented with either vehicle

(water) or M+6 labelled NMN (0.1 mM) and OD600 measured at time zero (before NMN), time zero (after NMN), and 140, 160 and 180 minutes after supplementation with NMN. The supernatant of cells was separated from the cells via centrifugation (5000 g for 10 minutes at

4℃) and stored immediately at -30℃. Meanwhile, the cell pellet was resuspended in cold (4°C) saline solution (0.9% NaCl) and centrifuged as above, to rinse away residual media before storage at -30℃. The OD600 was measured for each sample and used to normalise metabolite levels after LC-MS/MS analysis.

Primary hepatocyte culture

Primary hepatocytes were obtained as described previously 69. Male Sprague Dawley rats (250 grams, Animal Resources Centre, Perth, WA, Australia) were maintained on a 12:12 h day- night cycle, with water and food supplied ad libitum. Under deep non-recoverable general

266 APPENDIX

anaesthesia (75 mg/kg ketamine, 10 mg/kg xylazine, intraperitoneal administration) rats

underwent laparotomy. The portal vein was cannulated in situ and the liver perfused initially

with carbogen-saturated perfusion media (final: NaCl 138 mM, HEPES 25 mM, D-glucose 5.6

mM, KCl 5.4 mM, Na2HPO4, 0.34 mM, KH2PO4 0.44 mM, NaHCO3 4.17 mM, EDTA 0.5 mM, pH 7.4, 37°C, 25 ml/min flow rate). The inferior vena cava was cut to allow efflux. After 4 mins, the carbogen-saturated perfusion media was changed to the collagenase containing buffer

(final: NaCl 138 mM, HEPES 25 mM, D-glucose 5.6 mM, KCl 5.4 mM, Na2HPO4, 0.34 mM,

KH2PO4 0.44 mM, NaHCO3 4.17 mM, CaCl2 2 mM, collagenase II (Sigma, 15950-017), pH

7.4, 37°C, 25 ml/min flow rate) for 6 mins. The inferior vena cava was clamped at least 10

times during the collagenase digestion (preventing efflux) resulting in liver swelling that allows

a better digestion.

Following the collagenase digestion, the liver was removed and place on ice in 20 ml Williams’

Medium E (Life-technologies, Waltham, MA, USA). The hepatocytes were gently dispersed

in the medium and the cells filtered through a 100 µm cell strainer. Hepatocytes were washed

and diluted Williams’ Medium E and plated (6-well plates) at 106/2ml/well. After 4 hrs of

incubation the culture medium was changed to MOPS buffer (final, NaCl 128 mM, MOPS 23.9

mM, KCl 6 mM, MgSO4.7H2O 1.18 mM, CalCl2 1.29 mM, glucose 5 mM, BSA (FFA) 0.2 %,

pH 7.4) and cells incubated overnight. Following overnight incubation, cells were incubated in

M199 media without glutamine (Sigma M2154), supplemented with either unlabelled (Sigma)

or 15N-amide labelled glutamine (Cambridge Isotope Laboratories NLM-557) at 4 mM, in the presence or absence of NMN1 or NMN2 isotopes at 200 µM for 24 hr, following which samples were preserved for metabolomic analysis (Fig. 3).

267 APPENDIX

Preparation of NAD+ metabolite standards

NAD+ metabolites were serially diluted starting from a concentration of 100 µM to 0.39 µM.

The same volume (500 µL) of extraction buffer (acetonitrile:methanol:water) was added and

vortexed before centrifuging and transferring to new tube ready to be dried down as above. The subsequent steps were the same as preparing the tissue samples as above. All standards and samples were processed on the same day to reduce any experimental bias or variability.

Standard curves used to calculate absolute concentrations are shown on the following page

(Methods Fig. 2) and are available in supplementary raw data files.

268 APPENDIX

Above: NAD+ metabolite standard curves. (a) NMN (b) Nam (c) NAD+ (d) NR (e) Na (f) NaMN

(g) NaAD (h) NaR. Standard curves were serially diluted from 50 µM to 0.39 µM. Shown here

from 50 µM to 0.39 µM for all metabolites except for NR which is shown from 50 mM to 0.39

µM, due to the unexpectedly high concentrations of NR in the GIT (Fig. 1, 5, 6) and NaR from

100 µM to 0.39 µM.

Mass spectrometry

The LC-MS method was performed using 1260 Infinity LC System (Agilent) coupled to

QTRAP 5500 (AB Sciex) mass spectrometer. LC separation by gradient elution was

accomplished on an XBridge BEH amide column (100 mm x 2.1 mm, 3.5 µm particle size,

Waters Corporation) at room

temperature. For the mobile phase, Solvent A is 95%:5% H2O:acetonitrile containing 20 mM

ammonium acetate and 20 mM acetic acid, and solvent B is acetonitrile. The flow rate was 200

µL/min, with the percentage of solvent B set at 85% (0 min), 85% (0.1 min), 70% (10 min),

15% (13 min), 15% (17 min), 85% (17.5 min), 85% (30 min) (Supplementary Table 1).

Injection volume was 2.5 µL. Ion source was set at 350 °C and 4500 V with polarity switching.

Mass isotopologues of metabolites were acquired by MS2, using the unscheduled multiple reaction monitoring (MRM) mode with a dwell time of 40 ms. The MS parameters

(declustering potential, collision energy and cell exit potential) (Supplementary Table 2a) and

MRM transitions were calibrated based on the monoisotopic mass of chemical standards

(Supplementary Figure 11). Data processing was performed using MSConvert (version

3.0.18165-fd93202f5) and in-house MATLAB scripts. Deconvolution scripts were developed to resolve overlapping NAAD-NAD, NAR-NR and NAMN-NMN peaks using MATLAB’s

Optimisation Toolbox. Representative chromatograms are shown below.

269 APPENDIX

a Left: chromatograms represent individual peaks

(ion count) from 100µM

standard solutions at each

respective retention times for

b (a) NAM, NA, MeNAM,

NAD+ and NAAD, (b) NAMN

and NMN, (c) NAR and NR.

Statistical analysis for mass spectrometry

All data are presented as mean ± standard deviation (s.d.). Statistical significance was performed using a two-way ANOVA with a Sidak’s multiple comparisons test to determine differences between groups after removing outliers using the ROUT method (Q=1%). Data in

Fig. 7 were analysed by Mann-Whitney U-test between NMN isotope treated groups due to the absence of detection of labelled metabolites in animals that did not receive NMN isotopes. All statistics were performed on GraphPad Prism software (version 8.2.1). P values less than 0.05 were considered statistically significant. All data analyses are available as an .xml file available on our Mendeley data site. For in vivo experiments (Fig. 1, 5-8, Supp. Fig. 4-7), each data point

270 APPENDIX represents tissues from a separate animal, while each data point for in vitro experiments (Fig.

2, 3) represents an independent biological replicate.

DNA extraction from faecal pellets

(DeNovix®, DS-11 FX) and the purity of double-stranded DNA (dsDNA) was also determined by measuring the 260/280 ratio. All DNA extracts were stored at -80℃ until further processing by 16S rRNA sequencing.

16S Sequencing

Full length 16S rRNA genes were amplified by PCR using the Oxford Nanopore 16S

Barcoding Kit (SQK-RAB204; Oxford Nanopore Technologies, Oxford, UK). Briefly, 10 ng genomic DNA, 1 µL 16S Barcode (10 µM) and 25 µL LongAmp Taq 2X Master Mix (New

England Biolabs, Ipswich, MA, USA) were combined in a 50 µL reaction for PCR on a Bio-

PCR products were purified as per Oxford Nanopore Technologies (ONT) protocol using

AMPure XP magnetic beads (Beckman Coulter, Indianapolis, IN) and DNA quantified using the NanoDrop™ (DeNovix®, DS-11 FX). Barcodes were pooled to a total of 100 fmol in 10

μL of 10 mM Tris-HCl, pH 8.0 with 50 mM NaCl for library loading. Sequencing was

271 APPENDIX

performed using R9.4.1 ONT Flow Cells on the MinIONTM sequencing platform and data

acquired using MinKNOW software version 19.10.1 (Oxford Nanopore Technologies).

Data Analysis for 16S sequencing

Full length 16S sequencing reads acquired from MinION runs (i.e. FAST5 data) were base-

called to fastq files using Guppy software version 3.4.4 (Oxford Nanopore Technologies). Fastq

files were demultiplexed using Porechop (https://github.com/rrwick/Porechop) and trimmed to

1400bp with Trimmomatic version 0.39 70. Reads were imported to QIIME2 for dereplication

and chimeric reads screened and filtered from the dataset. Operational taxonomic unit

clustering was completed within QIIME2 version 2019.7.0 71 at 85% similarity to account for

typical sequencing errors obtained from long-read sequencing. Taxonomy was assigned to

reads using a pre-trained classifier on the SILVA 132 16S rRNA representative sequences.

Data was imported into R version 3.6.1 with qiime2R version 0.99.13

(https://github.com/jbisanz/qiime2R) for visualisation and alpha diversity analysis using raw

and rarefied data with the phyloseq version 1.30.0 72 package. Scripts for command line processing and analysis in R available in Supplementary Materials. Sequencing data has been deposited in the NCBI database Sequence Read Archive (SRA) under accession numbers

PRJNA635359.

Figures

Labelling schemes shown in Fig. 1-4, 7 were created using BioRender.com

272 APPENDIX

SUPPLEMENTARY TABLES

Supplementary Table 1.

Time (mins) Flow rate (µl/min) Buffer A (%) Buffer B (%) 0 200 15 85 0.1 200 15 85 10 200 30 70 13 200 85 30 17 200 85 30 17.5 200 15 85 30 200 15 85 Supplementary Table 2. Liquid chromatography (LC) separation gradient

Buffer A: 95:5 (v/v) HPLC H2O:Acetonitrile (CH3CN) with 20 mM ammonium acetate

(NH4OAc) + 20mM acetic acid (CH3COOH), pH 5. Buffer B: 100% Acetonitrile (CH3CN).

273 APPENDIX

Supplementary Table 2.

Metabolite_Q1_Q3 Q1 Q3 DP CE CXP RT (m/z) (m/z) (V) (V) (V) (mins) glutamine_0_0 147 44 51 73 10 13 glutamine_1_0 148 44 51 73 10 13 glutamine_1_1 148 45 51 73 10 13 glutamine_2_1 149 45 51 73 10 13 NA_0_0 124 78 70 25 10 4 NA_1_1 125 79 70 25 10 4 NaAD_0_0 665 428 139 35 38 17 NaAD_5_0 670 428 139 35 38 17 NaAD_6_0 671 428 139 35 38 17 NaAD_1_0 666 428 139 35 38 17 NaAD_5_5 670 433 139 35 38 17 NaAD_10_5 675 433 139 35 38 17 NaAD_11_5 676 433 139 35 38 17 NaAD_6_5 671 433 139 35 38 17 NaAD_6_6 671 434 139 35 38 17 NaAD_11_6 676 434 139 35 38 17 NaAD_12_6 677 434 139 35 38 17 NaAD_7_6 672 434 139 35 38 17 NaAD_1_1 666 429 139 35 38 17 NaAD_6_1 671 429 139 35 38 17 NaAD_7_1 672 429 139 35 38 17 NaAD_2_1 667 429 139 35 38 17 NaAD_7_7 672 435 139 35 38 17 NAD_0_0 664 428 33 36 31 17 NAD_5_0 669 428 33 36 31 17 NAD_6_0 670 428 33 36 31 17 NAD_7_0 671 428 33 36 31 17 NAD_1_0 665 428 33 36 31 17 NAD_2_0 666 428 33 36 31 17 NAD_5_5 669 433 33 36 31 17 NAD_6_6 670 434 33 36 31 17 NAD_7_7 671 435 33 36 31 17 NAD_1_1 665 429 33 36 31 17 NAD_2_2 666 430 33 36 31 17 NAD_10_5 674 433 33 36 31 17 NAD_11_5 675 433 33 36 31 17 NAD_12_5 676 433 33 36 31 17 NAD_6_1 670 429 33 36 31 17 NAD_7_1 671 429 33 36 31 17 NAD_8_1 672 429 33 36 31 17

274 APPENDIX

NAD_11_6 675 434 33 36 31 17 NAD_12_6 676 434 33 36 31 17 NAD_13_6 677 434 33 36 31 17 NAM_0_0 123 80 80 30 25 2 NAM_1_1 124 81 80 30 25 2 NAM_2_1 125 81 80 30 25 2 NAM_1_0 124 80 80 30 25 2 NaMN_0_0 336 124 66 28 11 19 NaMN_5_0 341 124 66 28 11 19 NaMN_6_1 342 125 66 28 11 19 NaMN_1_1 337 125 66 28 11 19 NaR_0_0 256 124 41 27 6 10 NaR_5_0 261 124 41 27 6 10 NaR_6_1 262 125 41 27 6 10 NaR_1_1 257 125 41 27 6 10 NMN_0_0 335 123 48 24 11 19 NMN_5_0 340 123 48 24 11 19 NMN_6_1 341 124 48 24 11 19 NMN_7_2 342 125 48 24 11 19 NMN_1_1 336 124 48 24 11 19 NMN_2_2 337 125 48 24 11 19 NR_0_0 255 123 64 30 13 11 NR_5_0 260 123 64 30 13 11 NR_6_1 261 124 64 30 13 11 NR_7_2 262 125 64 30 13 11 NR_1_1 256 124 64 30 13 11 NR_2_2 257 125 64 30 13 11 Thymine-d4 129 42 -115 -52 -11 2 CSA 231 80 -170 -40 -13 2 MES 196 100 140 31 25 4

Supplementary Table 2. MRM transitions and MS parameters of NAD+ metabolites and

MRM internal standards (Thymidine d4, CSA, MES). Q1: parent ion, Q3: fragment ion, MRM: multiple reaction monitoring, DP: declustering potential, CE: collision energy, CXP: collision cell exit potential.

275 APPENDIX

References (submitted work) 1 Ying, W. NAD+/NADH and NADP+/NADPH in cellular functions and cell death: regulation and biological consequences. Antioxidants & redox signaling 10, 179-206 (2008). 2 Xiao, W., Wang, R.-S., Handy, D. E. & Loscalzo, J. NAD (H) and NADP (H) redox couples and cellular energy metabolism. Antioxidants & redox signaling 28, 251-272 (2018). 3 Imai, S.-I., Armstrong, C. M., Kaeberlein, M. & Guarente, L. Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase. Nature 403, 795 (2000). 4 Kim, M. Y., Zhang, T. & Kraus, W. L. Poly (ADP-ribosyl) ation by PARP-1:PAR- laying'NAD+ into a nuclear signal. Genes & development 19, 1951-1967 (2005). 5 Haigis, M. C. & Sinclair, D. A. Mammalian sirtuins: biological insights and disease relevance. Annual Review of Pathology: Mechanisms of Disease 5, 253-295 (2010). 6 de Murcia, G. & de Murcia, J. M. Poly (ADP-ribose) polymerase: a molecular nick- sensor. Trends in biochemical sciences 19, 172-176 (1994). 7 Massudi, H. et al. Age-associated changes in oxidative stress and NAD+ metabolism in human tissue. PloS one 7, e42357 (2012). 8 Gomes, A. P. et al. Declining NAD+ induces a pseudohypoxic state disrupting nuclear- mitochondrial communication during aging. Cell 155, 1624-1638 (2013). 9 Camacho-Pereira, J. et al. CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism. Cell metabolism 23, 1127-1139 (2016). 10 Braidy, N. et al. Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats. PloS one 6, e19194 (2011). 11 Clement, J., Wong, M., Poljak, A., Sachdev, P. & Braidy, N. The plasma NAD+ metabolome is dysregulated in “normal” aging. Rejuvenation research 22, 121-130 (2019). 12 Bertoldo, M. J. et al. NAD(+) Repletion Rescues Female Fertility during Reproductive Aging. Cell Rep 30, 1670-1681 e1677, doi:10.1016/j.celrep.2020.01.058 (2020). 13 Frederick, D. W. et al. Loss of NAD homeostasis leads to progressive and reversible degeneration of skeletal muscle. Cell metabolism 24, 269-282 (2016). 14 Ryu, D. et al. NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation. Science translational medicine 8, 361ra139-361ra139 (2016). 15 Jukarainen, S. et al. Obesity is associated with low NAD+/SIRT pathway expression in adipose tissue of BMI-discordant monozygotic twins. The Journal of Clinical Endocrinology 101, 275-283 (2016). 16 Rajman, L., Chwalek, K. & Sinclair, D. A. Therapeutic potential of NAD-boosting molecules: the in vivo evidence. Cell metabolism 27, 529-547 (2018).

276 APPENDIX

17 Cantó, C. et al. The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity. Cell metabolism 15, 838-847 (2012). 18 Okabe, K., Yaku, K., Tobe, K. & Nakagawa, T. Implications of altered NAD metabolism in metabolic disorders. Journal of biomedical science 26, 34 (2019). 19 Yoshino, J., Baur, J. A. & Imai, S.-i. NAD+ intermediates: the biology and therapeutic potential of NMN and NR. Cell metabolism 27, 513-528 (2018). 20 Mills, K. F. et al. Long-term administration of nicotinamide mononucleotide mitigates age-associated physiological decline in mice. Cell metabolism 24, 795-806 (2016). 21 Tarantini, S. et al. Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice. Redox Biol 24, 101192, doi:10.1016/j.redox.2019.101192 (2019). 22 Das, A. et al. Impairment of an endothelial NAD+-H2S signaling network is a reversible cause of vascular aging. Cell 173, 74-89. e20 (2018). 23 Zhang, H. et al. NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice. Science 352, 1436-1443 (2016). 24 Revollo, J. R., Grimm, A. A. & Imai, S.-i. The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells. Journal of Biological Chemistry 279, 50754-50763 (2004). 25 Trammell, S. A. et al. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Nature communications 7, 12948 (2016). 26 Ratajczak, J. et al. NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells. Nature communications 7, 13103 (2016). 27 Bieganowski, P. & Brenner, C. Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans. Cell 117, 495-502 (2004). 28 Schweiger, M. et al. Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis. FEBS letters 492, 95-100 (2001). 29 Emanuelli, M. et al. Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase. Journal of Biological Chemistry 276, 406-412 (2001). 30 Garavaglia, S. et al. Structure of human NMN adenylyltransferase a key nuclear enzyme for NAD homeostasis. Journal of Biological Chemistry 277, 8524-8530 (2002). 31 Raffaelli, N. et al. Identification of a novel human nicotinamide mononucleotide adenylyltransferase. Biochemical and biophysical research communications 297, 835- 840 (2002).

277 APPENDIX

32 Zhang, X. et al. Structural characterization of a human cytosolic NMN/NaMN adenylyltransferase and implication in human NAD biosynthesis. Journal of Biological Chemistry 278, 13503-13511 (2003). 33 Berger, F., Lau, C., Dahlmann, M. & Ziegler, M. Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms. Journal of Biological Chemistry 280, 36334-36341 (2005). 34 Preiss, J. & Handler, P. Biosynthesis of diphosphopyridine nucleotide. I. Identification of intermediates. J Biol Chem 233, 488-492 (1958). 35 Preiss, J. & Handler, P. Biosynthesis of diphosphopyridine nucleotide II. Enzymatic aspects. Journal of Biological Chemistry 233, 493-500 (1958). 36 Galeazzi, L. et al. Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family. Journal of Biological Chemistry 286, 40365-40375 (2011). 37 Chen, X. C. et al. Development of a fluorescence resonance energy transfer assay for measuring the activity of Streptococcus pneumoniae DNA ligase, an enzyme essential for DNA replication, repair, and recombination. Analytical biochemistry 309, 232-240 (2002). 38 Geider, K. DNA Synthesis in Nucleotide‐Permeable Escherichia coli Cells: The Effects of Nucleotide Analogues on DNA Synthesis. European journal of biochemistry 27, 554-563 (1972). 39 Zimmerman, S. B. & Oshinsky, C. K. Enzymatic joining of deoxyribonucleic acid strands iii. Further purification of the deoxyribonucleic acid ligase from escherichia coli and multiple forms of the purified enzyme. Journal of Biological Chemistry 244, 4689- 4695 (1969). 40 Mori, V. et al. Metabolic profiling of alternative NAD biosynthetic routes in mouse tissues. PLoS One 9, e113939, doi:10.1371/journal.pone.0113939 (2014). 41 Shats, I. et al. Bacteria Boost Mammalian Host NAD Metabolism by Engaging the Deamidated Biosynthesis Pathway. Cell Metab 31, 564-579 e567, doi:10.1016/j.cmet.2020.02.001 (2020). 42 Grozio, A. et al. Slc12a8 is a nicotinamide mononucleotide transporter. Nature metabolism 1, 47 (2019). 43 Frothingham, R., Meeker-O'Connell, W. A., Talbot, E., George, J. W. & Kreuzer, K. N. Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA. Antimicrobial agents and chemotherapy 40, 1426-1431 (1996). 44 Olivera, B. M. & Lehman, I. Diphosphopyridine nucleotide: a cofactor for the polynucleotide-joining enzyme from Escherichia coli. Proceedings of the National Academy of Sciences of the United States of America 57, 1700 (1967). 45 Liu, L. et al. Quantitative analysis of NAD synthesis-breakdown fluxes. Cell metabolism 27, 1067-1080. e1065 (2018).

278 APPENDIX

46 Shimoyama, M. et al. Nicotinamide deamidation by microorganisms in rat stomach. Journal of bacteriology 108, 191-195 (1971). 47 Tanigawa, Y. et al. The role of microorganisms as a function of nicotinamide deamidation in rat stomach. Biochimica et Biophysica Acta (BBA)-General Subjects 201, 394-397 (1970). 48 Kawamura, T., Mori, N. & Shibata, K. beta-Nicotinamide Mononucleotide, an Anti- Aging Candidate Compound, Is Retained in the Body for Longer than Nicotinamide in Rats. J Nutr Sci Vitaminol (Tokyo) 62, 272-276, doi:10.3177/jnsv.62.272 (2016). 49 Diguet, N. et al. Nicotinamide Riboside Preserves Cardiac Function in a Mouse Model of Dilated Cardiomyopathy. Circulation 137, 2256-2273, doi:10.1161/CIRCULATIONAHA.116.026099 (2018). 50 Trammell, S. A. et al. Nicotinamide riboside is uniquely and orally bioavailable in mice and humans. Nat Commun 7, 12948, doi:10.1038/ncomms12948 (2016). 51 Wu, L. E. & Sinclair, D. A. The elusive NMN transporter is found. Nature Metabolism, 8-9 (2019). 52 Yoshino, J., Mills, K. F., Yoon, M. J. & Imai, S.-i. Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet-and age-induced diabetes in mice. Cell metabolism 14, 528-536 (2011). 53 Schultz, M. B. & Sinclair, D. A. Why NAD+ declines during aging: It’s destroyed. Cell metabolism 23, 965-966 (2016). 54 Essuman, K. et al. TIR Domain Proteins Are an Ancient Family of NAD(+)-Consuming Enzymes. Curr Biol 28, 421-430 e424, doi:10.1016/j.cub.2017.12.024 (2018). 55 Ratajczak, J. et al. NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells. Nat Commun 7, 13103, doi:10.1038/ncomms13103 (2016). 56 Belenky, P., Christensen, K. C., Gazzaniga, F., Pletnev, A. A. & Brenner, C. Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals. Quantitative basis for Urh1 and purine nucleoside phosphorylase function in NAD+ metabolism. J Biol Chem 284, 158-164, doi:10.1074/jbc.M807976200 (2009). 57 Gerdts, J., Brace, E. J., Sasaki, Y., DiAntonio, A. & Milbrandt, J. SARM1 activation triggers axon degeneration locally via NAD(+) destruction. Science 348, 453-457, doi:10.1126/science.1258366 (2015). 58 Sasaki, Y., Nakagawa, T., Mao, X., DiAntonio, A. & Milbrandt, J. NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD(+) depletion. Elife 5, doi:10.7554/eLife.19749 (2016). 59 Hara, N., Osago, H., Hiyoshi, M., Kobayashi-Miura, M. & Tsuchiya, M. Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry. PLoS One 14, e0214000, doi:10.1371/journal.pone.0214000 (2019). 60 Sorci, L. et al. Initial-rate kinetics of human NMN-adenylyltransferases: substrate and metal ion specificity, inhibition by products and multisubstrate analogues, and isozyme

279 APPENDIX

contributions to NAD+ biosynthesis. Biochemistry 46, 4912-4922, doi:10.1021/bi6023379 (2007). 61 Dong, W. R. et al. New function for Escherichia coli xanthosine phophorylase (xapA): genetic and biochemical evidences on its participation in NAD(+) salvage from nicotinamide. BMC Microbiol 14, 29, doi:10.1186/1471-2180-14-29 (2014). 62 Wielgus-Kutrowska, B., Kulikowska, E., Wierzchowski, J., Bzowska, A. & Shugar, D. Nicotinamide riboside, an unusual, non-typical, substrate of purified purine-nucleoside phosphorylases. Eur J Biochem 243, 408-414, doi:10.1111/j.1432-1033.1997.0408a.x (1997). 63 Grossman, L. & Kaplan, N. O. Nicotinamide riboside phosphorylase from human erythrocytes. II. Nicotinamide sensitivity. J Biol Chem 231, 727-740 (1958). 64 Moreschi, I. et al. Extracellular NAD+ is an agonist of the human P2Y11 purinergic receptor in human granulocytes. J Biol Chem 281, 31419-31429, doi:10.1074/jbc.M606625200 (2006). 65 Wise, A. et al. Molecular identification of high and low affinity receptors for nicotinic acid. J Biol Chem 278, 9869-9874, doi:10.1074/jbc.M210695200 (2003). 66 Macia, L. et al. Metabolite-sensing receptors GPR43 and GPR109A facilitate dietary fibre-induced gut homeostasis through regulation of the inflammasome. Nat Commun 6, 6734, doi:10.1038/ncomms7734 (2015). 67 Ubeda, C. et al. Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans. The Journal of clinical investigation 120, 4332-4341 (2010). 68 Rahman, M., Hasan, M. R., Oba, T. & Shimizu, K. Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations. Biotechnology and bioengineering 94, 585-595 (2006). 69 Chowdhury, M. K. et al. Niclosamide blocks glucagon phosphorylation of Ser552 on beta-catenin in primary rat hepatocytes via PKA signalling. Biochem J 473, 1247-1255, doi:10.1042/BCJ20160121 (2016). 70 Bolger, A. M., Lohse, M. & Usadel, B. Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30, 2114-2120 (2014). 71 Bolyen, E. et al. QIIME 2: Reproducible, interactive, scalable, and extensible microbiome data science. Report No. 2167-9843, (PeerJ Preprints, 2018). 72 McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PloS one 8 (2013).

280 References

References

Aarhus, Robert, Richard M Graeff, Deborah M Dickey, Timothy F Walseth, and Cheung Lee Hon. 1995. 'ADP-ribosyl Cyclase and CD38 Catalyze the Synthesis of a Calcium- mobilizing Metabolite from NADP+', Journal of Biological Chemistry, 270: 30327-33. Achor, Richard WP, Kenneth G Berge, Nelson W Barker, and Bernard F McKenzie. 1958. 'Treatment of hypercholesterolemia with nicotinic acid', Circulation, 17: 497-504. Agerholm, Marianne, Morten Dall, Benjamin AH Jensen, Clara Prats, Søren Madsen, Astrid L Basse, Anne-Sofie Graae, Steve Risis, Julie Goldenbaum, and Bjørn Quistorff. 2017. 'Perturbations of NAD+ salvage systems impact mitochondrial function and energy homeostasis in mouse myoblasts and intact skeletal muscle', American Journal of Physiology-Endocrinology and Metabolism, 314: E377-E95. Agledal, Line, Marc Niere, and Mathias Ziegler. 2010. 'The phosphate makes a difference: cellular functions of NADP', Redox Report, 15: 2-10. Ahel, Dragana, Zuzana Hořejší, Nicola Wiechens, Sophie E Polo, Elisa Garcia-Wilson, Ivan Ahel, Helen Flynn, Mark Skehel, Stephen C West, and Stephen P Jackson. 2009. 'Poly (ADP-ribose)–dependent regulation of DNA repair by the chromatin remodeling enzyme ALC1', Science, 325: 1240-43. Ahn, Bong-Hyun, Hyun-Seok Kim, Shiwei Song, In Hye Lee, Jie Liu, Athanassios Vassilopoulos, Chu-Xia Deng, and Toren Finkel. 2008. 'A role for the mitochondrial deacetylase Sirt3 in regulating energy homeostasis', Proceedings of the National Academy of Sciences, 105: 14447-52. Ahuja, Nidhi, Bjoern Schwer, Stefania Carobbio, David Waltregny, Brian J North, Vincenzo Castronovo, Pierre Maechler, and Eric Verdin. 2007. 'Regulation of insulin secretion by SIRT4, a mitochondrial ADP-ribosyltransferase', Journal of Biological Chemistry, 282: 33583-92. Airhart, Sophia E, Laura M Shireman, Linda J Risler, Gail D Anderson, GA Nagana Gowda, Daniel Raftery, Rong Tian, Danny D Shen, and Kevin D O’Brien. 2017a. 'An open- label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers', PloS one, 12. ———. 2017b. 'An open-label, non-randomized study of the pharmacokinetics of the nutritional supplement nicotinamide riboside (NR) and its effects on blood NAD+ levels in healthy volunteers', PloS one, 12: e0186459. Aksoy, Pinar, Thomas A White, Michael Thompson, and Eduardo N Chini. 2006. 'Regulation of intracellular levels of NAD: a novel role for CD38', Biochemical and biophysical research communications, 345: 1386-92. Al-Asmakh, Maha, and Fahad Zadjali. 2015. 'Use of germ-free animal models in microbiota- related research', J Microbiol Biotechnol, 25: 1583-88. Alano, Conrad C, Philippe Garnier, Weihai Ying, Youichirou Higashi, Tiina M Kauppinen, and Raymond A Swanson. 2010. 'NAD+ depletion is necessary and sufficient forPoly (ADP-ribose) polymerase-1-mediated neuronal death', Journal of Neuroscience, 30: 2967-78. Alberati‐Giani, Dsniela, Rico Buchli, Pari Malherbe, Clemens Broger, Gabrielle Lang, Christer Köhler, Hans‐Werner Lahm, and Andrea M Cesura. 1996. 'Isolation and expression of a cDNA clone encoding human kynureninase', European journal of biochemistry, 239: 460-68. Alhasson, Firas A, Suvarthi Das, Varun Chandrashekaran, Diptadip Dattaroy, Ratanesh Kumar Seth, Mitzi Nagarkatti, Prakash Nagarkatti, and Saurabh Chatterjee. 2017. "Adipokine

281 References

Leptin mediated NOX-2 promotes kidney inflammation in nonalcoholic fatty liver disease via miR21-dependent mesangial cell immune activation." In.: Am Assoc Immnol. Allen, Benjamin, Mark Kon, and Yaneer Bar-Yam. 2009. 'A new phylogenetic diversity measure generalizing the Shannon index and its application to phyllostomid bats', The American Naturalist, 174: 236-43. Almenara, Camila CP, José G Mill, Dalton V Vassallo, Marcelo P Baldo, and Alessandra S Padilha. 2015. 'In vitro fructose exposure overactivates NADPH oxidase and causes oxidative stress in the isolated rat aorta', Toxicology in Vitro, 29: 2030-37. Alport, A Cecil, P Ghalioungui, and G Hanna. 1938. 'Treatment of Pellagra with Nicotinamide', Lancet. Altschul, Rudolf, Abram Hoffer, and JD Stephen. 1955. 'Influence of nicotinic acid on serum cholesterol in man', Archives of Biochemistry, 54: 558-59. Alugoju, Phaniendra, Sudharshan Setra Janardhanshetty, Subasri Subaramanian, Latha Periyasamy, and Madhu Dyavaiah. 2018. 'Quercetin protects yeast Saccharomyces cerevisiae pep4 mutant from oxidative and apoptotic stress and extends chronological lifespan', Current microbiology, 75: 519-30. Aman, Yahyah, Yumin Qiu, Jun Tao, and Evandro F Fang. 2018. 'Therapeutic potential of boosting NAD+ in aging and age-related diseases', Translational Medicine of Aging, 2: 30-37. Anastasiou, Dimitrios, George Poulogiannis, John M Asara, Matthew B Boxer, Jian-kang Jiang, Min Shen, Gary Bellinger, Atsuo T Sasaki, Jason W Locasale, and Douglas S Auld. 2011. 'Inhibition of pyruvate kinase M2 by reactive oxygen species contributes to cellular antioxidant responses', Science, 334: 1278-83. Andersen, Christian, Elke Maier, Gabrielle Kemmer, Julia Blass, Anna-Karina Hilpert, Roland Benz, and Joachim Reidl. 2003. 'Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates', Journal of Biological Chemistry, 278: 24269-76. Anderson, Rozalyn M, Kevin J Bitterman, Jason G Wood, Oliver Medvedik, and David A Sinclair. 2003. 'Nicotinamide and PNC1 govern lifespan extension by calorie restriction in Saccharomyces cerevisiae', Nature, 423: 181-85. Andreoli, Anthony J, Thomas W Okita, Robert Bloom, and Thomas A Grover. 1972. 'The pyridine nucleotide cycle: Presence of a nicotinamide mononucleotide-specific glycohydrolase in Escherichiacoli', Biochemical and biophysical research communications, 49: 264-69. Angus, Annette A, Christina M Agapakis, Stephanie Fong, Shailaja Yerrapragada, Paulina Estrada-de Los Santos, Paul Yang, Nannie Song, Stephanie Kano, Jésus Caballero- Mellado, and Sergio M De Faria. 2014. 'Plant-associated symbiotic Burkholderia species lack hallmark strategies required in mammalian pathogenesis', PloS one, 9. Anisimov, Vladimir N, Lev M Berstein, Irina G Popovich, Mark A Zabezhinski, Peter A Egormin, Tatiana S Piskunova, Anna V Semenchenko, Margarita L Tyndyk, Maria N Yurova, and Irina G Kovalenko. 2011a. 'If started early in life, metformin treatment increases life span and postpones tumors in female SHR mice', Aging (Albany NY), 3: 148. Anisimov, Vladimir N, Mark A Zabezhinski, Irina G Popovich, Tatiana S Piskunova, Anna V Semenchenko, Margarita L Tyndyk, Maria N Yurova, Marina P Antoch, and Mikhail V Blagosklonny. 2010. 'Rapamycin extends maximal lifespan in cancer-prone mice', The American journal of pathology, 176: 2092-97. Anisimov, Vladimir N, Mark A Zabezhinski, Irina G Popovich, Tatiana S Piskunova, Anna V Semenchenko, Margarita L Tyndyk, Maria N Yurova, Svetlana V Rosenfeld, and

282 References

Mikhail V Blagosklonny. 2011b. 'Rapamycin increases lifespan and inhibits spontaneous tumorigenesis in inbred female mice', Cell cycle, 10: 4230-36. Annesley, Thomas M. 2003. 'Ion suppression in mass spectrometry', Clinical chemistry, 49: 1041-44. Ansari, Hifzur R, and Gajendra PS Raghava. 2010. 'Identification of NAD interacting residues in proteins', BMC bioinformatics, 11: 160. Anzenbacher, Pavel, and Eva Anzenbacherova. 2001. 'Cytochromes P450 and metabolism of xenobiotics', Cellular and Molecular Life Sciences CMLS, 58: 737-47. Arkblad, Eva L, Maxim Egorov, Michail Shakhparonov, Ludmila Romanova, Mikhail Polzikov, and Jan Rydström. 2002. 'Expression of proton-pumping nicotinamide nucleotide transhydrogenase in mouse, human brain and C. elegans', Comparative Biochemistry and Physiology Part B: Biochemistry and Molecular Biology, 133: 13- 21. Asara, John M, Jason Locasale, Xuemei Yang, Rami Rahal, Norbert Perrimon, Lewis C Cantley, Eric T Wong, and Matthew G Vander Heiden. 2009. 'A Mass Spectrometry Platform to Quantitatively Profile Cancer Cell Metabolism from Cell Lines to Tissues', Nature Biotechnology, 27: 916-17. Audebert, Marc, Bernard Salles, and Patrick Calsou. 2004. 'Involvement of poly (ADP-ribose) polymerase-1 and XRCC1/DNA ligase III in an alternative route for DNA double- strand breaks rejoining', Journal of Biological Chemistry, 279: 55117-26. ———. 2008. 'Effect of double-strand break DNA sequence on the PARP-1 NHEJ pathway', Biochemical and biophysical research communications, 369: 982-88. Audrito, Valentina, Tiziana Vaisitti, Davide Rossi, Daniela Gottardi, Giovanni D'Arena, Luca Laurenti, Gianluca Gaidano, Fabio Malavasi, and Silvia Deaglio. 2011. 'Nicotinamide blocks proliferation and induces apoptosis of chronic lymphocytic leukemia cells through activation of the p53/miR-34a/SIRT1 tumor suppressor network', Cancer research, 71: 4473-83. Avalos, José L, Katherine M Bever, and Cynthia Wolberger. 2005. 'Mechanism of sirtuin inhibition by nicotinamide: altering the NAD+ cosubstrate specificity of a Sir2 enzyme', Molecular cell, 17: 855-68. Babu, Dinesh, Georges Leclercq, Vera Goossens, Tom Vanden Berghe, Evelien Van Hamme, Peter Vandenabeele, and Romain A Lefebvre. 2015. 'Mitochondria and NADPH oxidases are the major sources of TNF-α/cycloheximide-induced oxidative stress in murine intestinal epithelial MODE-K cells', Cellular signalling, 27: 1141-58. Bahn, Andrew, Yohannes Hagos, Stefan Reuter, Daniela Balen, Hrvoje Brzica, Wolfgang Krick, Birgitta C Burckhardt, Ivan Sabolić, and Gerhard Burckhardt. 2008. 'Identification of a new urate and high affinity nicotinate transporter, hOAT10 (SLC22A13)', Journal of Biological Chemistry, 283: 16332-41. Bai, Péter, Carles Cantó, Hugues Oudart, Attila Brunyánszki, Yana Cen, Charles Thomas, Hiroyasu Yamamoto, Aline Huber, Borbála Kiss, and Riekelt H Houtkooper. 2011. 'PARP-1 inhibition increases mitochondrial metabolism through SIRT1 activation', Cell metabolism, 13: 461-68. Baldwin, Stephen A, Paul R Beal, Sylvia YM Yao, Anne E King, Carol E Cass, and James D Young. 2004. 'The equilibrative nucleoside transporter family, SLC29', Pflügers Archiv, 447: 735-43. Banks, Alexander S, Ning Kon, Colette Knight, Michihiro Matsumoto, Roger Gutiérrez- Juárez, Luciano Rossetti, Wei Gu, and Domenico Accili. 2008. 'SirT1 gain of function increases energy efficiency and prevents diabetes in mice', Cell metabolism, 8: 333-41.

283 References

Bao, Renyue, Tania Christova, Siyuan Song, Stephane Angers, Xiaojun Yan, and Liliana Attisano. 2012. 'Inhibition of tankyrases induces Axin stabilization and blocks Wnt signalling in breast cancer cells', PloS one, 7. Barbosa, Maria Thereza P, Sandra M Soares, Colleen M Novak, David Sinclair, James A Levine, Pinar Aksoy, and Eduardo Nunes Chini. 2007. 'The enzyme CD38 (a NAD glycohydrolase, EC 3.2. 2.5) is necessary for the development of diet-induced obesity', The FASEB Journal, 21: 3629-39. Barman, Scott A, Feng Chen, Yunchao Su, Christiana Dimitropoulou, Yusi Wang, John D Catravas, Weihong Han, Laszlo Orfi, Csaba Szantai-Kis, and Gyorgy Keri. 2014. 'NADPH oxidase 4 is expressed in pulmonary artery adventitia and contributes to hypertensive vascular remodeling', Arteriosclerosis, thrombosis, and vascular biology, 34: 1704-15. Baumeier, Christian, Daniel Kaiser, Jörg Heeren, Ludger Scheja, Clara John, Christoph Weise, Murat Eravci, Merit Lagerpusch, Gunnar Schulze, and Hans-Georg Joost. 2015. 'Caloric restriction and intermittent fasting alter hepatic lipid droplet proteome and diacylglycerol species and prevent diabetes in NZO mice', Biochimica et Biophysica Acta (BBA)-Molecular and Cell Biology of Lipids, 1851: 566-76. Beck, Carole, Christian Boehler, Josée Guirouilh Barbat, Marie-Elise Bonnet, Giuditta Illuzzi, Philippe Ronde, Laurent R Gauthier, Najat Magroun, Anbazhagan Rajendran, and Bernard S Lopez. 2014a. 'PARP3 affects the relative contribution of homologous recombination and nonhomologous end-joining pathways', Nucleic acids research, 42: 5616-32. Beck, Carole, Isabelle Robert, Bernardo Reina-San-Martin, Valérie Schreiber, and Françoise Dantzer. 2014b. 'Poly (ADP-ribose) polymerases in double-strand break repair: focus on PARP1, PARP2 and PARP3', Experimental cell research, 329: 18-25. Bedalov, Antonio, Maki Hirao, Jeffrey Posakony, Melisa Nelson, and Julian A Simon. 2003. 'NAD+-dependent deacetylase Hst1p controls biosynthesis and cellular NAD+ levels in Saccharomyces cerevisiae', Molecular and cellular biology, 23: 7044-54. Belenky, Peter, Kathryn C Christensen, Francesca Gazzaniga, Alexandre A Pletnev, and Charles Brenner. 2009. 'Nicotinamide riboside and nicotinic acid riboside salvage in fungi and mammals quantitative basis for urh1 and purine nucleoside phosphorylase function in NAD+ metabolism', Journal of Biological Chemistry, 284: 158-64. Belenky, Peter, Frances G Racette, Katrina L Bogan, Julie M McClure, Jeffrey S Smith, and Charles Brenner. 2007. 'Nicotinamide riboside promotes Sir2 silencing and extends lifespan via Nrk and Urh1/Pnp1/Meu1 pathways to NAD+', Cell, 129: 473-84. Bell, Charles E, Todd O Yeates, and David Eisenberg. 1997. 'Unusual conformation of nicotinamide adenine dinucleotide (NAD) bound to diphtheria toxin: a comparison with NAD bound to the oxidoreductase enzymes', Protein Science, 6: 2084-96. Bell, EL, BM Emerling, SJH Ricoult, and L Guarente. 2011. 'SirT3 suppresses hypoxia inducible factor 1α and tumor growth by inhibiting mitochondrial ROS production', Oncogene, 30: 2986-96. Benítez-Páez, Alfonso, Kevin J Portune, and Yolanda Sanz. 2016. 'Species-level resolution of 16S rRNA gene amplicons sequenced through the MinION™ portable nanopore sequencer', Gigascience, 5: 4. Benyó, Zoltán, Andreas Gille, Jukka Kero, Marion Csiky, Marie Catherine Suchánková, Rolf M Nüsing, Alexandra Moers, Klaus Pfeffer, and Stefan Offermanns. 2005. 'GPR109A (PUMA-G/HM74A) mediates nicotinic acid–induced flushing', The Journal of clinical investigation, 115: 3634-40. Berg, Rodney D. 1996. 'The indigenous gastrointestinal microflora', Trends in microbiology, 4: 430-35.

284 References

Berger, Felicitas, Corinna Lau, Mathias Dahlmann, and Mathias Ziegler. 2005. 'Subcellular compartmentation and differential catalytic properties of the three human nicotinamide mononucleotide adenylyltransferase isoforms', Journal of Biological Chemistry, 280: 36334-41. Bernofsky, Carl, and Marcella Swan. 1973. 'An improved cycling assay for nicotinamide adenine dinucleotide', Analytical biochemistry, 53: 452-58. Bertoldo, Michael J, Dave R Listijono, Wing-Hong Jonathan Ho, Angelique H Riepsamen, Dale M Goss, Dulama Richani, Xing L Jin, Saabah Mahbub, Jared M Campbell, and Abbas Habibalahi. 2020. 'NAD+ Repletion Rescues Female Fertility during Reproductive Aging', Cell reports, 30: 1670-81. e7. Bharathi, Sivakama S, Yuxun Zhang, Al-Walid Mohsen, Radha Uppala, Manimalha Balasubramani, Emanuel Schreiber, Guy Uechi, Megan E Beck, Matthew J Rardin, and Jerry Vockley. 2013. 'Sirtuin 3 (SIRT3) protein regulates long-chain acyl-CoA dehydrogenase by deacetylating conserved lysines near the active site', Journal of Biological Chemistry, 288: 33837-47. Bhatia, Rajula, and KC Calvo. 1996. 'The Sequencing, Expression, Purification, and Steady- State Kinetic Analysis of Quinolinate Phosphoribosyl Transferase from Escherichia coli', Archives of biochemistry and biophysics, 325: 270-78. Bhattarai, Sanjay, Marianne Freundlieb, Jan Pippel, Anne Meyer, Aliaa Abdelrahman, Amelie Fiene, Sang-Yong Lee, Herbert Zimmermann, Gennady G Yegutkin, and Norbert Sträter. 2015. 'α, β-methylene-ADP (AOPCP) derivatives and analogues: development of potent and selective ecto-5′-nucleotidase (CD73) inhibitors', Journal of medicinal chemistry, 58: 6248-63. Bhutia, Yangzom D, Ellappan Babu, and Vadivel Ganapathy. 2015. 'Interferon-γ induces a tryptophan-selective amino acid transporter in human colonic epithelial cells and mouse dendritic cells', Biochimica et Biophysica Acta (BBA)-Biomembranes, 1848: 453-62. Biagi, Elena, Lotta Nylund, Marco Candela, Rita Ostan, Laura Bucci, Elisa Pini, Janne Nikkïla, Daniela Monti, Reetta Satokari, and Claudio Franceschi. 2010. 'Through ageing, and beyond: gut microbiota and inflammatory status in seniors and centenarians', PloS one, 5. Bieganowski, Pawel, and Charles Brenner. 2004. 'Discoveries of nicotinamide riboside as a nutrient and conserved NRK genes establish a Preiss-Handler independent route to NAD+ in fungi and humans', Cell, 117: 495-502. Billington, Richard A, Cristina Travelli, Emanuela Ercolano, Ubaldina Galli, Cintia Blasi Roman, Ambra A Grolla, Pier Luigi Canonico, Fabrizio Condorelli, and Armando A Genazzani. 2008. 'Characterization of NAD uptake in mammalian cells', Journal of Biological Chemistry, 283: 6367-74. Bindesbøll, Christian, Susanna Tan, Debbie Bott, Tiffany Cho, Laura Tamblyn, Laura MacPherson, Line Grønning-Wang, Hilde Irene Nebb, and Jason Matthews. 2016. 'TCDD-inducible poly-ADP-ribose polymerase (TIPARP/PARP7) mono-ADP- ribosylates and co-activates liver X receptors', Biochemical Journal, 473: 899-910. Bitterman, Kevin J, Rozalyn M Anderson, Haim Y Cohen, Magda Latorre-Esteves, and David A Sinclair. 2002. 'Inhibition of silencing and accelerated aging by nicotinamide, a putative negative regulator of yeast sir2 and human SIRT1', Journal of Biological Chemistry, 277: 45099-107. Bitto, Alessandro, Takashi K Ito, Victor V Pineda, Nicolas J LeTexier, Heather Z Huang, Elissa Sutlief, Herman Tung, Nicholas Vizzini, Belle Chen, and Kaleb Smith. 2016. 'Transient rapamycin treatment can increase lifespan and healthspan in middle-aged mice', Elife, 5: e16351.

285 References

Blacher, Eran, Bar Ben Baruch, Ayelet Levy, Nurit Geva, Keith D Green, Sylvie Garneau‐ Tsodikova, Micha Fridman, and Reuven Stein. 2015. 'Inhibition of glioma progression by a newly discovered CD38 inhibitor', International journal of cancer, 136: 1422-33. Blakley, RL, and E Vitols. 1968. 'The control of nucleotide biosynthesis', Annual review of biochemistry, 37: 201-24. Bock, Florian J, Tanya T Todorova, and Paul Chang. 2015. 'RNA regulation by poly (ADP- ribose) polymerases', Molecular cell, 58: 959-69. Boehler, Christian, Laurent R Gauthier, Oliver Mortusewicz, Denis S Biard, Jean-Michel Saliou, Anne Bresson, Sarah Sanglier-Cianferani, Susan Smith, Valérie Schreiber, and François Boussin. 2011. 'Poly (ADP-ribose) polymerase 3 (PARP3), a newcomer in cellular response to DNA damage and mitotic progression', Proceedings of the National Academy of Sciences, 108: 2783-88. Bogan, Katrina L, and Charles Brenner. 2010. '5′-Nucleotidases and their new roles in NAD+ and phosphate metabolism', New Journal of Chemistry, 34: 845-53. Bogan, Katrina L, Charles Evans, Peter Belenky, Peng Song, Charles F Burant, Robert Kennedy, and Charles Brenner. 2009. 'Identification of Isn1 and Sdt1 as glucose-and vitamin-regulated nicotinamide mononucleotide and nicotinic acid mononucleotide 5′- nucleotidases responsible for production of nicotinamide riboside and nicotinic acid riboside', Journal of Biological Chemistry, 284: 34861-69. Bolger, Anthony M, Marc Lohse, and Bjoern Usadel. 2014. 'Trimmomatic: a flexible trimmer for Illumina sequence data', Bioinformatics, 30: 2114-20. Bolyen, Evan, Jai Ram Rideout, Matthew R Dillon, Nicholas A Bokulich, Christian Abnet, Gabriel A Al-Ghalith, Harriet Alexander, Eric J Alm, Manimozhiyan Arumugam, and Francesco Asnicar. 2018. "QIIME 2: Reproducible, interactive, scalable, and extensible microbiome data science." In.: PeerJ Preprints. Borea, Pier Andrea, Stefania Gessi, Stefania Merighi, Fabrizio Vincenzi, and Katia Varani. 2018. 'Pharmacology of adenosine receptors: the state of the art', Physiological reviews, 98: 1591-625. Boslett, James, Craig Hemann, Yong Juan Zhao, Hon-Cheung Lee, and Jay L Zweier. 2017. 'Luteolinidin protects the postischemic heart through CD38 inhibition with preservation of NAD (P)(H)', Journal of Pharmacology and Experimental Therapeutics, 361: 99- 108. Boylston, Jennifer A, Junhui Sun, Yong Chen, Marjan Gucek, Michael N Sack, and Elizabeth Murphy. 2015. 'Characterization of the cardiac succinylome and its role in ischemia– reperfusion injury', Journal of molecular and cellular cardiology, 88: 73-81. Brachmann, Carrie Baker, Joyce M Sherman, Scott E Devine, Elizabeth E Cameron, Lorraine Pillus, and Jef D Boeke. 1995. 'The SIR2 gene family, conserved from bacteria to humans, functions in silencing, cell cycle progression, and chromosome stability', Genes & development, 9: 2888-902. Braidy, Nady, Jade Berg, James Clement, Fatemeh Khorshidi, Anne Poljak, Tharusha Jayasena, Ross Grant, and Perminder Sachdev. 2018. 'Role of nicotinamide adenine dinucleotide and related precursors as therapeutic targets for age-related degenerative diseases: rationale, biochemistry, pharmacokinetics, and outcomes', Antioxidants & redox signaling, 30: 251-94. ———. 2019. 'Role of nicotinamide adenine dinucleotide and related precursors as therapeutic targets for age-related degenerative diseases: rationale, biochemistry, pharmacokinetics, and outcomes', Antioxidants & redox signaling, 30: 251-94. Braidy, Nady, Gilles J Guillemin, Hussein Mansour, Tailoi Chan-Ling, Anne Poljak, and Ross Grant. 2011a. 'Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats', PloS one, 6: e19194.

286 References

———. 2011b. 'Age related changes in NAD+ metabolism oxidative stress and Sirt1 activity in wistar rats', PloS one, 6. Braidy, Nady, Anne Poljak, Ross Grant, Tharusha Jayasena, Hussein Mansour, Tailoi Chan- Ling, Gilles J Guillemin, George Smythe, and Perminder Sachdev. 2014. 'Mapping NAD+ metabolism in the brain of ageing Wistar rats: potential targets for influencing brain senescence', Biogerontology, 15: 177-98. Brázda, Václav, Rob C Laister, Eva B Jagelská, and Cheryl Arrowsmith. 2011. 'Cruciform structures are a common DNA feature important for regulating biological processes', BMC molecular biology, 12: 33. Breton, Jérôme, Nilla Avanzi, Simona Magagnin, Nevie Covini, Giovanni Magistrelli, Liviana Cozzi, and Antonella Isacchi. 2000. 'Functional characterization and mechanism of action of recombinant human kynurenine 3‐hydroxylase', European journal of biochemistry, 267: 1092-99. Brown, B Greg, and Xue-Qiao Zhao. 2008. 'Nicotinic acid, alone and in combinations, for reduction of cardiovascular risk', The American journal of cardiology, 101: S58-S62. Brown, Kevin D, Sadia Maqsood, Jing-Yi Huang, Yong Pan, William Harkcom, Wei Li, Anthony Sauve, Eric Verdin, and Samie R Jaffrey. 2014. 'Activation of SIRT3 by the NAD+ precursor nicotinamide riboside protects from noise-induced hearing loss', Cell metabolism, 20: 1059-68. Bruce-Keller, Annadora J, Christy L White, Sunita Gupta, Alecia G Knight, Paul J Pistell, Donald K Ingram, Christopher D Morrison, and Jeffrey N Keller. 2010. 'NOX activity in brain aging: exacerbation by high fat diet', Free Radical Biology and Medicine, 49: 22-30. Bruins, Andries P. 1998. 'Mechanistic aspects of electrospray ionization', Journal of Chromatography A, 794: 345-57. Bruzzone, Santina, Lucrezia Guida, Elena Zocchi, Luisa Franco, and Antonio De Flora. 2001. 'Connexin 43 hemi channels mediate Ca2+-regulated transmembrane NAD+ fluxes in intact cells', The FASEB Journal, 15: 10-12. Burger, Joep MS, Séverine D Buechel, and Tadeusz J Kawecki. 2010. 'Dietary restriction affects lifespan but not cognitive aging in Drosophila melanogaster', Aging cell, 9: 327- 35. Burns, Brendan P, George L Mendz, and Stuart L Hazell. 1998. 'Methods for the measurement of a bacterial enzyme activity in cell lysates and extracts', Biological procedures online, 1: 17. Burns, Dennis M, and Ifor R Beacham. 1986. 'Nucleotide sequence and transcriptional analysis of the E. coli ush A gene, encoding periplasmic UDP-sugar hydrolase (5'-nucleotidase): regulation of the ushA gene, and the signal sequence of its encoded protein product', Nucleic acids research, 14: 4325-42. Bustamante, Sonia, Tharusha Jayasena, Dulama Richani, Robert Bruce Gilchrist, Lindsay E Wu, David A Sinclair, Perminder Singh Sachdev, and Nady Braidy. 2018. 'Quantifying the cellular NAD+ metabolome using a tandem liquid chromatography mass spectrometry approach', Metabolomics, 14: 15. Bütepage, Mareike, Laura Eckei, Patricia Verheugd, and Bernhard Lüscher. 2015. 'Intracellular mono-ADP-ribosylation in signaling and disease', Cells, 4: 569-95. Cabreiro, Filipe, Catherine Au, Kit-Yi Leung, Nuria Vergara-Irigaray, Helena M Cochemé, Tahereh Noori, David Weinkove, Eugene Schuster, Nicholas DE Greene, and David Gems. 2013. 'Metformin retards aging in C. elegans by altering microbial folate and methionine metabolism', Cell, 153: 228-39.

287 References

Cadoni, Erika, Fabio Marongiu, Maura Fanti, Monica Serra, and Ezio Laconi. 2017. 'Caloric restriction delays early phases of carcinogenesis via effects on the tissue microenvironment', Oncotarget, 8: 36020. Calcraft, Peter J, Margarida Ruas, Zui Pan, Xiaotong Cheng, Abdelilah Arredouani, Xuemei Hao, Jisen Tang, Katja Rietdorf, Lydia Teboul, and Kai-Ting Chuang. 2009. 'NAADP mobilizes calcium from acidic organelles through two-pore channels', Nature, 459: 596-600. Camacho-Pereira, Juliana, Mariana G Tarragó, Claudia CS Chini, Veronica Nin, Carlos Escande, Gina M Warner, Amrutesh S Puranik, Renee A Schoon, Joel M Reid, and Antonio Galina. 2016. 'CD38 dictates age-related NAD decline and mitochondrial dysfunction through an SIRT3-dependent mechanism', Cell metabolism, 23: 1127-39. Cang, Chunlei, Yandong Zhou, Betsy Navarro, Young-jun Seo, Kimberly Aranda, Lucy Shi, Shyuefang Battaglia-Hsu, Itzhak Nissim, David E Clapham, and Dejian Ren. 2013. 'mTOR regulates lysosomal ATP-sensitive two-pore Na+ channels to adapt to metabolic state', Cell, 152: 778-90. Cantó, Carles, Zachary Gerhart-Hines, Jerome N Feige, Marie Lagouge, Lilia Noriega, Jill C Milne, Peter J Elliott, Pere Puigserver, and Johan Auwerx. 2009. 'AMPK regulates energy expenditure by modulating NAD+ metabolism and SIRT1 activity', Nature, 458: 1056-60. Cantó, Carles, and Riekelt H Houtkooper. 2016. 'Sirtuins and aging.' in, Sirtuins (Springer). Cantó, Carles, Riekelt H Houtkooper, Eija Pirinen, Dou Y Youn, Maaike H Oosterveer, Yana Cen, Pablo J Fernandez-Marcos, Hiroyasu Yamamoto, Pénélope A Andreux, and Philippe Cettour-Rose. 2012. 'The NAD+ precursor nicotinamide riboside enhances oxidative metabolism and protects against high-fat diet-induced obesity', Cell metabolism, 15: 838-47. Canto, Carles, Keir J Menzies, and Johan Auwerx. 2015. 'NAD+ metabolism and the control of energy homeostasis: a balancing act between mitochondria and the nucleus', Cell metabolism, 22: 31-53. Cappelli, Enrico, Richard Taylor, Michela Cevasco, Angelo Abbondandolo, Keith Caldecott, and Guido Frosina. 1997. 'Involvement of XRCC1 and DNA ligase III gene products in DNA base excision repair', Journal of Biological Chemistry, 272: 23970-75. Carlier, Aurelien, Kirsty Agnoli, Gabriella Pessi, Angela Suppiger, Christian Jenul, Nadine Schmid, Burkhard Tümmler, Marta Pinto-Carbo, and Leo Eberl. 2014. 'Genome sequence of Burkholderia cenocepacia H111, a cystic fibrosis airway isolate', Genome Announc., 2: e00298-14. Carmody, Rachel N, and Peter J Turnbaugh. 2014. 'Host-microbial interactions in the metabolism of therapeutic and diet-derived xenobiotics', The Journal of clinical investigation, 124: 4173-81. Caroline, H Yi, Heling Pan, Jan Seebacher, Il-Ho Jang, Sven G Hyberts, Gregory J Heffron, Matthew G Vander Heiden, Renliang Yang, Fupeng Li, and Jason W Locasale. 2011. 'Metabolic regulation of protein N-alpha-acetylation by Bcl-xL promotes cell survival', Cell, 146: 607-20. Caron, Annabelle Z, Xiaohong He, Walid Mottawea, Erin L Seifert, Karen Jardine, Danielle Dewar-Darch, Greg O Cron, Mary-Ellen Harper, Alain Stintzi, and Michael W McBurney. 2014. 'The SIRT1 deacetylase protects mice against the symptoms of metabolic syndrome', The FASEB Journal, 28: 1306-16. Carson, DA, S Seto, and DB Wasson. 1987. 'Pyridine nucleotide cycling and poly (ADP- ribose) synthesis in resting human lymphocytes', The Journal of Immunology, 138: 1904-07.

288 References

Carter-O’Connell, Ian, Haihong Jin, Rory K Morgan, Roko Zaja, Larry L David, Ivan Ahel, and Michael S Cohen. 2016. 'Identifying family-member-specific targets of mono- ARTDs by using a chemical genetics approach', Cell reports, 14: 621-31. Carter-O’Connell, Ian, Anke Vermehren-Schmaedick, Haihong Jin, Rory K Morgan, Larry L David, and Michael S Cohen. 2018. 'Combining Chemical Genetics with Proximity- Dependent Labeling Reveals Cellular Targets of Poly (ADP-ribose) Polymerase 14 (PARP14)', ACS chemical biology, 13: 2841-48. Casabona, G, L Sturiale, MR L'Episcopo, G Raciti, A Fazzio, MG Sarpietro, AA Genazzani, A Cambria, and F Nicoletti. 1995. 'HPLC analysis of cyclic adenosine diphosphate ribose and adenosine diphosphate ribose: determination of NAD+ metabolites in hippocampal membranes', The Italian journal of biochemistry, 44: 258-68. Catara, Giuliana, Giovanna Grimaldi, Laura Schembri, Daniela Spano, Gabriele Turacchio, Matteo Lo Monte, Andrea Rosario Beccari, Carmen Valente, and Daniela Corda. 2017. 'PARP1-produced poly-ADP-ribose causes the PARP12 translocation to stress granules and impairment of Golgi complex functions', Scientific reports, 7: 1-17. Caton, PW, J Kieswich, MM Yaqoob, MJ Holness, and MC Sugden. 2011. 'Nicotinamide mononucleotide protects against pro-inflammatory cytokine-mediated impairment of mouse islet function', Diabetologia, 54: 3083-92. Catterson, James H, Mobina Khericha, Miranda C Dyson, Alec J Vincent, Rebecca Callard, Steven M Haveron, Arjunan Rajasingam, Mumtaz Ahmad, and Linda Partridge. 2018. 'Short-term, intermittent fasting induces long-lasting gut health and TOR-independent lifespan extension', Current Biology, 28: 1714-24. e4. Ceciliani, Fabrizio, Tiziana Caramori, Severino Ronchi, Gabriella Tedeschi, Michele Mortarino, and Alessandro Galizzi. 2000. 'Cloning, overexpression, and purification of Escherichia coli quinolinate synthetase', Protein expression and purification, 18: 64- 70. Cerutti, Raffaele, Eija Pirinen, Costanza Lamperti, Silvia Marchet, Anthony A Sauve, Wei Li, Valerio Leoni, Eric A Schon, Françoise Dantzer, and Johan Auwerx. 2014. 'NAD+- dependent activation of Sirt1 corrects the phenotype in a mouse model of mitochondrial disease', Cell metabolism, 19: 1042-49. Cha, Eun Jung, Sang Jae Noh, Keun Sang Kwon, Chan Young Kim, Byung-Hyun Park, Ho Sung Park, Ho Lee, Myoung Ja Chung, Myoung Jae Kang, and Dong Geun Lee. 2009. 'Expression of DBC1 and SIRT1 is associated with poor prognosis of gastric carcinoma', Clinical Cancer Research, 15: 4453-59. Cha, Hye-Ran, Sun-Young Chang, Jae-Hoon Chang, Jae-Ouk Kim, Jin-Young Yang, Chang- Hoon Kim, and Mi-Na Kweon. 2010. 'Downregulation of Th17 cells in the small intestine by disruption of gut flora in the absence of retinoic acid', The Journal of Immunology, 184: 6799-806. Cha, Jin Joo, Hye Sook Min, Ki Tae Kim, Jung Eun Kim, Jung Yeon Ghee, Hyun Wook Kim, Ji Eun Lee, Jee Young Han, Gayoung Lee, and Hun Joo Ha. 2017. 'APX-115, a first- in-class pan-NADPH oxidase (Nox) inhibitor, protects db/db mice from renal injury', Laboratory Investigation, 97: 419-31. Chalkiadaki, Angeliki, and Leonard Guarente. 2012. 'High-fat diet triggers inflammation- induced cleavage of SIRT1 in adipose tissue to promote metabolic dysfunction', Cell metabolism, 16: 180-88. Chambon, P, JD Weill, and P Mandel. 1963. 'Nicotinamide mononucleotide activation of a new DNA-dependent polyadenylic acid synthesizing nuclear enzyme', Biochemical and biophysical research communications, 11: 39-43.

289 References

Chasovskikh, Sergey, Alexandre Dimtchev, Mark Smulson, and Anatoly Dritschilo. 2005. 'DNA transitions induced by binding of PARP‐1 to cruciform structures in supercoiled plasmids', Cytometry Part A, 68: 21-27. Chen, Jin-Ran, Oxana P Lazarenko, Kartik Shankar, Michael L Blackburn, Charles K Lumpkin, Thomas M Badger, and Martin JJ Ronis. 2011a. 'Inhibition of NADPH oxidases prevents chronic ethanol-induced bone loss in female rats', Journal of Pharmacology and Experimental Therapeutics, 336: 734-42. Chen, Jun-Jie, Daniel Silver, Sharon Cantor, David M Livingston, and Ralph Scully. 1999. 'BRCA1, BRCA2, and Rad51 operate in a common DNA damage response pathway', Cancer research, 59: 1752s-56s. Chen, Limo, Lixia Diao, Yongbin Yang, Xiaohui Yi, B Leticia Rodriguez, Yanli Li, Pamela A Villalobos, Tina Cascone, Xi Liu, and Lin Tan. 2018. 'CD38-mediated immunosuppression as a mechanism of tumor cell escape from PD-1/PD-L1 blockade', Cancer discovery, 8: 1156-75. Chen, Wen Yong, David H Wang, RayWhay Chiu Yen, Jianyuan Luo, Wei Gu, and Stephen B Baylin. 2005. 'Tumor suppressor HIC1 directly regulates SIRT1 to modulate p53- dependent DNA-damage responses', Cell, 123: 437-48. Chen, Xinyi Cynthia, Nathaniel G Hentz, Fredrick Hubbard, Timothy I Meier, Sitta Sittampalam, and Genshi Zhao. 2002. 'Development of a fluorescence resonance energy transfer assay for measuring the activity of Streptococcus pneumoniae DNA ligase, an enzyme essential for DNA replication, repair, and recombination', Analytical biochemistry, 309: 232-40. Chen, Xiqun, Pauline Wales, Luisa Quinti, Fuxing Zuo, Sébastien Moniot, Fanny Herisson, Nazifa Abdul Rauf, Hua Wang, Richard B Silverman, and Cenk Ayata. 2015. 'The sirtuin-2 inhibitor AK7 is neuroprotective in models of Parkinson’s disease but not amyotrophic lateral sclerosis and cerebral ischemia', PloS one, 10. Chen, Yaohui, Jinye Zhang, Yan Lin, Qunying Lei, Kun‐Liang Guan, Shimin Zhao, and Yue Xiong. 2011b. 'Tumour suppressor SIRT3 deacetylates and activates manganese superoxide dismutase to scavenge ROS', EMBO reports, 12: 534-41. Cheng, Qiao, Nadia Barboule, Philippe Frit, Dennis Gomez, Oriane Bombarde, Bettina Couderc, Guo-Sheng Ren, Bernard Salles, and Patrick Calsou. 2011. 'Ku counteracts mobilization of PARP1 and MRN in chromatin damaged with DNA double-strand breaks', Nucleic acids research, 39: 9605-19. Chi, Yuling, and Anthony A Sauve. 2013. 'Nicotinamide riboside, a trace nutrient in foods, is a vitamin B3 with effects on energy metabolism and neuroprotection', Current Opinion in Clinical Nutrition & Metabolic Care, 16: 657-61. Chini, Claudia CS, Mariana G Tarragó, and Eduardo N Chini. 2017. 'NAD and the aging process: Role in life, death and everything in between', Molecular and cellular endocrinology, 455: 62-74. Chini, Claudia, Kelly A Hogan, Gina M Warner, Mariana G Tarragó, Thais R Peclat, Tamar Tchkonia, James L Kirkland, and Eduardo Chini. 2019. 'The NADase CD38 is induced by factors secreted from senescent cells providing a potential link between senescence and age-related cellular NAD+ decline', Biochemical and biophysical research communications, 513: 486-93. Chini, Eduardo N. 2009. 'CD38 as a regulator of cellular NAD: a novel potential pharmacological target for metabolic conditions', Current pharmaceutical design, 15: 57-63. Chini, Eduardo N, and Thomas P Dousa. 1995. 'Enzymatic-synthesis and degradation of nicotinate adenine dinucleotide phosphate (NAADP), a Ca2+-releasing agonist, in rat tissues', Biochemical and biophysical research communications, 209: 167-74.

290 References

Choi, Jinah, Nicole LB Corder, Bhargav Koduru, and Yiyan Wang. 2014. 'Oxidative stress and hepatic Nox proteins in chronic hepatitis C and hepatocellular carcinoma', Free Radical Biology and Medicine, 72: 267-84. Choi, Sung‐E, Ting Fu, Sunmi Seok, Dong‐Hyun Kim, Eunkyung Yu, Kwan‐Woo Lee, Yup Kang, Xiaoling Li, Byron Kemper, and Jongsook K Kemper. 2013. 'Elevated micro RNA‐34a in obesity reduces NAD+ levels and SIRT 1 activity by directly targeting NAMPT', Aging cell, 12: 1062-72. Chopra, Vanita, Luisa Quinti, Jinho Kim, Lorraine Vollor, K Lakshmi Narayanan, Christina Edgerly, Patricia M Cipicchio, Molly A Lauver, Soo Hyuk Choi, and Richard B Silverman. 2012. 'The sirtuin 2 inhibitor AK-7 is neuroprotective in Huntington’s disease mouse models', Cell reports, 2: 1492-97. Chortis, Vasileios, Angela E Taylor, Craig L Doig, Mark D Walsh, Eirini Meimaridou, Carl Jenkinson, Giovanny Rodriguez-Blanco, Cristina L Ronchi, Alisha Jafri, and Louise A Metherell. 2018. 'Nicotinamide Nucleotide Transhydrogenase as a novel treatment target in adrenocortical carcinoma', Endocrinology, 159: 2836-49. Chou, Han-Yi E, Han Ting Chou, and Sheng-Chung Lee. 2006. 'CDK-dependent activation of poly (ADP-ribose) polymerase member 10 (PARP10)', Journal of Biological Chemistry, 281: 15201-07. Civitarese, Anthony E, Stacy Carling, Leonie K Heilbronn, Mathew H Hulver, Barbara Ukropcova, Walter A Deutsch, Steven R Smith, and Eric Ravussin. 2007. 'Calorie restriction increases muscle mitochondrial biogenesis in healthy humans', PLoS medicine, 4. Clark, Robert SB, Vincent A Vagni, Paula D Nathaniel, Larry W Jenkins, C Edward Dixon, and Csaba Szabó. 2007. 'Local administration of the poly (ADP-ribose) polymerase inhibitor INO-1001 prevents NAD+ depletion and improves water maze performance after traumatic brain injury in mice', Journal of neurotrauma, 24: 1399-405. Clarke, Gerard, Kiran V Sandhu, Brendan T Griffin, Timothy G Dinan, John F Cryan, and Niall P Hyland. 2019. 'Gut reactions: breaking down xenobiotic–microbiome interactions', Pharmacological reviews, 71: 198-224. Clement, James, Matthew Wong, Anne Poljak, Perminder Sachdev, and Nady Braidy. 2019. 'The plasma NAD+ metabolome is dysregulated in “normal” aging', Rejuvenation research, 22: 121-30. Cohen, Haim Y, Christine Miller, Kevin J Bitterman, Nathan R Wall, Brian Hekking, Benedikt Kessler, Konrad T Howitz, Myriam Gorospe, Rafael de Cabo, and David A Sinclair. 2004. 'Calorie restriction promotes mammalian cell survival by inducing the SIRT1 deacetylase', Science, 305: 390-92. Cohen, Keren, Maayan Waldman, Nader G Abraham, Michal Laniado-Schwartzman, Danny Gurfield, Dan Aravot, Michael Arad, and Edith Hochhauser. 2017. 'Caloric restriction ameliorates cardiomyopathy in animal model of diabetes', Experimental cell research, 350: 147-53. Cohen, Michael S. 2020. 'Interplay between compartmentalized NAD+ synthesis and consumption: a focus on the PARP family', Genes & development, 34: 254-62. Collins, Stephanie L, and Andrew D Patterson. 2019. 'The gut microbiome: an orchestrator of xenobiotic metabolism', Acta Pharmaceutica Sinica B. Colman, Ricki J, Rozalyn M Anderson, Sterling C Johnson, Erik K Kastman, Kristopher J Kosmatka, T Mark Beasley, David B Allison, Christina Cruzen, Heather A Simmons, and Joseph W Kemnitz. 2009. 'Caloric restriction delays disease onset and mortality in rhesus monkeys', Science, 325: 201-04.

291 References

Colman, Ricki J, T Mark Beasley, Joseph W Kemnitz, Sterling C Johnson, Richard Weindruch, and Rozalyn M Anderson. 2014. 'Caloric restriction reduces age-related and all-cause mortality in rhesus monkeys', Nature communications, 5: 1-5. Connell, Niels J, Riekelt H Houtkooper, and Patrick Schrauwen. 2019. 'NAD+ metabolism as a target for metabolic health: have we found the silver bullet?', Diabetologia, 62: 888- 99. Conze, Dietrich, Charles Brenner, and Claire L Kruger. 2019. 'Safety and metabolism of long- term administration of NIAGEN (nicotinamide riboside chloride) in a randomized, double-blind, placebo-controlled clinical trial of healthy overweight adults', Scientific reports, 9: 1-13. Cook, Brandoch D, Jasmin N Dynek, William Chang, Grigoriy Shostak, and Susan Smith. 2002. 'Role for the related poly (ADP-Ribose) polymerases tankyrase 1 and 2 at human telomeres', Molecular and cellular biology, 22: 332-42. Cooney, Sean J, Sara L Bermudez-Sabogal, and Kimberly R Byrnes. 2013. 'Cellular and temporal expression of NADPH oxidase (NOX) isotypes after brain injury', Journal of neuroinflammation, 10: 917. Cosker, François, Nathalie Cheviron, Michiko Yamasaki, Alexis Menteyne, Frances E Lund, Marie-Jo Moutin, Antony Galione, and Jose-Manuel Cancela. 2010. 'The ecto-enzyme CD38 is a nicotinic acid adenine dinucleotide phosphate (NAADP) synthase that couples receptor activation to Ca2+ mobilization from lysosomes in pancreatic acinar cells', Journal of Biological Chemistry, 285: 38251-59. Cresci, Gail, Laura E Nagy, and Vadivel Ganapathy. 2013. 'Lactobacillus GG and tributyrin supplementation reduce antibiotic‐induced intestinal injury', Journal of Parenteral and Enteral Nutrition, 37: 763-74. Crimmins, Eileen M. 2015. 'Lifespan and healthspan: past, present, and promise', The Gerontologist, 55: 901-11. Cupp, Jill R, and Lee McAlister-Henn. 1992. 'Cloning and characterization of the gene encoding the IDH1 subunit of NAD (+)-dependent isocitrate dehydrogenase from Saccharomyces cerevisiae', Journal of Biological Chemistry, 267: 16417-23. D’Argenio, Valeria, and Francesco Salvatore. 2015. 'The role of the gut microbiome in the healthy adult status', Clinica chimica acta, 451: 97-102. Dantzer, Françoise, Valérie Schreiber, Claude Niedergang, Carlotta Trucco, Eric Flatter, Guadalupe De La Rubia, Javier Oliver, Véronique Rolli, Josiane Ménissier-de Murcia, and Gilbert de Murcia. 1999. 'Involvement of poly (ADP-ribose) polymerase in base excision repair', Biochimie, 81: 69-75. Das, Abhirup, George X Huang, Michael S Bonkowski, Alban Longchamp, Catherine Li, Michael B Schultz, Lynn-Jee Kim, Brenna Osborne, Sanket Joshi, and Yuancheng Lu. 2018. 'Impairment of an endothelial NAD+-H2S signaling network is a reversible cause of vascular aging', Cell, 173: 74-89. e20. Davignon, Jean, Ghislaine Roederer, Martine Montigny, Michael R Hayden, Meng-Hee Tan, Philip W Connelly, Robert Hegele, Ruth McPherson, Paul J Lupien, and Claude Gagné. 1994. 'Comparative efficacy and safety of pravastatin, nicotinic acid and the two combined in patients with hypercholesterolemia', The American journal of cardiology, 73: 339-45. Davila, Antonio, Ling Liu, Karthikeyani Chellappa, Philip Redpath, Eiko Nakamaru-Ogiso, Lauren M Paolella, Zhigang Zhang, Marie E Migaud, Joshua D Rabinowitz, and Joseph A Baur. 2018. 'Nicotinamide adenine dinucleotide is transported into mammalian mitochondria', Elife, 7: e33246. Dawicki-McKenna, Jennine M, Marie-France Langelier, Jamie E DeNizio, Amanda A Riccio, Connie D Cao, Kelly R Karch, Michael McCauley, Jamin D Steffen, Ben E Black, and

292 References

John M Pascal. 2015. 'PARP-1 activation requires local unfolding of an autoinhibitory domain', Molecular cell, 60: 755-68. De Ingeniis, Jessica, Marat D Kazanov, Konstantin Shatalin, Mikhail S Gelfand, Andrei L Osterman, and Leonardo Sorci. 2012. 'Glutamine versus ammonia utilization in the NAD synthetase family', PloS one, 7. de Murcia, Gilbert, and Josiane Ménissier de Murcia. 1994. 'Poly (ADP-ribose) polymerase: a molecular nick-sensor', Trends in biochemical sciences, 19: 172-76. de Picciotto, Natalie E, Lindsey B Gano, Lawrence C Johnson, Christopher R Martens, Amy L Sindler, Kathryn F Mills, Shin‐ichiro Imai, and Douglas R Seals. 2016. 'Nicotinamide mononucleotide supplementation reverses vascular dysfunction and oxidative stress with aging in mice', Aging cell, 15: 522-30. Deléens, E, JB Cliquet, and JL Prioul. 1994. 'Use of 13C and 15N plant label near natural abundance for monitoring carbon and nitrogen partitioning', Functional Plant Biology, 21: 133-46. Dellinger, Ryan W, Santiago Roel Santos, Mark Morris, Mal Evans, Dan Alminana, Leonard Guarente, and Eric Marcotulli. 2017. 'Repeat dose NRPT (nicotinamide riboside and pterostilbene) increases NAD+ levels in humans safely and sustainably: a randomized, double-blind, placebo-controlled study', npj Aging and Mechanisms of Disease, 3: 17. Demarest, Tyler G, Mansi Babbar, Mustafa N Okur, Xiuli Dan, Deborah L Croteau, Nima B Fakouri, Mark P Mattson, and Vilhelm A Bohr. 2019. 'NAD+ metabolism in aging and cancer', Annual Review of Cancer Biology, 3: 105-30. Deshpande, Deepak A, Timothy F Walseth, Reynold A Panettieri, and Mathur S Kannan. 2003. 'CD38/cyclic ADP-ribose-mediated Ca2+ signaling contributes to airway smooth muscle hyper-responsiveness', The FASEB Journal, 17: 452-54. Di Lisa, Fabio, Roberta Menabò, Marcella Canton, Maria Barile, and Paolo Bernardi. 2001. 'Opening of the mitochondrial permeability transition pore causes depletion of mitochondrial and cytosolic NAD+ and is a causative event in the death of myocytes in postischemic reperfusion of the heart', Journal of Biological Chemistry, 276: 2571-75. Di Lisa, Fabio, and Mathias Ziegler. 2001. 'Pathophysiological relevance of mitochondria in NAD+ metabolism', FEBS letters, 492: 4-8. Di Luccio, Eric, and David K Wilson. 2008. 'Comprehensive X-ray structural studies of the quinolinate phosphoribosyl transferase (BNA6) from Saccharomyces cerevisiae', Biochemistry, 47: 4039-50. Di Marco, Elyse, Stephen Peter Gray, Phyliss Chew, Christine Koulis, A Ziegler, Cedric Szyndralewiez, Rhian M Touyz, HHHW Schmidt, Mark Emmanuel Cooper, and R Slattery. 2014. 'Pharmacological inhibition of NOX reduces atherosclerotic lesions, vascular ROS and immune–inflammatory responses in diabetic Apoe−/− mice', Diabetologia, 57: 633-42. Di Paola, Simone, Massimo Micaroni, Giuseppe Di Tullio, Roberto Buccione, and Maria Di Girolamo. 2012. 'PARP16/ARTD15 is a novel endoplasmic-reticulum-associated mono-ADP-ribosyltransferase that interacts with, and modifies karyopherin-ß1', PloS one, 7. Di Stefano, M, I Nascimento-Ferreira, G Orsomando, V Mori, Jonathan Gilley, R Brown, L Janeckova, ME Vargas, LA Worrell, and Andrea Loreto. 2015. 'A rise in NAD precursor nicotinamide mononucleotide (NMN) after injury promotes axon degeneration', Cell Death & Differentiation, 22: 731-42. Di Stefano, Michele, Andrea Loreto, Giuseppe Orsomando, Valerio Mori, Federica Zamporlini, Richard P Hulse, Jamie Webster, Lucy F Donaldson, Martin Gering, and Nadia Raffaelli. 2017. 'NMN deamidase delays Wallerian degeneration and rescues axonal defects caused by NMNAT2 deficiency in vivo', Current Biology, 27: 784-94.

293 References

Dianova, Irina I, Kate M Sleeth, Sarah L Allinson, Jason L Parsons, Claire Breslin, Keith W Caldecott, and Grigory L Dianov. 2004. 'XRCC1–DNA polymerase β interaction is required for efficient base excision repair', Nucleic acids research, 32: 2550-55. Diguet, Nicolas, Samuel AJ Trammell, Cynthia Tannous, Robin Deloux, Jérôme Piquereau, Nathalie Mougenot, Anne Gouge, Mélanie Gressette, Boris Manoury, and Jocelyne Blanc. 2018. 'Nicotinamide riboside preserves cardiac function in a mouse model of dilated cardiomyopathy', Circulation, 137: 2256-73. Dijkstra, Paul, Ayaka Ishizu, Richard Doucett, Stephen C Hart, Egbert Schwartz, Oleg V Menyailo, and Bruce A Hungate. 2006. '13C and 15N natural abundance of the soil microbial biomass', Soil Biology and Biochemistry, 38: 3257-66. Djamali, A, A Vidyasagar, M Adulla, D Hullett, and S Reese. 2009. 'Nox‐2 is a modulator of fibrogenesis in kidney allografts', American Journal of Transplantation, 9: 74-82. Dodd, Antony N, Michael J Gardner, Carlos T Hotta, Katharine E Hubbard, Neil Dalchau, John Love, Jean-Maurice Assie, Fiona C Robertson, Mia Kyed Jakobsen, and Jorge Gonçalves. 2007. 'The Arabidopsis circadian clock incorporates a cADPR-based feedback loop', Science, 318: 1789-92. Dogan, Soner, Anna C Johannsen, Joseph P Grande, and Margot P Cleary. 2011. 'Effects of intermittent and chronic calorie restriction on mammalian target of rapamycin (mTOR) and IGF-I signaling pathways in mammary fat pad tissues and mammary tumors', Nutrition and cancer, 63: 389-401. Dolopikou, CF, Ioannis A Kourtzidis, Nikos V Margaritelis, Ioannis S Vrabas, Irene Koidou, Antonios Kyparos, Anastasios A Theodorou, Vassilis Paschalis, and Michalis G Nikolaidis. 2019. 'Acute nicotinamide riboside supplementation improves redox homeostasis and exercise performance in old individuals: a double-blind cross-over study', European journal of nutrition: 1-11. Dominy Jr, John E, Yoonjin Lee, Zachary Gerhart-Hines, and Pere Puigserver. 2010. 'Nutrient- dependent regulation of PGC-1α's acetylation state and metabolic function through the enzymatic activities of Sirt1/GCN5', Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804: 1676-83. Dregalla, Ryan C, Junqing Zhou, Rupa R Idate, Christine LR Battaglia, Howard L Liber, and Susan M Bailey. 2010. 'Regulatory roles of tankyrase 1 at telomeres and in DNA repair: suppression of T-SCE and stabilization of DNA-PKcs', Aging (Albany NY), 2: 691. Du, Jintang, Yeyun Zhou, Xiaoyang Su, Jiu Jiu Yu, Saba Khan, Hong Jiang, Jungwoo Kim, Jimin Woo, Jun Huyn Kim, and Brian Hyun Choi. 2011. 'Sirt5 is a NAD-dependent protein lysine demalonylase and desuccinylase', Science, 334: 806-09. Du, Lina, Xiaopeng Zhang, Yong Y Han, Nancy A Burke, Patrick M Kochanek, Simon C Watkins, Steven H Graham, Joseph A Carcillo, Csaba Szabó, and Robert SB Clark. 2003. 'Intra-mitochondrial poly (ADP-ribosylation) contributes to NAD+ depletion and cell death induced by oxidative stress', Journal of Biological Chemistry, 278: 18426- 33. Ealick, SE, SA Rule, DC Carter, TJ Greenhough, YS Babu, WJ Cook, J Habash, JR Helliwell, JD Stoeckler, and RE Parks. 1990. 'Three-dimensional structure of human erythrocytic purine nucleoside phosphorylase at 3.2 A resolution', Journal of Biological Chemistry, 265: 1812-20. Eisenberg, Tobias, Mahmoud Abdellatif, Sabrina Schroeder, Uwe Primessnig, Slaven Stekovic, Tobias Pendl, Alexandra Harger, Julia Schipke, Andreas Zimmermann, and Albrecht Schmidt. 2016. 'Cardioprotection and lifespan extension by the natural polyamine spermidine', Nature medicine, 22: 1428. Eisenberg, Tobias, Heide Knauer, Alexandra Schauer, Sabrina Büttner, Christoph Ruckenstuhl, Didac Carmona-Gutierrez, Julia Ring, Sabrina Schroeder, Christoph

294 References

Magnes, and Lucia Antonacci. 2009. 'Induction of autophagy by spermidine promotes longevity', Nature cell biology, 11: 1305-14. El‐Benna, Jamel, Margarita Hurtado‐Nedelec, Viviana Marzaioli, Jean‐Claude Marie, Marie‐ Anne Gougerot‐Pocidalo, and Pham My‐Chan Dang. 2016. 'Priming of the neutrophil respiratory burst: role in host defense and inflammation', Immunological reviews, 273: 180-93. El‐Khamisy, Sherif F, Mitsuko Masutani, Hiroshi Suzuki, and Keith W Caldecott. 2003. 'A requirement for PARP‐1 for the assembly or stability of XRCC1 nuclear foci at sites of oxidative DNA damage', Nucleic acids research, 31: 5526-33. Elhassan, Yasir S, Katarina Kluckova, Rachel S Fletcher, Mark S Schmidt, Antje Garten, Craig L Doig, David M Cartwright, Lucy Oakey, Claire V Burley, and Ned Jenkinson. 2019. 'Nicotinamide riboside augments the aged human skeletal muscle NAD+ metabolome and induces transcriptomic and anti-inflammatory signatures', Cell reports, 28: 1717- 28. e6. ElRakaiby, Marwa, Bas E Dutilh, Mariam R Rizkallah, Annemarie Boleij, Jason N Cole, and Ramy K Aziz. 2014. 'Pharmacomicrobiomics: the impact of human microbiome variations on systems pharmacology and personalized therapeutics', Omics: a journal of integrative biology, 18: 402-14. Emanuelli, Monica, Adolfo Amici, Francesco Carnevali, Francesca Pierella, Nadia Raffaelli, and Giulio Magni. 2003. 'Identification and characterization of a second NMN adenylyltransferase gene in Saccharomyces cerevisiae', Protein expression and purification, 27: 357-64. Emanuelli, Monica, Francesco Carnevali, Maria Lorenzi, Nadia Raffaelli, Adolfo Amici, Silverio Ruggieri, and Giulio Magni. 1999. 'Identification and characterization of YLR328W, the Saccharomyces cerevisiae structural gene encoding NMN adenylyltransferase. Expression and characterization of the recombinant enzyme', FEBS letters, 455: 13-17. Emanuelli, Monica, Francesco Carnevali, Franca Saccucci, Francesca Pierella, Adolfo Amici, Nadia Raffaelli, and Giulio Magni. 2001. 'Molecular cloning, chromosomal localization, tissue mRNA levels, bacterial expression, and enzymatic properties of human NMN adenylyltransferase', Journal of Biological Chemistry, 276: 406-12. Erburu, Mercedes, Irene Muñoz-Cobo, Teresa Diaz-Perdigon, Paolo Mellini, Takayoshi Suzuki, Elena Puerta, and Rosa M Tordera. 2017. 'SIRT2 inhibition modulate glutamate and serotonin systems in the prefrontal cortex and induces antidepressant-like action', Neuropharmacology, 117: 195-208. Esposito, Luke A, Jason E Kokoszka, Katrina G Waymire, Barbara Cottrell, Grant R MacGregor, and Douglas C Wallace. 2000. 'Mitochondrial oxidative stress in mice lacking the glutathione peroxidase-1 gene', Free Radical Biology and Medicine, 28: 754-66. Essuman, Kow, Daniel W Summers, Yo Sasaki, Xianrong Mao, Aaron DiAntonio, and Jeffrey Milbrandt. 2017. 'The SARM1 Toll/interleukin-1 receptor domain possesses intrinsic NAD+ cleavage activity that promotes pathological axonal degeneration', Neuron, 93: 1334-43. e5. Estrella, Michael A, Jin Du, Li Chen, Sneha Rath, Eliza Prangley, Alisha Chitrakar, Tsutomu Aoki, Paul Schedl, Joshua Rabinowitz, and Alexei Korennykh. 2019. 'The metabolites NADP+ and NADPH are the targets of the circadian protein Nocturnin (Curled)', Nature communications, 10: 1-10. Evans, Charles, Katrina L Bogan, Peng Song, Charles F Burant, Robert T Kennedy, and Charles Brenner. 2010. 'NAD+ metabolite levels as a function of vitamins and calorie

295 References

restriction: evidence for different mechanisms of longevity', BMC chemical biology, 10: 2. Evans, Jason, Tao-Chin Wang, Melvyn P Heyes, and SP Markey. 2002. 'LC/MS analysis of NAD biosynthesis using stable isotope pyridine precursors', Analytical biochemistry, 306: 197-203. Fan, Wei, and Jianyuan Luo. 2010. 'SIRT1 regulates UV-induced DNA repair through deacetylating XPA', Molecular cell, 39: 247-58. Fang, Cheng, Ting Li, Ying Li, Guan Jie Xu, Qi Wen Deng, Ya Jie Chen, Yun Nan Hou, Hon Cheung Lee, and Yong Juan Zhao. 2018. 'CD38 produces nicotinic acid adenosine dinucleotide phosphate in the lysosome', Journal of Biological Chemistry, 293: 8151- 60. Fang, Evandro F, Sofie Lautrup, Yujun Hou, Tyler G Demarest, Deborah L Croteau, Mark P Mattson, and Vilhelm A Bohr. 2017. 'NAD+ in aging: molecular mechanisms and translational implications', Trends in molecular medicine, 23: 899-916. Fang, Evandro Fei, Henok Kassahun, Deborah L Croteau, Morten Scheibye-Knudsen, Krisztina Marosi, Huiming Lu, Raghavendra A Shamanna, Sumana Kalyanasundaram, Ravi Chand Bollineni, and Mark A Wilson. 2016. 'NAD+ replenishment improves lifespan and healthspan in ataxia telangiectasia models via mitophagy and DNA repair', Cell metabolism, 24: 566-81. Fang, Evandro Fei, Morten Scheibye-Knudsen, Lear E Brace, Henok Kassahun, Tanima SenGupta, Hilde Nilsen, James R Mitchell, Deborah L Croteau, and Vilhelm A Bohr. 2014. 'Defective mitophagy in XPA via PARP-1 hyperactivation and NAD+/SIRT1 reduction', Cell, 157: 882-96. Fattah, Farjana, Eu Han Lee, Natalie Weisensel, Yongbao Wang, Natalie Lichter, and Eric A Hendrickson. 2010. 'Ku regulates the non-homologous end joining pathway choice of DNA double-strand break repair in human somatic cells', PLoS genetics, 6. Fedorenko, Andriy, Polina V Lishko, and Yuriy Kirichok. 2012. 'Mechanism of fatty-acid- dependent UCP1 uncoupling in brown fat mitochondria', Cell, 151: 400-13. Fehr, Anthony R, Sasha A Singh, Catherine M Kerr, Shin Mukai, Hideyuki Higashi, and Masanori Aikawa. 2020. 'The impact of PARPs and ADP-ribosylation on inflammation and host–pathogen interactions', Genes & development, 34: 341-59. Feigin, Michael E, Tyler Garvin, Peter Bailey, Nicola Waddell, David K Chang, David R Kelley, Shimin Shuai, Steven Gallinger, John D McPherson, and Sean M Grimmond. 2017. 'Recurrent noncoding regulatory mutations in pancreatic ductal adenocarcinoma', Nature genetics, 49: 825. Fenech, Michael. 2001. 'The role of folic acid and vitamin B12 in genomic stability of human cells', Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 475: 57-67. Feng, Lin, Jun Huang, and Junjie Chen. 2009. 'MERIT40 facilitates BRCA1 localization and DNA damage repair', Genes & development, 23: 719-28. Finley, Lydia WS, Arkaitz Carracedo, Jaewon Lee, Amanda Souza, Ainara Egia, Jiangwen Zhang, Julie Teruya-Feldstein, Paula I Moreira, Sandra M Cardoso, and Clary B Clish. 2011. 'SIRT3 opposes reprogramming of cancer cell metabolism through HIF1α destabilization', Cancer cell, 19: 416-28. Fischer, Jan MF, Oliver Popp, Daniel Gebhard, Sebastian Veith, Arthur Fischbach, Sascha Beneke, Alfred Leitenstorfer, Jörg Bergemann, Martin Scheffner, and Elisa Ferrando‐ May. 2014. 'Poly (ADP‐ribose)‐mediated interplay of XPA and PARP 1 leads to reciprocal regulation of protein function', The FEBS journal, 281: 3625-41. Flachmann, Ralf, Norbert Kunz, Jochen Seifert, Markus Gütlich, Franz‐Josef Wientjes, Albrecht Läufer, and Hans Günter Gassen. 1988. 'Molecular biology of pyridine

296 References

nucleotide biosynthesis in Escherichia coli: cloning and characterization of quinolinate synthesis genes nadA and nadB', European journal of biochemistry, 175: 221-28. Fletcher, Rachel S, Joanna Ratajczak, Craig L Doig, Lucy A Oakey, Rebecca Callingham, Gabriella Da Silva Xavier, Antje Garten, Yasir S Elhassan, Philip Redpath, and Marie E Migaud. 2017. 'Nicotinamide riboside kinases display redundancy in mediating nicotinamide mononucleotide and nicotinamide riboside metabolism in skeletal muscle cells', Molecular metabolism, 6: 819-32. Foster, JOHN W, and ANNE M Baskowsky-Foster. 1980. 'Pyridine nucleotide cycle of Salmonella typhimurium: in vivo recycling of nicotinamide adenine dinucleotide', Journal of bacteriology, 142: 1032-35. Fouts, Paul J, OM Helmer, Samuel Lepkovsky, and TH Jukes. 1937. 'Treatment of human pellagra with nicotinic acid', Proceedings of the Society for Experimental Biology and Medicine, 37: 405-07. Franceschi, Claudio, and Judith Campisi. 2014. 'Chronic inflammation (inflammaging) and its potential contribution to age-associated diseases', Journals of Gerontology Series A: Biomedical Sciences and Medical Sciences, 69: S4-S9. Frasca, Loredana, Giorgio Fedele, Silvia Deaglio, Cristina Capuano, Raffaella Palazzo, Tiziana Vaisitti, Fabio Malavasi, and Clara Maria Ausiello. 2006. 'CD38 orchestrates migration, survival, and Th1 immune response of human mature dendritic cells', Blood, 107: 2392-99. Frederick, David W, Emanuele Loro, Ling Liu, Antonio Davila Jr, Karthikeyani Chellappa, Ian M Silverman, William J Quinn III, Sager J Gosai, Elisia D Tichy, and James G Davis. 2016. 'Loss of NAD homeostasis leads to progressive and reversible degeneration of skeletal muscle', Cell metabolism, 24: 269-82. Frothingham, Richard, Winifred A Meeker-O'Connell, EA Talbot, James W George, and Kenneth N Kreuzer. 1996. 'Identification, cloning, and expression of the Escherichia coli pyrazinamidase and nicotinamidase gene, pncA', Antimicrobial agents and chemotherapy, 40: 1426-31. Frye, Roy A. 1999. 'Characterization of five human cDNAs with homology to the yeast SIR2 gene: Sir2-like proteins (sirtuins) metabolize NAD and may have protein ADP- ribosyltransferase activity', Biochemical and biophysical research communications, 260: 273-79. Fukuda, Shinji, Hidehiro Toh, Koji Hase, Kenshiro Oshima, Yumiko Nakanishi, Kazutoshi Yoshimura, Toru Tobe, Julie M Clarke, David L Topping, and Tohru Suzuki. 2011. 'Bifidobacteria can protect from enteropathogenic infection through production of acetate', Nature, 469: 543-47. Fukuoka, Shin-Ichi, Cornelio M Nyaruhucha, and Katsumi Shibata. 1998. 'Characterization and functional expression of the cDNA encoding human brain quinolinate phosphoribosyltransferase', Biochimica et Biophysica Acta (BBA)-Gene Structure and Expression, 1395: 192-201. Furukawa, Ayako, Saeko Tada-Oikawa, Shosuke Kawanishi, and Shinji Oikawa. 2007. 'H2O2 accelerates cellular senescence by accumulation of acetylated p53 via decrease in the function of SIRT1 by NAD+ depletion', Cellular Physiology and Biochemistry, 20: 045-54. Galassi, Lucia, Michele Di Stefano, Lucia Brunetti, Giuseppe Orsomando, Adolfo Amici, Silverio Ruggieri, and Giulio Magni. 2012. 'Characterization of human nicotinate phosphoribosyltransferase: Kinetic studies, structure prediction and functional analysis by site-directed mutagenesis', Biochimie, 94: 300-09. Galeazzi, Luca, Paola Bocci, Adolfo Amici, Lucia Brunetti, Silverio Ruggieri, Margaret Romine, Samantha Reed, Andrei L Osterman, Dmitry A Rodionov, and Leonardo

297 References

Sorci. 2011. 'Identification of nicotinamide mononucleotide deamidase of the bacterial pyridine nucleotide cycle reveals a novel broadly conserved amidohydrolase family', Journal of Biological Chemistry, 286: 40365-75. Gao, Daming, Hiroyuki Inuzuka, Meng-Kwang Marcus Tan, Hidefumi Fukushima, Jason W Locasale, Pengda Liu, Lixin Wan, Bo Zhai, Y Rebecca Chin, and Shavali Shaik. 2011. 'mTOR drives its own activation via SCFβTrCP-dependent degradation of the mTOR inhibitor DEPTOR', Molecular cell, 44: 290-303. Gao, Li, Wei-Feng Wu, Lei Dong, Gui-Ling Ren, Hai-Di Li, Qin Yang, Xiao-Feng Li, Tao Xu, Zeng Li, and Bao-Ming Wu. 2016. 'Protocatechuic aldehyde attenuates cisplatin- induced acute kidney injury by suppressing nox-mediated oxidative stress and renal inflammation', Frontiers in pharmacology, 7: 479. Garavaglia, Silvia, Igor D'Angelo, Monica Emanuelli, Francesco Carnevali, Francesca Pierella, Giulio Magni, and Menico Rizzi. 2002. 'Structure of human NMN adenylyltransferase a key nuclear enzyme for NAD homeostasis', Journal of Biological Chemistry, 277: 8524-30. Garg, Abhimanyu, and Scott M Grundy. 1990. 'Nicotinic acid as therapy for dyslipidemia in non—insulin-dependent diabetes mellitus', Jama, 264: 723-26. Gariani, Karim, Keir J Menzies, Dongryeol Ryu, Casey J Wegner, Xu Wang, Eduardo R Ropelle, Norman Moullan, Hongbo Zhang, Alessia Perino, and Vera Lemos. 2016. 'Eliciting the mitochondrial unfolded protein response by nicotinamide adenine dinucleotide repletion reverses fatty liver disease in mice', Hepatology, 63: 1190-204. Garten, Antje, Susanne Schuster, Melanie Penke, Theresa Gorski, Tommaso De Giorgis, and Wieland Kiess. 2015. 'Physiological and pathophysiological roles of NAMPT and NAD metabolism', Nature Reviews Endocrinology, 11: 535-46. Gazzaniga, Francesca, Rebecca Stebbins, Sheila Z Chang, Mark A McPeek, and Charles Brenner. 2009. 'Microbial NAD metabolism: lessons from comparative genomics', Microbiol. Mol. Biol. Rev., 73: 529-41. Geider, Klaus. 1972. 'DNA Synthesis in Nucleotide‐Permeable Escherichia coli Cells: The Effects of Nucleotide Analogues on DNA Synthesis', European journal of biochemistry, 27: 554-63. Geisbrecht, Brian V, and Stephen J Gould. 1999. 'The human PICD gene encodes a cytoplasmic and peroxisomal NADP+-dependent isocitrate dehydrogenase', Journal of Biological Chemistry, 274: 30527-33. Geisler, Stefanie, Shay X Huang, Amy Strickland, Ryan A Doan, Daniel W Summers, Xianrong Mao, Jiwoong Park, Aaron DiAntonio, and Jeffrey Milbrandt. 2019. 'Gene therapy targeting SARM1 blocks pathological axon degeneration in mice', Journal of Experimental Medicine, 216: 294-303. Gerdts, Josiah, EJ Brace, Yo Sasaki, Aaron DiAntonio, and Jeffrey Milbrandt. 2015. 'SARM1 activation triggers axon degeneration locally via NAD+ destruction', Science, 348: 453- 57. Gerdts, Josiah, Daniel W Summers, Yo Sasaki, Aaron DiAntonio, and Jeffrey Milbrandt. 2013. 'Sarm1-mediated axon degeneration requires both SAM and TIR interactions', Journal of Neuroscience, 33: 13569-80. Gholson, RK, I Ueda, N Ogasawara, and LM Henderson. 1964. 'The enzymatic conversion of quinolinate to nicotinic acid mononucleotide in mammalian liver', Journal of Biological Chemistry, 239: 1208-14. Gilley, Jonathan, Giuseppe Orsomando, Isabel Nascimento-Ferreira, and Michael P Coleman. 2015. 'Absence of SARM1 rescues development and survival of NMNAT2-deficient axons', Cell reports, 10: 1974-81.

298 References

Gilley, Jonathan, Richard R Ribchester, and Michael P Coleman. 2017. 'Sarm1 deletion, but not WldS, confers lifelong rescue in a mouse model of severe axonopathy', Cell reports, 21: 10-16. Giralt, Albert, Elayne Hondares, Josep A Villena, Francesc Ribas, Julieta Díaz-Delfín, Marta Giralt, Roser Iglesias, and Francesc Villarroya. 2011. 'Peroxisome proliferator- activated receptor-γ coactivator-1α controls transcription of the Sirt3 gene, an essential component of the thermogenic brown adipocyte phenotype', Journal of Biological Chemistry, 286: 16958-66. Giroud-Gerbetant, Judith, Magali Joffraud, Maria Pilar Giner, Angelique Cercillieux, Simona Bartova, Mikhail V Makarov, Rubén Zapata-Pérez, José L Sánchez-García, Riekelt H Houtkooper, and Marie E Migaud. 2019. 'A reduced form of nicotinamide riboside defines a new path for NAD+ biosynthesis and acts as an orally bioavailable NAD+ precursor', Molecular metabolism, 30: 192-202. Göckel, A, and C Richert. 2015. 'Synthesis of an oligonucleotide with a nicotinamide mononucleotide residue and its molecular recognition in DNA helices', Organic & biomolecular chemistry, 13: 10303-09. Goldberg, Michal, Manuel Stucki, Jacob Falck, Damien D'Amours, Dinah Rahman, Darryl Pappin, Jiri Bartek, and Stephen P Jackson. 2003. 'MDC1 is required for the intra-S- phase DNA damage checkpoint', Nature, 421: 952-56. Goldsmith, Grace A, Herbert P Sarett, UD Register, and Janis Gibbens. 1952. 'Studies of niacin requirement in man. I. Experimental pellagra in subjects on corn diets low in niacin and tryptophan', The Journal of clinical investigation, 31: 533-42. Gomes, Ana P, Nathan L Price, Alvin JY Ling, Javid J Moslehi, Magdalene K Montgomery, Luis Rajman, James P White, João S Teodoro, Christiane D Wrann, and Basil P Hubbard. 2013. 'Declining NAD+ induces a pseudohypoxic state disrupting nuclear- mitochondrial communication during aging', Cell, 155: 1624-38. Gomez, Alvin, Christian Bindesbøll, Somisetty V Satheesh, Giulia Grimaldi, David Hutin, Laura MacPherson, Shaimaa Ahmed, Laura Tamblyn, Tiffany Cho, and Hilde Irene Nebb. 2018. 'Characterization of TCDD-inducible poly-ADP-ribose polymerase (TIPARP/ARTD14) catalytic activity', Biochemical Journal, 475: 3827-46. Gong, Bing, Yong Pan, Prashant Vempati, Wei Zhao, Lindsay Knable, Lap Ho, Jun Wang, Magdalena Sastre, Kenjiro Ono, and Anthony A Sauve. 2013. 'Nicotinamide riboside restores cognition through an upregulation of proliferator-activated receptor-γ coactivator 1α regulated β-secretase 1 degradation and mitochondrial gene expression in Alzheimer's mouse models', Neurobiology of aging, 34: 1581-88. González-González, Marianela, Camilo Díaz-Zepeda, Johana Eyzaguirre-Velásquez, Camila González-Arancibia, Javier A Bravo, and Marcela Julio-Pieper. 2019. 'Investigating gut permeability in animal models of disease', Frontiers in physiology, 9: 1962. Gopal, Elangovan, You-Jun Fei, Seiji Miyauchi, Lina Zhuang, Puttur D Prasad, and Vadivel Ganapathy. 2005. 'Sodium-coupled and electrogenic transport of B-complex vitamin nicotinic acid by slc5a8, a member of the Na/glucose co-transporter gene family', Biochemical Journal, 388: 309-16. Gräff, Johannes, Martin Kahn, Alireza Samiei, Jun Gao, Kristie T Ota, Damien Rei, and Li- Huei Tsai. 2013. 'A dietary regimen of caloric restriction or pharmacological activation of SIRT1 to delay the onset of neurodegeneration', Journal of Neuroscience, 33: 8951- 60. Greer, Eric L, Dara Dowlatshahi, Max R Banko, Judit Villen, Kimmi Hoang, Daniel Blanchard, Steven P Gygi, and Anne Brunet. 2007. 'An AMPK-FOXO pathway mediates longevity induced by a novel method of dietary restriction in C. elegans', Current Biology, 17: 1646-56.

299 References

Grose, Julianne H, Ulfar Bergthorsson, and John R Roth. 2005. 'Regulation of NAD synthesis by the trifunctional NadR protein of Salmonella enterica', Journal of bacteriology, 187: 2774-82. Grozio, Alessia, Kathryn F Mills, Jun Yoshino, Santina Bruzzone, Giovanna Sociali, Kyohei Tokizane, Hanyue Cecilia Lei, Richard Cunningham, Yo Sasaki, and Marie E Migaud. 2019a. 'Slc12a8 is a nicotinamide mononucleotide transporter', Nature metabolism, 1: 47. Grozio, Alessia, Kathryn Mills, Jun Yoshino, Santina Bruzzone, Giovanna Sociali, Kyohei Tokizane, Hanyue Cecilia Lei, Yo Sasaki, Marie Migaud, and Shin-ichiro Imai. 2019b. 'Reply to: Absence of evidence that Slc12a8 encodes a nicotinamide mononucleotide transporter', Nature metabolism, 1: 662-65. Grozio, Alessia, Giovanna Sociali, Laura Sturla, Irene Caffa, Debora Soncini, Annalisa Salis, Nadia Raffaelli, Antonio De Flora, Alessio Nencioni, and Santina Bruzzone. 2013. 'CD73 protein as a source of extracellular precursors for sustained NAD+ biosynthesis in FK866-treated tumor cells', Journal of Biological Chemistry, 288: 25938-49. Guan, Xiao‐Hui, Xuan Hong, Ning Zhao, Xiao‐Hong Liu, Yun‐Fei Xiao, Ting‐Tao Chen, Li‐ Bin Deng, Xiao‐Lei Wang, Jian‐Bin Wang, and Guang‐Ju Ji. 2017a. 'CD38 promotes angiotensin II‐induced cardiac hypertrophy', Journal of cellular and molecular medicine, 21: 1492-502. Guan, Yi, Su-Rong Wang, Xin-Zhong Huang, Qiong-hong Xie, Yun-Yu Xu, Da Shang, and Chuan-Ming Hao. 2017b. 'Nicotinamide Mononucleotide, an NAD+ Precursor, Rescues Age-Associated Susceptibility to AKI in a Sirtuin 1–Dependent Manner', Journal of the American Society of Nephrology, 28: 2337-52. Guo, Tingting, Yibo Zuo, Liping Qian, Jin Liu, Yukang Yuan, Kailin Xu, Ying Miao, Qian Feng, Xiangjie Chen, and Lincong Jin. 2019. 'ADP-ribosyltransferase PARP11 modulates the interferon antiviral response by mono-ADP-ribosylating the ubiquitin E3 ligase β-TrCP', Nature microbiology, 4: 1872-84. Guo, Xiumei, Jason G Williams, Thaddeus T Schug, and Xiaoling Li. 2010. 'DYRK1A and DYRK3 promote cell survival through phosphorylation and activation of SIRT1', Journal of Biological Chemistry, 285: 13223-32. Guthrie, Leah, Sarah Wolfson, and Libusha Kelly. 2019. 'The human gut chemical landscape predicts microbe-mediated biotransformation of foods and drugs', Elife, 8: e42866. Ha, Hyo Chol, and Solomon H Snyder. 1999. 'Poly (ADP-ribose) polymerase is a mediator of necrotic cell death by ATP depletion', Proceedings of the National Academy of Sciences, 96: 13978-82. Haak, T, T Meinicke, R Jones, S Weber, M von Eynatten, and H‐J Woerle. 2012. 'Initial combination of linagliptin and metformin improves glycaemic control in type 2 diabetes: a randomized, double‐blind, placebo‐controlled study', Diabetes, Obesity and Metabolism, 14: 565-74. Haferkamp, Ilka, Stephan Schmitz-Esser, Nicole Linka, Claude Urbany, Astrid Collingro, Michael Wagner, Matthias Horn, and H Ekkehard Neuhaus. 2004. 'A candidate NAD+ transporter in an intracellular bacterial symbiont related to Chlamydiae', Nature, 432: 622-25. Hägele, Edgar, Jurgen Neeff, and Dieter Mecke. 1978. 'The malate dehydrogenase isoenzymes of Saccharomyces cerevisiae. Purification, characterisation and studies on their regulation', European journal of biochemistry, 83: 67-76. Haigis, Marcia C, Raul Mostoslavsky, Kevin M Haigis, Kamau Fahie, Danos C Christodoulou, Andrew J Murphy, David M Valenzuela, George D Yancopoulos, Margaret Karow, and Gil Blander. 2006. 'SIRT4 inhibits glutamate dehydrogenase and opposes the effects of calorie restriction in pancreatic β cells', Cell, 126: 941-54.

300 References

Haigis, Marcia C, and David A Sinclair. 2010. 'Mammalian sirtuins: biological insights and disease relevance', Annual Review of Pathology: Mechanisms of Disease, 5: 253-95. Haince, Jean-François, Darin McDonald, Amélie Rodrigue, Ugo Déry, Jean-Yves Masson, Michael J Hendzel, and Guy G Poirier. 2008. 'PARP1-dependent kinetics of recruitment of MRE11 and NBS1 proteins to multiple DNA damage sites', Journal of Biological Chemistry, 283: 1197-208. Hallows, William C, Wei Yu, Brian C Smith, Mark K Devires, James J Ellinger, Shinichi Someya, Michael R Shortreed, Tomas Prolla, John L Markley, and Lloyd M Smith. 2011. 'Sirt3 promotes the urea cycle and fatty acid oxidation during dietary restriction', Molecular cell, 41: 139-49. Halperin-Sheinfeld, Meital, Asaf Gertler, Eitan Okun, Benjamin Sredni, and Haim Y Cohen. 2012. 'The Tellurium compound, AS101, increases SIRT1 level and activity and prevents type 2 diabetes', Aging (Albany NY), 4: 436. Hammer, Sebastiaan, Marieke Snel, Hildo J Lamb, Ingrid M Jazet, Rutger W van der Meer, Hanno Pijl, Edo A Meinders, Johannes A Romijn, Albert de Roos, and Johannes WA Smit. 2008. 'Prolonged caloric restriction in obese patients with type 2 diabetes mellitus decreases myocardial triglyceride content and improves myocardial function', Journal of the American College of Cardiology, 52: 1006-12. Han, Myung-Kwan, Eun-Kyung Song, Ying Guo, Xuan Ou, Charlie Mantel, and Hal E Broxmeyer. 2008. 'SIRT1 regulates apoptosis and Nanog expression in mouse embryonic stem cells by controlling p53 subcellular localization', Cell stem cell, 2: 241- 51. Hanzlikova, Hana, William Gittens, Katerina Krejcikova, Zhihong Zeng, and Keith W Caldecott. 2017. 'Overlapping roles for PARP1 and PARP2 in the recruitment of endogenous XRCC1 and PNKP into oxidized chromatin', Nucleic acids research, 45: 2546-57. Hara, Nobumasa, Harumi Osago, Mineyoshi Hiyoshi, Mikiko Kobayashi-Miura, and Mikako Tsuchiya. 2019. 'Quantitative analysis of the effects of nicotinamide phosphoribosyltransferase induction on the rates of NAD+ synthesis and breakdown in mammalian cells using stable isotope-labeling combined with mass spectrometry', PloS one, 14: e0214000. Hara, Nobumasa, Kazuo Yamada, Tomoko Shibata, Harumi Osago, and Mikako Tsuchiya. 2011. 'Nicotinamide phosphoribosyltransferase/visfatin does not catalyze nicotinamide mononucleotide formation in blood plasma', PloS one, 6. Hara, Nobumasa, Kazuo Yamada, Masaharu Terashima, Harumi Osago, Makoto Shimoyama, and Mikako Tsuchiya. 2003. 'Molecular identification of human glutamine-and ammonia-dependent NAD synthetases carbon-nitrogen hydrolase domain confers glutamine dependency', Journal of Biological Chemistry, 278: 10914-21. Harden, Arthur, and William John Young. 1906a. 'The alcoholic ferment of yeast-juice', Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character, 77: 405-20. ———. 1906b. 'The alcoholic ferment of yeast-juice. Part II.—The coferment of yeast-juice', Proceedings of the Royal Society of London. Series B, Containing Papers of a Biological Character, 78: 369-75. Hardie, D Grahame, Fiona A Ross, and Simon A Hawley. 2012. 'AMPK: a nutrient and energy sensor that maintains energy homeostasis', Nature reviews Molecular cell biology, 13: 251-62. Harris, J Ieuan. 1972. 'Glyceraldehyde 3-phosphate dehydrogenase.' in, Structure and Function of Oxidation–Reduction Enzymes (Elsevier).

301 References

Harrison, David E, Randy Strong, Zelton Dave Sharp, James F Nelson, Clinton M Astle, Kevin Flurkey, Nancy L Nadon, J Erby Wilkinson, Krystyna Frenkel, and Christy S Carter. 2009. 'Rapamycin fed late in life extends lifespan in genetically heterogeneous mice', Nature, 460: 392-95. Harrison, Ian F, Andrew D Smith, and David T Dexter. 2018. 'Pathological histone acetylation in Parkinson’s disease: Neuroprotection and inhibition of microglial activation through SIRT 2 inhibition', Neuroscience letters, 666: 48-57. Hasegawa, Takafumi, Toru Baba, Michiko Kobayashi, Masatoshi Konno, Naoto Sugeno, Akio Kikuchi, Yasuto Itoyama, and Atsushi Takeda. 2010. 'Role of TPPP/p25 on α- synuclein-mediated oligodendroglial degeneration and the protective effect of SIRT2 inhibition in a cellular model of multiple system atrophy', Neurochemistry international, 57: 857-66. He, Shenghui, and Norman E Sharpless. 2017. 'Senescence in health and disease', Cell, 169: 1000-11. Heale, Jason T, Alexander R Ball Jr, John A Schmiesing, Jong-Soo Kim, Xiangduo Kong, Sharleen Zhou, Damien F Hudson, William C Earnshaw, and Kyoko Yokomori. 2006. 'Condensin I interacts with the PARP-1-XRCC1 complex and functions in DNA single- strand break repair', Molecular cell, 21: 837-48. Hegyi, Juraj, Robert A Schwartz, and Vladimir Hegyi. 2004. 'Pellagra: dermatitis, dementia, and diarrhea', International journal of dermatology, 43: 1-5. Henninger, Nils, James Bouley, Elif M Sikoglu, Jiyan An, Constance M Moore, Jean A King, Robert Bowser, Marc R Freeman, and Robert H Brown Jr. 2016. 'Attenuated traumatic axonal injury and improved functional outcome after traumatic brain injury in mice lacking Sarm1', Brain, 139: 1094-105. Henrique Mazucanti, Caio, Joao Victor Cabral-Costa, Andrea Rodrigues Vasconcelos, Diana Zukas Andreotti, Cristoforo Scavone, and Elisa Mitiko Kawamoto. 2015. 'Longevity pathways (mTOR, SIRT, Insulin/IGF-1) as key modulatory targets on aging and neurodegeneration', Current topics in medicinal chemistry, 15: 2116-38. Herman, Timothy F, and Cynthia Santos. 2019. 'First Pass Effect.' in, StatPearls [Internet] (StatPearls Publishing). Herranz, Daniel, Maribel Muñoz-Martin, Marta Cañamero, Francisca Mulero, Barbara Martinez-Pastor, Oscar Fernandez-Capetillo, and Manuel Serrano. 2010. 'Sirt1 improves healthy ageing and protects from metabolic syndrome-associated cancer', Nature communications, 1: 1-8. Hirschey, Matthew D, Tadahiro Shimazu, Eric Goetzman, Enxuan Jing, Bjoern Schwer, David B Lombard, Carrie A Grueter, Charles Harris, Sudha Biddinger, and Olga R Ilkayeva. 2010. 'SIRT3 regulates mitochondrial fatty-acid oxidation by reversible enzyme deacetylation', Nature, 464: 121-25. Ho, Ch S, CWK Lam, MHM Chan, RCK Cheung, LK Law, LCW Lit, KF Ng, MWM Suen, and HL Tai. 2003. 'Electrospray ionisation mass spectrometry: principles and clinical applications', The Clinical Biochemist Reviews, 24: 3. Ho, Hung-Yao, Yu-Ting Lin, Gigin Lin, Pei-Ru Wu, and Mei-Ling Cheng. 2017. 'Nicotinamide nucleotide transhydrogenase (NNT) deficiency dysregulates mitochondrial retrograde signaling and impedes proliferation', Redox biology, 12: 916- 28. Hoffmann, Gesine, Frank Breitenbücher, Martin Schuler, and Ann E Ehrenhofer-Murray. 2014. 'A novel sirtuin 2 (SIRT2) inhibitor with p53-dependent pro-apoptotic activity in non-small cell lung cancer', Journal of Biological Chemistry, 289: 5208-16.

302 References

Hogan, Kelly A, Claudia Chini, and Eduardo N Chini. 2019. 'The multi-faceted ecto-enzyme CD38: roles in immunomodulation, cancer, aging and metabolic diseases', Frontiers in immunology, 10: 1187. Honjoh, Sakiko, Takuya Yamamoto, Masaharu Uno, and Eisuke Nishida. 2009. 'Signalling through RHEB-1 mediates intermittent fasting-induced longevity in C. elegans', Nature, 457: 726-30. Hooper, Lora V, Tore Midtvedt, and Jeffrey I Gordon. 2002. 'How host-microbial interactions shape the nutrient environment of the mammalian intestine', Annual review of nutrition, 22: 283-307. Horenstein, Alberto L, Antonella Chillemi, Gianluca Zaccarello, Santina Bruzzone, Valeria Quarona, Andrea Zito, Sara Serra, and Fabio Malavasi. 2013. 'A CD38/CD203a/CD73 ectoenzymatic pathway independent of CD39 drives a novel adenosinergic loop in human T lymphocytes', Oncoimmunology, 2: e26246. Horenstein, Alberto L, Valeria Quarona, Denise Toscani, Federica Costa, Antonella Chillemi, Vito Pistoia, Nicola Giuliani, and Fabio Malavasi. 2016. 'Adenosine generated in the bone marrow niche through a CD38-mediated pathway correlates with progression of human myeloma', Molecular medicine, 22: 694-704. Hotta, Nigishi, R Kawamori, M Fukuda, Y Shigeta, and Aldose Reductase Inhibitor–Diabetes Complications Trial Study Group. 2012. 'Long‐term clinical effects of epalrestat, an aldose reductase inhibitor, on progression of diabetic neuropathy and other microvascular complications: multivariate epidemiological analysis based on patient background factors and severity of diabetic neuropathy', Diabetic medicine, 29: 1529- 33. Hou, Wei, Shiying Lu, Han Zhao, Yan Yu, Haodong Xu, Biao Yu, Lin Su, Chenshui Lin, and Benfang Helen Ruan. 2019. 'Propylselen inhibits cancer cell growth by targeting glutamate dehydrogenase at the NADP+ binding site', Biochemical and biophysical research communications, 509: 262-67. Hou, Yujun, Sofie Lautrup, Stephanie Cordonnier, Yue Wang, Deborah L Croteau, Eduardo Zavala, Yongqing Zhang, Kanako Moritoh, Jennifer F O’Connell, and Beverly A Baptiste. 2018. 'NAD+ supplementation normalizes key Alzheimer’s features and DNA damage responses in a new AD mouse model with introduced DNA repair deficiency', Proceedings of the National Academy of Sciences, 115: E1876-E85. Houthoofd, Koen, Bart P Braeckman, Thomas E Johnson, and Jacques R Vanfleteren. 2003. 'Life extension via dietary restriction is independent of the Ins/IGF-1 signalling pathway in Caenorhabditis elegans', Experimental gerontology, 38: 947-54. Houtkooper, Riekelt H, Eija Pirinen, and Johan Auwerx. 2012. 'Sirtuins as regulators of metabolism and healthspan', Nature reviews Molecular cell biology, 13: 225-38. Howard, Maureen, J Christopher Grimaldi, J Fernando Bazan, Frances E Lund, Leopoldo Santos-Argumedo, RM Parkhouse, Timothy F Walseth, and Hon Cheung Lee. 1993. 'Formation and hydrolysis of cyclic ADP-ribose catalyzed by lymphocyte antigen CD38', Science, 262: 1056-59. Howitz, Konrad T, Kevin J Bitterman, Haim Y Cohen, Dudley W Lamming, Siva Lavu, Jason G Wood, Robert E Zipkin, Phuong Chung, Anne Kisielewski, and Li-Li Zhang. 2003. 'Small molecule activators of sirtuins extend Saccharomyces cerevisiae lifespan', Nature, 425: 191-96. Hu, Gang, Alexander B Taylor, Lee McAlister-Henn, and P John Hart. 2007. 'Crystal structure of the yeast nicotinamidase Pnc1p', Archives of biochemistry and biophysics, 461: 66- 75.

303 References

Huang, Jeffrey Y, Kang Wang, Anke Vermehren-Schmaedick, John P Adelman, and Michael S Cohen. 2016. 'PARP6 is a regulator of hippocampal dendritic morphogenesis', Scientific reports, 6: 18512. Huang, Shih-Min A, Yuji M Mishina, Shanming Liu, Atwood Cheung, Frank Stegmeier, Gregory A Michaud, Olga Charlat, Elizabeth Wiellette, Yue Zhang, and Stephanie Wiessner. 2009. 'Tankyrase inhibition stabilizes axin and antagonizes Wnt signalling', Nature, 461: 614-20. Hughes, DE, and DH Williamson. 1953. 'The deamidation of nicotinamide by bacteria', Biochemical Journal, 55: 851-56. Hussain, Alamgir MM, Hon Cheung Lee, and Chan Fong Chang. 1998. 'Functional expression of secreted mouse BST-1 in yeast', Protein expression and purification, 12: 133-37. Huttenhower, Curtis, Dirk Gevers, Rob Knight, Sahar Abubucker, Jonathan H Badger, Asif T Chinwalla, Heather H Creasy, Ashlee M Earl, Michael G FitzGerald, and Robert S Fulton. 2012. 'Structure, function and diversity of the healthy human microbiome', Nature, 486: 207. Imai, Shin-Ichiro, Christopher M Armstrong, Matt Kaeberlein, and Leonard Guarente. 2000a. 'Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase', Nature, 403: 795-800. ———. 2000b. 'Transcriptional silencing and longevity protein Sir2 is an NAD-dependent histone deacetylase', Nature, 403: 795. Imai, Shin-ichiro, and Leonard Guarente. 2016. 'It takes two to tango: NAD+ and sirtuins in aging/longevity control', npj Aging and Mechanisms of Disease, 2: 1-6. Imai, Tadayoshi. 1973. 'Purification and properties of nicotinamide mononucleotide amidohydrolase from Azotobacter vinelandii', The Journal of Biochemistry, 73: 139- 53. Imsande, John. 1961. 'Pathway of diphosphopyridine nucleotide biosynthesis in Escherichia coli', Journal of Biological Chemistry, 236: 1494-97. Innes, David, Ifor R Beacham, and Dennis M Burns. 2001. 'The role of the intracellular inhibitor of periplasmic UDP‐sugar hydrolase (5′‐nucleotidase) in Escherichia coli: cytoplasmic localisation of 5′‐nucleotidase is conditionally lethal', Journal of Basic Microbiology: An International Journal on Biochemistry, Physiology, Genetics, Morphology, and Ecology of Microorganisms, 41: 329-37. Irie, Junichiro, Emi Inagaki, Masataka Fujita, Hideaki Nakaya, Masanori Mitsuishi, Shintaro Yamaguchi, Kazuya Yamashita, Shuhei Shigaki, Takashi Ono, and Hideo Yukioka. 2019. 'Effect of oral administration of nicotinamide mononucleotide on clinical parameters and nicotinamide metabolite levels in healthy Japanese men', Endocrine journal: EJ19-0313. ———. 2020. 'Effect of oral administration of nicotinamide mononucleotide on clinical parameters and nicotinamide metabolite levels in healthy Japanese men', Endocrine journal, 67: 153-60. Iwamoto, Yoshiki, In Sook Matsui Lee, Ryo Kido, and Motonari Tsubaki. 1995. 'Tryptophan 2, 3-dioxygenase in Saccharomyces cerevisiae', Canadian journal of microbiology, 41: 19-26. Iwata, Hiroshi, Masanori Aikawa, Takuya Hara, Sasha Singh, Piero Ricchiuto, Hideo Yoshida, and Iwao Yamada. 2015. "Parp9 and parp14 as key regulators of macrophage activation." In.: Google Patents. Iwata, Hiroshi, Claudia Goettsch, Amitabh Sharma, Piero Ricchiuto, Wilson Wen Bin Goh, Arda Halu, Iwao Yamada, Hideo Yoshida, Takuya Hara, and Mei Wei. 2016. 'PARP9 and PARP14 cross-regulate macrophage activation via STAT1 ADP-ribosylation', Nature communications, 7: 1-19.

304 References

Iwata, Hiroshi, Hideo Yoshida, Piero Ricchiuto, Takuya Hara, Iwao Yamada, Alexander Mojcher, Peter Libby, Mark Boothby, Elena Aikawa, and Sasha Singh. 2014. 'PARP9 and PARP14 are Novel Regulators of Macrophage Activation', Circulation, 130: A18687-A87. Jacquot, Jean-Pierre, and Mirko Zaffagnini. 2019. Thioredoxin and Glutaredoxin Systems (MDPI-Multidisciplinary Digital Publishing Institute). Jadeja, Ravirajsinh N, Folami L Powell, Malita A Jones, Jasmine Fuller, Ethan Joseph, Menaka C Thounaojam, Manuela Bartoli, and Pamela M Martin. 2018. 'Loss of NAMPT in aging retinal pigment epithelium reduces NAD+ availability and promotes cellular senescence', Aging (Albany NY), 10: 1306. Jaehme, Michael, Albert Guskov, and Dirk Jan Slotboom. 2014. 'Crystal structure of the vitamin B 3 transporter PnuC, a full-length SWEET homolog', nature structural & molecular biology, 21: 1013-15. Jaehme, Michael, and Dirk Jan Slotboom. 2015. 'Diversity of membrane transport proteins for vitamins in bacteria and archaea', Biochimica et Biophysica Acta (BBA)-General Subjects, 1850: 565-76. Jang, So-young, Hyun Tae Kang, and Eun Seong Hwang. 2012. 'Nicotinamide-induced mitophagy event mediated by high NAD+/NADH ratio and SIRT1 protein activation', Journal of Biological Chemistry, 287: 19304-14. Jarvis, SIMON M. 1986. 'Nitrobenzylthioinosine-sensitive nucleoside transport system: mechanism of inhibition by dipyridamole', Molecular pharmacology, 30: 659-65. Jeanguenin, Linda, Aurora Lara-Núñez, Dmitry A Rodionov, Andrei L Osterman, Nataliya Y Komarova, Doris Rentsch, Jesse F Gregory, and Andrew D Hanson. 2012. 'Comparative genomics and functional analysis of the NiaP family uncover nicotinate transporters from bacteria, plants, and mammals', Functional & integrative genomics, 12: 25-34. Jeong, Jaemin, Kyungmi Juhn, Hansoo Lee, Sang-Hoon Kim, Bon-Hong Min, Kyung-Mi Lee, Myung-Haeng Cho, Gil-Hong Park, and Kee-Ho Lee. 2007. 'SIRT1 promotes DNA repair activity and deacetylation of Ku70', Experimental & molecular medicine, 39: 8- 13. Jeong, Seung Min, Annie Lee, Jaewon Lee, and Marcia C Haigis. 2014. 'SIRT4 protein suppresses tumor formation in genetic models of Myc-induced B cell lymphoma', Journal of Biological Chemistry, 289: 4135-44. Jeong, Seung Min, Cuiying Xiao, Lydia WS Finley, Tyler Lahusen, Amanda L Souza, Kerry Pierce, Ying-Hua Li, Xiaoxu Wang, Gaëlle Laurent, and Natalie J German. 2013. 'SIRT4 has tumor-suppressive activity and regulates the cellular metabolic response to DNA damage by inhibiting mitochondrial glutamine metabolism', Cancer cell, 23: 450- 63. Jęśko, Henryk, Przemysław Wencel, Robert P Strosznajder, and Joanna B Strosznajder. 2017. 'Sirtuins and their roles in brain aging and neurodegenerative disorders', Neurochemical research, 42: 876-90. Jilani, Arshad, Dindial Ramotar, Carolyn Slack, Colin Ong, Xiao Ming Yang, Stephen W Scherer, and Dana D Lasko. 1999. 'Molecular cloning of the human gene, PNKP, encoding a polynucleotide kinase 3′-phosphatase and evidence for its role in repair of DNA strand breaks caused by oxidative damage', Journal of Biological Chemistry, 274: 24176-86. Jimeno, Rebeca, Phillip M Brailey, and Patricia Barral. 2018. 'Quantitative Polymerase Chain Reaction-based Analyses of Murine Intestinal Microbiota After Oral Antibiotic Treatment', JoVE (Journal of Visualized Experiments): e58481.

305 References

Jin, Duo, Hong-Xiang Liu, Hirokazu Hirai, Takashi Torashima, Taku Nagai, Olga Lopatina, Natalia A Shnayder, Kiyofumi Yamada, Mami Noda, and Toshihiro Seike. 2007a. 'CD38 is critical for social behaviour by regulating oxytocin secretion', Nature, 446: 41-45. Jin, Lingtao, Dan Li, Gina N Alesi, Jun Fan, Hee-Bum Kang, Zhou Lu, Titus J Boggon, Peng Jin, Hong Yi, and Elizabeth R Wright. 2015. 'Glutamate dehydrogenase 1 signals through antioxidant glutathione peroxidase 1 to regulate redox homeostasis and tumor growth', Cancer cell, 27: 257-70. Jin, Qihuang, Tingting Yan, Xinjian Ge, Cheng Sun, Xianglin Shi, and Qiwei Zhai. 2007b. 'Cytoplasm‐localized SIRT1 enhances apoptosis', Journal of cellular physiology, 213: 88-97. Jin, Richard C, Christopher E Mahoney, Laura Anderson, Filomena Ottaviano, Kevin Croce, Jane A Leopold, Ying-Yi Zhang, Shiow-Shih Tang, Diane E Handy, and Joseph Loscalzo. 2011. 'Glutathione peroxidase-3 deficiency promotes platelet-dependent thrombosis in vivo', Circulation, 123: 1963-73. Jing, Enxuan, Stephane Gesta, and C Ronald Kahn. 2007. 'SIRT2 regulates adipocyte differentiation through FoxO1 acetylation/deacetylation', Cell metabolism, 6: 105-14. Jing, Hui, Jing Hu, Bin He, Yashira L Negrón Abril, Jack Stupinski, Keren Weiser, Marisa Carbonaro, Ying-Ling Chiang, Teresa Southard, and Paraskevi Giannakakou. 2016. 'A SIRT2-selective inhibitor promotes c-Myc oncoprotein degradation and exhibits broad anticancer activity', Cancer cell, 29: 297-310. John, CM, R Ramasamy, G Al Naqeeb, AHD Al-Nuaimi, and A Adam. 2012. 'Nicotinamide supplementation protects gestational diabetic rats by reducing oxidative stress and enhancing immune responses', Current medicinal chemistry, 19: 5181-86. Johnson, Michael DL, Haley Echlin, Tina H Dao, and Jason W Rosch. 2015. 'Characterization of NAD salvage pathways and their role in virulence in Streptococcus pneumoniae', Microbiology, 161: 2127. Johnson, Sean, and Shin–ichiro Imai. 2018. 'NAD+ biosynthesis, aging, and disease', F1000Research, 7. Johnson, Sean, David F Wozniak, and S Imai. 2018. 'CA1 Nampt knockdown recapitulates hippocampal cognitive phenotypes in old mice which nicotinamide mononucleotide improves', npj Aging and Mechanisms of Disease, 4: 10. Jwa, Miri, and Paul Chang. 2012. 'PARP16 is a tail-anchored endoplasmic reticulum protein required for the PERK-and IRE1α-mediated unfolded protein response', Nature cell biology, 14: 1223-30. Kaeberlein, Matt, Kathryn T Kirkland, Stanley Fields, and Brian K Kennedy. 2004. 'Sir2- independent life span extension by calorie restriction in yeast', PLoS biology, 2. Kaeberlein, Matt, Mitch McVey, and Leonard Guarente. 1999. 'The SIR2/3/4 complex and SIR2 alone promote longevity in Saccharomyces cerevisiae by two different mechanisms', Genes & development, 13: 2570-80. Kaidi, Abderrahmane, Brian T Weinert, Chunaram Choudhary, and Stephen P Jackson. 2010. 'Human SIRT6 promotes DNA end resection through CtIP deacetylation', Science, 329: 1348-53. Kanfi, Yariv, Shoshana Naiman, Gail Amir, Victoria Peshti, Guy Zinman, Liat Nahum, Ziv Bar-Joseph, and Haim Y Cohen. 2012. 'The sirtuin SIRT6 regulates lifespan in male mice', Nature, 483: 218-21. Kang, Chil-sung, Mingi Ban, Eun-Jeong Choi, Hyung-Geun Moon, Jun-Sung Jeon, Dae-Kyum Kim, Soo-Kyung Park, Seong Gyu Jeon, Tae-Young Roh, and Seung-Jae Myung. 2013a. 'Extracellular vesicles derived from gut microbiota, especially Akkermansia

306 References

muciniphila, protect the progression of dextran sulfate sodium-induced colitis', PloS one, 8. Kang, Hyeog, Jae-Won Jung, Myung K Kim, and Jay H Chung. 2009. 'CK2 is the regulator of SIRT1 substrate-binding affinity, deacetylase activity and cellular response to DNA- damage', PloS one, 4. Kang, Mi Jeong, Hyung Gyun Kim, Jin Sung Kim, Do Gyeong Oh, Yeon Ji Um, Chae Shin Seo, Ji Won Han, Hyun Ji Cho, Ghee Hwan Kim, and Tae Cheon Jeong. 2013b. 'The effect of gut microbiota on drug metabolism', Expert opinion on drug metabolism & toxicology, 9: 1295-308. Kato, Michiko, and Su-Ju Lin. 2014. 'YCL047C/POF1 is a novel nicotinamide mononucleotide adenylyltransferase (NMNAT) in Saccharomyces cerevisiae', Journal of Biological Chemistry, 289: 15577-87. Katsyuba, Elena, Mario Romani, Dina Hofer, and Johan Auwerx. 2020. 'NAD+ homeostasis in health and disease', Nature metabolism: 1-23. Kawahara, Tiara LA, Eriko Michishita, Adam S Adler, Mara Damian, Elisabeth Berber, Meihong Lin, Ron A McCord, Kristine CL Ongaigui, Lisa D Boxer, and Howard Y Chang. 2009. 'SIRT6 links histone H3 lysine 9 deacetylation to NF-κB-dependent gene expression and organismal life span', Cell, 136: 62-74. Kawai, Toshihide, Izumi Takei, Mikiya Tokui, Osamu Funae, Kazunori Miyamoto, Mitsuhisa Tabata, Takumi Hirata, Takao Saruta, Akira Shimada, and Hiroshi Itoh. 2010. 'Effects of epalrestat, an aldose reductase inhibitor, on diabetic peripheral neuropathy in patients with type 2 diabetes, in relation to suppression of Nɛ-carboxymethyl lysine', Journal of Diabetes and its Complications, 24: 424-32. Kawamura, Tomoyo, Noriyuki Mori, and Katsumi Shibata. 2016. 'β-Nicotinamide mononucleotide, an anti-aging candidate compound, is retained in the body for longer than nicotinamide in rats', Journal of nutritional science and vitaminology, 62: 272-76. Kelly, Andrew D, Susanne B Breitkopf, Min Yuan, Jeffrey Goldsmith, Dimitrios Spentzos, and John M Asara. 2011. 'Metabolomic profiling from formalin-fixed, paraffin- embedded tumor tissue using targeted LC/MS/MS: application in sarcoma', PloS one, 6: e25357. Khalsa, Jag, Linda C Duffy, Gabriela Riscuta, Pamela Starke‐Reed, and Van S Hubbard. 2017. 'Omics for Understanding the Gut‐Liver‐Microbiome Axis and Precision Medicine', Clinical pharmacology in drug development, 6: 176-85. Khan, Javed A, Song Xiang, and Liang Tong. 2007. 'Crystal structure of human nicotinamide riboside kinase', Structure, 15: 1005-13. Khan, Nahid A, Mari Auranen, Ilse Paetau, Eija Pirinen, Liliya Euro, Saara Forsström, Lotta Pasila, Vidya Velagapudi, Christopher J Carroll, and Johan Auwerx. 2014. 'Effective treatment of mitochondrial myopathy by nicotinamide riboside, a vitamin B3', EMBO molecular medicine, 6: 721-31. Kilianski, Andy, Jamie L Haas, Elizabeth J Corriveau, Alvin T Liem, Kristen L Willis, Dana R Kadavy, C Nicole Rosenzweig, and Samuel S Minot. 2015. 'Bacterial and viral identification and differentiation by amplicon sequencing on the MinION nanopore sequencer', Gigascience, 4: 12. Kim, Byung-Ju, Kwang-Hyun Park, Chang-Yeol Yim, Shin Takasawa, Hiroshi Okamoto, Mie- Jae Im, and Uh-Hyun Kim. 2008. 'Generation of NAADP and cADPR by glucagon-like peptide-1 evokes Ca2+ signal that Is essential for insulin secretion in mouse pancreatic islets', Diabetes. Kim, Hyun-Seok, Krish Patel, Kristi Muldoon-Jacobs, Kheem S Bisht, Nukhet Aykin-Burns, J Daniel Pennington, Riet van der Meer, Phuongmai Nguyen, Jason Savage, and Kjerstin M Owens. 2010. 'SIRT3 is a mitochondria-localized tumor suppressor required

307 References

for maintenance of mitochondrial integrity and metabolism during stress', Cancer cell, 17: 41-52. Kim, Hyuntae, Elaine L Jacobson, and Myron K Jacobson. 1993. 'Synthesis and degradation of cyclic ADP-ribose by NAD glycohydrolases', Science, 261: 1330-33. Kim, Ja-Eun, Junjie Chen, and Zhenkun Lou. 2008. 'DBC1 is a negative regulator of SIRT1', Nature, 451: 583-86. Kim, Ji-Eun, Jaesung Choi, JooYong Park, Chulbum Park, Se Mi Lee, Seong Eun Park, Nan Song, Seokang Chung, Hyuna Sung, and Wonshik Han. 2018. 'Associations between genetic polymorphisms of membrane transporter genes and prognosis after chemotherapy: meta-analysis and finding from Seoul Breast Cancer Study (SEBCS)', The pharmacogenomics journal, 18: 633-45. Kim, Jung Ryul, Young Jae Moon, Keun Sang Kwon, Jun Sang Bae, Sajeev Wagle, Taek Kyun Yu, Kyoung Min Kim, Ho Sung Park, Ju-Hyung Lee, and Woo Sung Moon. 2013a. 'Expression of SIRT1 and DBC1 is associated with poor prognosis of soft tissue sarcomas', PloS one, 8. Kim, Kwang Seok, Young Bae Seu, Suk-Hwan Baek, Mi Jin Kim, Keuk Jun Kim, Jung Hye Kim, and Jae-Ryong Kim. 2007. 'Induction of cellular senescence by insulin-like growth factor binding protein-5 through a p53-dependent mechanism', Molecular biology of the cell, 18: 4543-52. Kim, Mi Jin, Dae Won Kim, Jung Hwan Park, Sang Jin Kim, Chi Hern Lee, Ji In Yong, Eun Ji Ryu, Su Bin Cho, Hyeon Ji Yeo, and Jiye Hyeon. 2013b. 'PEP-1–SIRT2 inhibits inflammatory response and oxidative stress-induced cell death via expression of antioxidant enzymes in murine macrophages', Free Radical Biology and Medicine, 63: 432-45. Kim, Mi Young, Tong Zhang, and W Lee Kraus. 2005. 'Poly (ADP-ribosyl) ation by PARP- 1:PAR-laying'NAD+ into a nuclear signal', Genes & development, 19: 1951-67. King, Brenee S, Karen L Cooper, Ke Jian Liu, and Laurie G Hudson. 2012. 'Poly (ADP-ribose) contributes to an association between poly (ADP-ribose) polymerase-1 and xeroderma pigmentosum complementation group A in nucleotide excision repair', Journal of Biological Chemistry, 287: 39824-33. Kirby, Ilsa T, Ana Kojic, Moriah R Arnold, Ann-Gerd Thorsell, Tobias Karlberg, Anke Vermehren-Schmaedick, Raashi Sreenivasan, Carsten Schultz, Herwig Schüler, and Michael S Cohen. 2018. 'A potent and selective PARP11 inhibitor suggests coupling between cellular localization and catalytic activity', Cell chemical biology, 25: 1547- 53. e12. Kirchner, Julius, J Gary Watson, and Sterling Chaykin. 1966. 'Nicotinamide deamidase from rabbit liver', Journal of Biological Chemistry, 241: 953-60. Kirkman, Henry N. 1962. 'Glucose 6-phosphate dehydrogenase from human erythrocytes I. Further purification and characterization', Journal of Biological Chemistry, 237: 2364- 70. Kita, K, CR Vibat, S Meinhardt, John R Guest, and Robert B Gennis. 1989. 'One-step purification from Escherichia coli of complex II (succinate: ubiquinone oxidoreductase) associated with succinate-reducible cytochrome b556', Journal of Biological Chemistry, 264: 2672-77. Klar, AJ, and Seymour Fogel. 1979. 'Activation of mating type genes by transposition in Saccharomyces cerevisiae', Proceedings of the National Academy of Sciences, 76: 4539-43. Koh, Ho-Jin, Su-Min Lee, Byung-Gap Son, Soh-Hyun Lee, Zae Young Ryoo, Kyu-Tae Chang, Jeen-Woo Park, Dong-Chan Park, Byoung J Song, and Richard L Veech. 2004.

308 References

'Cytosolic NADP+-dependent isocitrate dehydrogenase plays a key role in lipid metabolism', Journal of Biological Chemistry, 279: 39968-74. Kong, Shanshan, Yanhui H Zhang, and Weiqiang Zhang. 2018. 'Regulation of intestinal epithelial cells properties and functions by amino acids', BioMed research international, 2018. Kong, Xingxing, Rui Wang, Yuan Xue, Xiaojun Liu, Huabing Zhang, Yong Chen, Fude Fang, and Yongsheng Chang. 2010. 'Sirtuin 3, a new target of PGC-1α, plays an important role in the suppression of ROS and mitochondrial biogenesis', PloS one, 5. Koppel, Nitzan, Vayu Maini Rekdal, and Emily P Balskus. 2017. 'Chemical transformation of xenobiotics by the human gut microbiota', Science, 356: eaag2770. Kops, Geert JPL, Tobias B Dansen, Paulien E Polderman, Ingrid Saarloos, Karel WA Wirtz, Paul J Coffer, Ting-T Huang, Johannes L Bos, René H Medema, and Boudewijn MT Burgering. 2002. 'Forkhead transcription factor FOXO3a protects quiescent cells from oxidative stress', Nature, 419: 316-21. Kornberg, Arthur. 1950. 'Enzymatic synthesis of triphosphopyridine nucleotide', J. biol. Chem, 182: 805-13. Kourtzidis, Ioannis A, Andreas T Stoupas, Ioannis S Gioris, Aristidis S Veskoukis, Nikos V Margaritelis, Maria Tsantarliotou, Ioannis Taitzoglou, Ioannis S Vrabas, Vassilis Paschalis, and Antonios Kyparos. 2016. 'The NAD+ precursor nicotinamide riboside decreases exercise performance in rats', Journal of the International Society of Sports Nutrition, 13: 32. Kraus, W Lee. 2020. "PARPs and ADP-ribosylation: 60 years on." In.: Cold Spring Harbor Lab. Krauss, Raul, Todd Bosanac, Rajesh Devraj, Thomas Engber, and Robert O Hughes. 2020. 'Axons Matter: The Promise of Treating Neurodegenerative Disorders by Targeting SARM1-Mediated Axonal Degeneration', Trends in Pharmacological Sciences. Krehl, WA de, J De La Huerga, CA Elvehjem, and EB Hart. 1946. 'The distribution of niacin amide and niacin in natural materials', Journal of Biological Chemistry, 166: 53-57. Krokan, Hans E, Rune Standal, and Geir Slupphaug. 1997. 'DNA glycosylases in the base excision repair of DNA', Biochemical Journal, 325: 1-16. Kubota, Yoshiko, Rachel A Nash, Arne Klungland, P Schär, DE Barnes, and T Lindahl. 1996. 'Reconstitution of DNA base excision‐repair with purified human proteins: interaction between DNA polymerase beta and the XRCC1 protein', The EMBO journal, 15: 6662- 70. Kucharczyk, Roza, Marek Zagulski, Joanna Rytka, and Christopher J Herbert. 1998. 'The yeast gene YJR025c encodes a 3‐hydroxyanthranilic acid dioxygenase and is involved in nicotinic acid biosynthesis', FEBS letters, 424: 127-30. Kulikova, Veronika, Konstantin Shabalin, Kirill Nerinovski, Christian Dölle, Marc Niere, Alexander Yakimov, Philip Redpath, Mikhail Khodorkovskiy, Marie E Migaud, and Mathias Ziegler. 2015. 'Generation, release, and uptake of the NAD precursor nicotinic acid riboside by human cells', Journal of Biological Chemistry, 290: 27124-37. Kulikova, Veronika, Konstantin Shabalin, Kirill Nerinovski, Alexander Yakimov, Maria Svetlova, Ljudmila Solovjeva, Andrey Kropotov, Mikhail Khodorkovskiy, Marie E Migaud, and Mathias Ziegler. 2019. 'Degradation of Extracellular NAD+ Intermediates in Cultures of Human HEK293 Cells', Metabolites, 9: 293. Kulkarni, Chaitanya A, and Paul S Brookes. 2019. 'Cellular Compartmentation and the Redox/Nonredox Functions of NAD+', Antioxidants & redox signaling, 31: 623-42. Kuny, Chad V, and Christopher S Sullivan. 2016. 'Virus–host interactions and the ARTD/PARP family of enzymes', PLoS pathogens, 12.

309 References

Kurnasov, Oleg, Vasiliy Goral, Keri Colabroy, Svetlana Gerdes, Shubha Anantha, Andrei Osterman, and Tadhg P Begley. 2003. 'NAD biosynthesis: identification of the tryptophan to quinolinate pathway in bacteria', Chemistry & biology, 10: 1195-204. Kurnasov, Oleg V, Boris M Polanuyer, Shubha Ananta, Roman Sloutsky, Annie Tam, Svetlana Y Gerdes, and Andrei L Osterman. 2002. 'Ribosylnicotinamide kinase domain of NadR protein: identification and implications in NAD biosynthesis', Journal of bacteriology, 184: 6906-17. Kwon, Guideock, Md Jamal Uddin, Gayoung Lee, Songling Jiang, Ahreum Cho, Jung Hwa Lee, Sae Rom Lee, Yun Soo Bae, Sung Hwan Moon, and Soo Jin Lee. 2017. 'A novel pan-Nox inhibitor, APX-115, protects kidney injury in streptozotocin-induced diabetic mice: possible role of peroxisomal and mitochondrial biogenesis', Oncotarget, 8: 74217. Lagouge, Marie, Carmen Argmann, Zachary Gerhart-Hines, Hamid Meziane, Carles Lerin, Frederic Daussin, Nadia Messadeq, Jill Milne, Philip Lambert, and Peter Elliott. 2006. 'Resveratrol improves mitochondrial function and protects against metabolic disease by activating SIRT1 and PGC-1α', Cell, 127: 1109-22. Landry, Joseph, Ann Sutton, Stefan T Tafrov, Ryan C Heller, John Stebbins, Lorraine Pillus, and Rolf Sternglanz. 2000. 'The silencing protein SIR2 and its homologs are NAD- dependent protein deacetylases', Proceedings of the National Academy of Sciences, 97: 5807-11. Langelier, Marie-France, Jamie L Planck, Swati Roy, and John M Pascal. 2012. 'Structural basis for DNA damage–dependent poly (ADP-ribosyl) ation by human PARP-1', Science, 336: 728-32. Langelier, Marie-France, Levani Zandarashvili, Pedro M Aguiar, Ben E Black, and John M Pascal. 2018. 'NAD+ analog reveals PARP-1 substrate-blocking mechanism and allosteric communication from catalytic center to DNA-binding domains', Nature communications, 9: 1-13. Lanza-Jacoby, Susan, Guang Yan, Glenn Radice, Christopher LePhong, Jeffrey Baliff, and Rachael Hess. 2013. 'Calorie restriction delays the progression of lesions to pancreatic cancer in the LSL-KrasG12D; Pdx-1/Cre mouse model of pancreatic cancer', Experimental Biology and Medicine, 238: 787-97. Laurent, Gaëlle, Natalie J German, Asish K Saha, Vincent CJ de Boer, Michael Davies, Timothy R Koves, Noah Dephoure, Frank Fischer, Gina Boanca, and Bhavapriya Vaitheesvaran. 2013. 'SIRT4 coordinates the balance between lipid synthesis and catabolism by repressing malonyl CoA decarboxylase', Molecular cell, 50: 686-98. Le Chatelier, Emmanuelle, Trine Nielsen, Junjie Qin, Edi Prifti, Falk Hildebrand, Gwen Falony, Mathieu Almeida, Manimozhiyan Arumugam, Jean-Michel Batto, and Sean Kennedy. 2013. 'Richness of human gut microbiome correlates with metabolic markers', Nature, 500: 541-46. Le Page, Florence, Valérie Schreiber, Claudine Dhérin, Gilbert de Murcia, and Serge Boiteux. 2003. 'Poly (ADP-ribose) polymerase-1 (PARP-1) is required in murine cell lines for base excision repair of oxidative DNA damage in the absence of DNA polymerase β', Journal of Biological Chemistry, 278: 18471-77. Lee, Cha-Uk, Eun-Kyung Song, Chae-Hwa Yoo, Yong-Keun Kwak, and Myung-Kwan Han. 2012. 'Lipopolysaccharide induces CD38 expression and solubilization in J774 macrophage cells', Molecules and cells, 34: 573-76. Lee, Cheol-Koo, Roger G Klopp, Richard Weindruch, and Tomas A Prolla. 1999. 'Gene expression profile of aging and its retardation by caloric restriction', Science, 285: 1390- 93.

310 References

Lee, Chi Fung, Juan D Chavez, Lorena Garcia-Menendez, Yongseon Choi, Nathan D Roe, Ying Ann Chiao, John S Edgar, Young Ah Goo, David R Goodlett, and James E Bruce. 2016a. 'Normalization of NAD+ redox balance as a therapy for heart failure', Circulation, 134: 883-94. Lee, Garrick D, Mark A Wilson, Min Zhu, Catherine A Wolkow, Rafael De Cabo, Donald K Ingram, and Sige Zou. 2006. 'Dietary deprivation extends lifespan in Caenorhabditis elegans', Aging cell, 5: 515-24. Lee, Ho, Kyung Ryoul Kim, Sang Jae Noh, Ho Sung Park, Keun Sang Kwon, Byung-Hyun Park, Sung Hoo Jung, Hyun Jo Youn, Byoung Kil Lee, and Myoung Ja Chung. 2011. 'Expression of DBC1 and SIRT1 is associated with poor prognosis for breast carcinoma', Human pathology, 42: 204-13. Lee, Min Kyung, Hyun Sub Cheong, Youngil Koh, Kwang-Sung Ahn, Sung-Soo Yoon, and Hyoung Doo Shin. 2016b. 'Genetic association of PARP15 polymorphisms with clinical outcome of acute myeloid leukemia in a Korean population', Genetic testing and molecular biomarkers, 20: 696-701. Lehnman, I Robert. 1974. 'DNA ligase: structure, mechanism, and function', Science, 186: 790- 97. Leidecker, Orsolya, Juan José Bonfiglio, Thomas Colby, Qi Zhang, Ilian Atanassov, Roko Zaja, Luca Palazzo, Anna Stockum, Ivan Ahel, and Ivan Matic. 2016. 'Serine is a new target residue for endogenous ADP-ribosylation on histones', Nature chemical biology, 12: 998. Lerner, Felicitas, Marc Niere, Antje Ludwig, and Mathias Ziegler. 2001. 'Structural and functional characterization of human NAD kinase', Biochemical and biophysical research communications, 288: 69-74. Lewis-Ballester, Ariel, Farhad Forouhar, Sung-Mi Kim, Scott Lew, YongQiang Wang, Shay Karkashon, Jayaraman Seetharaman, Dipanwita Batabyal, Bing-Yu Chiang, and Munif Hussain. 2016. 'Molecular basis for catalysis and substrate-mediated cellular stabilization of human tryptophan 2, 3-dioxygenase', Scientific reports, 6: 35169. Ley, Ruth E, Peter J Turnbaugh, Samuel Klein, and Jeffrey I Gordon. 2006. 'Human gut microbes associated with obesity', Nature, 444: 1022-23. Li, Da, Yan-Jie Tian, Jing Guo, Wu-Ping Sun, Yong-Zhi Lun, Ming Guo, Ning Luo, Yu Cao, Ji-Min Cao, and Xiao-Jie Gong. 2013. 'Nicotinamide supplementation induces detrimental metabolic and epigenetic changes in developing rats', British journal of nutrition, 110: 2156-64. Li, H, and W Jia. 2013. 'Cometabolism of microbes and host: implications for drug metabolism and drug‐induced toxicity', Clinical Pharmacology & Therapeutics, 94: 574-81. Li, Jiaxin, Xiaobing Dou, Songtao Li, Ximei Zhang, Yong Zeng, and Zhenyuan Song. 2015. 'Nicotinamide ameliorates palmitate-induced ER stress in hepatocytes via cAMP/PKA/CREB pathway-dependent Sirt1 upregulation', Biochimica et Biophysica Acta (BBA)-Molecular Cell Research, 1853: 2929-36. Li, Li, Yu-cai Fu, Jin-jie Xu, Xiao-chun Chen, Xuan-hao Lin, and Li-li Luo. 2011. 'Caloric restriction promotes the reproductive capacity of female rats via modulating the level of insulin-like growth factor-1 (IGF-1)', General and comparative endocrinology, 174: 232-37. Li, Mo, and Xiaochun Yu. 2013. 'Function of BRCA1 in the DNA damage response is mediated by ADP-ribosylation', Cancer cell, 23: 693-704. Li, Shuai, Zhuonan Zhuang, Teng Wu, Jie-Chun Lin, Ze-Xian Liu, Li-Fen Zhou, Ting Dai, Lei Lu, and Huai-Qiang Ju. 2018. 'Nicotinamide nucleotide transhydrogenase-mediated redox homeostasis promotes tumor growth and metastasis in gastric cancer', Redox biology, 18: 246-55.

311 References

Li, Wan-Yu, Ji-Hua Ren, Na-Na Tao, Long-Kuan Ran, Xiang Chen, Hong-Zhong Zhou, Bo Liu, Xiao-Song Li, Ai-Long Huang, and Juan Chen. 2016. 'The SIRT1 inhibitor, nicotinamide, inhibits hepatitis B virus replication in vitro and in vivo', Archives of virology, 161: 621-30. Liang, Mingyu, Eduardo N Chini, Jingfei Cheng, and Thomas P Dousa. 1999. 'Synthesis of NAADP and cADPR in mitochondria', Archives of biochemistry and biophysics, 371: 317-25. Liang, Xiaorong, Lulu Yang, Ann R Qin, Justin Ly, Bianca M Liederer, Kirsten Messick, Shuguang Ma, Mark Zak, Peter S Dragovich, and Brian J Dean. 2014. 'Measuring NAD+ levels in mouse blood and tissue samples via a surrogate matrix approach using LC–MS/MS', Bioanalysis, 6: 1445-57. Lima, Wanessa C, Alessandro M Varani, and Carlos FM Menck. 2009. 'NAD biosynthesis evolution in bacteria: lateral gene transfer of kynurenine pathway in Xanthomonadales and Flavobacteriales', Molecular biology and evolution, 26: 399-406. Lin, Leu-Fen Hou, Shih-Jung Lan, Arlan H Richardson, and LaVell M Henderson. 1972. 'Pyridine nucleotide synthesis purification of nicotinamide mononucleotide pyrophosphorylase from rat erythrocytes', Journal of Biological Chemistry, 247: 8016- 22. Lin, Su-Ju, Ethan Ford, Marcia Haigis, Greg Liszt, and Leonard Guarente. 2004. 'Calorie restriction extends yeast life span by lowering the level of NADH', Genes & development, 18: 12-16. Lin, Zhi-Feng, Hong-Bing Xu, Jian-Yun Wang, Qiang Lin, Zhen Ruan, Fa-Bing Liu, Wang Jin, Hai-Hua Huang, and Xi Chen. 2013. 'SIRT5 desuccinylates and activates SOD1 to eliminate ROS', Biochemical and biophysical research communications, 441: 191-95. Lindahl, Tomas. 1993. 'Instability and decay of the primary structure of DNA', Nature, 362: 709-15. Liu, Ban, Wenliang Che, Jinsong Xue, Changzhu Zheng, Kai Tang, Jingying Zhang, Jing Wen, and Yawei Xu. 2013. 'SIRT4 prevents hypoxia-induced apoptosis in H9c2 cardiomyoblast cells', Cellular Physiology and Biochemistry, 32: 655-62. Liu, Dong, Robert Gharavi, Michael Pitta, Marc Gleichmann, and Mark P Mattson. 2009. 'Nicotinamide prevents NAD+ depletion and protects neurons against excitotoxicity and cerebral ischemia: NAD+ consumption by SIRT1 may endanger energetically compromised neurons', Neuromolecular medicine, 11: 28-42. Liu, Jun, Yong Juan Zhao, Wan Hua Li, Yun Nan Hou, Ting Li, Zhi Ying Zhao, Cheng Fang, Song Lu Li, and Hon Cheung Lee. 2017a. 'Cytosolic interaction of type III human CD38 with CIB1 modulates cellular cyclic ADP-ribose levels', Proceedings of the National Academy of Sciences, 114: 8283-88. Liu, Lili, Muwen Kong, Natalie R Gassman, Bret D Freudenthal, Rajendra Prasad, Stephanie Zhen, Simon C Watkins, Samuel H Wilson, and Bennett Van Houten. 2017b. 'PARP1 changes from three-dimensional DNA damage searching to one-dimensional diffusion after auto-PARylation or in the presence of APE1', Nucleic acids research, 45: 12834- 47. Liu, Ling, Xiaoyang Su, William J Quinn III, Sheng Hui, Kristin Krukenberg, David W Frederick, Philip Redpath, Le Zhan, Karthikeyani Chellappa, and Eileen White. 2018. 'Quantitative analysis of NAD synthesis-breakdown fluxes', Cell metabolism, 27: 1067- 80. e5. Liu, Ling, Ping Wang, Xinwei Liu, Dongwei He, Canxin Liang, and Ying Yu. 2014. 'Exogenous NAD+ supplementation protects H 9c2 cardiac myoblasts against hypoxia/reoxygenation injury via S irt1‐p53 pathway', Fundamental & clinical pharmacology, 28: 180-89.

312 References

Liu, Qun, Irina A Kriksunov, Hong Jiang, Richard Graeff, Hening Lin, Hon Cheung Lee, and Quan Hao. 2008. 'Covalent and noncovalent intermediates of an NAD utilizing enzyme, human CD38', Chemistry & biology, 15: 1068-78. Liu, Xueni, Elphire Ehmed, Boyao Li, Jianming Dou, Xiaojing Qiao, Wenyong Jiang, Xi Yang, Shouyi Qiao, and Yanhua Wu. 2016. 'Breast cancer metastasis suppressor 1 modulates SIRT1-dependent p53 deacetylation through interacting with DBC1', American journal of cancer research, 6: 1441. Locasale, Jason W, Alexandra R Grassian, Tamar Melman, Costas A Lyssiotis, Katherine R Mattaini, Adam J Bass, Gregory Heffron, Christian M Metallo, Taru Muranen, and Hadar Sharfi. 2011. 'Phosphoglycerate dehydrogenase diverts glycolytic flux and contributes to oncogenesis', Nature genetics, 43: 869. Locasale, Jason W, Tamar Melman, Susan Song, Xuemei Yang, Kenneth D Swanson, Lewis C Cantley, Eric T Wong, and John M Asara. 2012. 'Metabolomics of human cerebrospinal fluid identifies signatures of malignant glioma', Molecular & Cellular Proteomics, 11: M111. 014688. Loeber, G, AA Infante, I Maurer-Fogy, E Krystek, and MB Dworkin. 1991. 'Human NAD (+)- dependent mitochondrial malic enzyme. cDNA cloning, primary structure, and expression in Escherichia coli', Journal of Biological Chemistry, 266: 3016-21. Loeber, Gerhard, Mark B Dworkin, Anthony Infante, and Horst Ahorn. 1994. 'Characterization of cytosolic malic enzyme in human tumor cells', FEBS letters, 344: 181-86. Lombard, David B, Frederick W Alt, Hwei-Ling Cheng, Jakob Bunkenborg, Ryan S Streeper, Raul Mostoslavsky, Jennifer Kim, George Yancopoulos, David Valenzuela, and Andrew Murphy. 2007. 'Mammalian Sir2 homolog SIRT3 regulates global mitochondrial lysine acetylation', Molecular and cellular biology, 27: 8807-14. Long, Aaron, Ji H Park, Nina Klimova, Carol Fowler, David J Loane, and Tibor Kristian. 2017. 'CD38 knockout mice show significant protection against ischemic brain damage despite high level poly-ADP-ribosylation', Neurochemical research, 42: 283-93. López-Otín, Carlos, Maria A Blasco, Linda Partridge, Manuel Serrano, and Guido Kroemer. 2013. 'The hallmarks of aging', Cell, 153: 1194-217. Lord, Christopher J, and Alan Ashworth. 2017. 'PARP inhibitors: Synthetic lethality in the clinic', Science, 355: 1152-58. Lou, Zhenkun, Katherine Minter-Dykhouse, Sonia Franco, Monica Gostissa, Melissa A Rivera, Arkady Celeste, John P Manis, Jan Van Deursen, André Nussenzweig, and Tanya T Paull. 2006. 'MDC1 maintains genomic stability by participating in the amplification of ATM-dependent DNA damage signals', Molecular cell, 21: 187-200. Lou, Zhenkun, Katherine Minter-Dykhouse, Xianglin Wu, and Junjie Chen. 2003. 'MDC1 is coupled to activated CHK2 in mammalian DNA damage response pathways', Nature, 421: 957-61. Love, Michael I, Wolfgang Huber, and Simon Anders. 2014. 'Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2', Genome biology, 15: 550. Lowry, Oliver H, Janet V Passonneau, and Martha K Rock. 1961. 'The stability of pyridine nucleotides', J Biol Chem, 236: 2756-9. Lu, Lei, Le Tang, Wenshi Wei, Yunyi Hong, Heyu Chen, Weihai Ying, and Shengdi Chen. 2014a. 'Nicotinamide mononucleotide improves energy activity and survival rate in an in vitro model of Parkinson's disease', Experimental and therapeutic medicine, 8: 943- 50. Lu, Ming, Cunjin Su, Chen Qiao, Yaqi Bian, Jianhua Ding, and Gang Hu. 2016. 'Metformin prevents dopaminergic neuron death in MPTP/P-induced mouse model of Parkinson’s disease via autophagy and mitochondrial ROS clearance', International Journal of Neuropsychopharmacology, 19: pyw047.

313 References

Lu, Xin-Yu, Han-Dong Wang, Jian-Guo Xu, Ke Ding, and Tao Li. 2014b. 'NADPH oxidase inhibition improves neurological outcome in experimental traumatic brain injury', Neurochemistry international, 69: 14-19. Lu, Yingying, Bai-Xia Hao, Richard Graeff, Connie WM Wong, Wu-Tian Wu, and Jianbo Yue. 2013a. 'Two pore channel 2 (TPC2) inhibits autophagosomal-lysosomal fusion by alkalinizing lysosomal pH', Journal of Biological Chemistry, 288: 24247-63. Lu, Yingying, Baixia Hao, Richard Graeff, and Jianbo Yue. 2013b. 'NAADP/TPC2/Ca2+ signaling inhibits autophagy', Communicative & integrative biology, 6: e27595. Luckey, Thomas D. 1972. "Introduction to intestinal microecology." In.: Oxford University Press. Luo, Hongyu, Xiaochuan Shan, and Jiangping Wu. 1996. 'Expression of human mitochondrial NADP‐dependent isocitrate dehydrogenase during lymphocyte activation', Journal of cellular biochemistry, 60: 495-507. Luo, Jianyuan, Anatoly Y Nikolaev, Shin-ichiro Imai, Delin Chen, Fei Su, Ariel Shiloh, Leonard Guarente, and Wei Gu. 2001. 'Negative control of p53 by Sir2α promotes cell survival under stress', Cell, 107: 137-48. Luo, Xiaoting, Rongrong Li, and Liang-Jun Yan. 2015. 'Roles of pyruvate, NADH, and mitochondrial complex I in redox balance and imbalance in β cell function and dysfunction', Journal of diabetes research, 2015. Luo, Yu-Xuan, Xiaoqiang Tang, Xi-Zhou An, Xue-Min Xie, Xiao-Feng Chen, Xiang Zhao, De-Long Hao, Hou-Zao Chen, and De-Pei Liu. 2017. 'SIRT4 accelerates Ang II- induced pathological cardiac hypertrophy by inhibiting manganese superoxide dismutase activity', European heart journal, 38: 1389-98. Luthi-Carter, Ruth, David M Taylor, Judit Pallos, Emmanuel Lambert, Allison Amore, Alex Parker, Hilary Moffitt, Donna L Smith, Heike Runne, and Ozgun Gokce. 2010. 'SIRT2 inhibition achieves neuroprotection by decreasing sterol biosynthesis', Proceedings of the National Academy of Sciences, 107: 7927-32. Lynch, Susan V, and Oluf Pedersen. 2016. 'The human intestinal microbiome in health and disease', New England Journal of Medicine, 375: 2369-79. Lynn, Edward G, Christopher J McLeod, Jeffrey P Gordon, Jianjun Bao, and Michael N Sack. 2008. 'SIRT2 is a negative regulator of anoxia–reoxygenation tolerance via regulation of 14‐3‐3 ζ and BAD in H9c2 cells', FEBS letters, 582: 2857-62. Ma, Cao, Chenchen Pi, Yue Yang, Lin Lin, Yingai Shi, Yan Li, Yulin Li, and Xu He. 2017. 'Nampt expression decreases age-related senescence in rat bone marrow mesenchymal stem cells by targeting Sirt1', PloS one, 12. Ma, L, W Maruwge, A Strambi, P D'arcy, P Pellegrini, L Kis, A De Milito, S Lain, and B Brodin. 2014. 'SIRT1 and SIRT2 inhibition impairs pediatric soft tissue sarcoma growth', Cell death & disease, 5: e1483-e83. Macfarlane, Sandra, and George T Macfarlane. 2004. 'Bacterial diversity in the human gut', Advances in applied microbiology, 54: 261-90. Mack, Till GA, Michael Reiner, Bogdan Beirowski, Weiqian Mi, Monica Emanuelli, Diana Wagner, Derek Thomson, Tom Gillingwater, Felipe Court, and Laura Conforti. 2001. 'Wallerian degeneration of injured axons and synapses is delayed by a Ube4b/Nmnat chimeric gene', Nature neuroscience, 4: 1199-206. MacMillan-Crow, Lee Ann, and John A Thompson. 1999. 'Tyrosine modifications and inactivation of active site manganese superoxide dismutase mutant (Y34F) by peroxynitrite', Archives of biochemistry and biophysics, 366: 82-88. Madeo, Frank, Didac Carmona-Gutierrez, Sebastian J Hofer, and Guido Kroemer. 2019. 'Caloric restriction mimetics against age-associated disease: targets, mechanisms, and therapeutic potential', Cell metabolism, 29: 592-610.

314 References

Maggio-Hall, Lori A, and Jorge C Escalante-Semerena. 2003. 'α-5, 6-Dimethylbenzimidazole adenine dinucleotide (α-DAD), a putative new intermediate of coenzyme B12 biosynthesis in Salmonella typhimurium', Microbiology, 149: 983-90. Magni, Giulio, Giuseppe Orsomando, Nadia Raffelli, and Silverio Ruggieri. 2008. 'Enzymology of mammalian NAD metabolism in health and disease', Front Biosci, 13: 6135-54. Malavasi, Fabio, Silvia Deaglio, Ada Funaro, Enza Ferrero, Alberto L Horenstein, Erika Ortolan, Tiziana Vaisitti, and Semra Aydin. 2008. 'Evolution and function of the ADP ribosyl cyclase/CD38 gene family in physiology and pathology', Physiological reviews, 88: 841-86. Malherbe, Pari, Ch Köhler, MOSE Da Prada, Gabrielle Lang, Vivian Kiefer, Robert Schwarcz, Hans-Werner Lahm, and Andrea M Cesura. 1994. 'Molecular cloning and functional expression of human 3-hydroxyanthranilic-acid dioxygenase', Journal of Biological Chemistry, 269: 13792-97. Maltseva, Ekaterina A, Nadejda I Rechkunova, Maria V Sukhanova, and Olga I Lavrik. 2015. 'Poly (ADP-ribose) polymerase 1 modulates interaction of the nucleotide excision repair factor XPC-RAD23B with DNA via poly (ADP-ribosyl) ation', Journal of Biological Chemistry, 290: 21811-20. Mandal, Santi M, Muralidhar L Hegde, Arpita Chatterjee, Pavana M Hegde, Bartosz Szczesny, Dibyendu Banerjee, Istvan Boldogh, Rui Gao, Maria Falkenberg, and Claes M Gustafsson. 2012. 'Role of human DNA glycosylase Nei-like 2 (NEIL2) and single strand break repair protein polynucleotide kinase 3′-phosphatase in maintenance of mitochondrial genome', Journal of Biological Chemistry, 287: 2819-29. Mangravite, Lara M, Guangqing Xiao, and Kathleen M Giacomini. 2003. 'Localization of human equilibrative nucleoside transporters, hENT1 and hENT2, in renal epithelial cells', American Journal of Physiology-Renal Physiology, 284: F902-F10. Manlapaz-Fernandez, Purita, and Baldomero M Olivera. 1973. 'Pyridine Nucleotide Metabolism in Escherichia coli IV. TURNOVER', Journal of Biological Chemistry, 248: 5150-55. Mann, T, D Ben-David, A Zlotkin, D Shachar, N Keller, A Toren, Arnon Nagler, G Smollan, A Barzilai, and G Rahav. 2010. 'An outbreak of Burkholderia cenocepacia bacteremia in immunocompromised oncology patients', Infection, 38: 187-94. Manning, David R, BA Fraser, Richard A Kahn, and AG Gilman. 1984. 'ADP-ribosylation of transducin by islet-activation protein. Identification of asparagine as the site of ADP- ribosylation', Journal of Biological Chemistry, 259: 749-56. Mansour, Wael Y, Tim Rhein, and Jochen Dahm-Daphi. 2010. 'The alternative end-joining pathway for repair of DNA double-strand breaks requires PARP1 but is not dependent upon microhomologies', Nucleic acids research, 38: 6065-77. Mao, Zhiyong, Michael Bozzella, Andrei Seluanov, and Vera Gorbunova. 2008. 'DNA repair by nonhomologous end joining and homologous recombination during cell cycle in human cells', Cell cycle, 7: 2902-06. Mao, Zhiyong, Christopher Hine, Xiao Tian, Michael Van Meter, Matthew Au, Amita Vaidya, Andrei Seluanov, and Vera Gorbunova. 2011. 'SIRT6 promotes DNA repair under stress by activating PARP1', Science, 332: 1443-46. Mariat, Denis, O Firmesse, F Levenez, VD Guimarăes, H Sokol, J Doré, G Corthier, and JP Furet. 2009. 'The Firmicutes/Bacteroidetes ratio of the human microbiota changes with age', BMC microbiology, 9: 123. Marion, Christina M, Dennis P McDaniel, and Regina C Armstrong. 2019. 'Sarm1 deletion reduces axon damage, demyelination, and white matter atrophy after experimental traumatic brain injury', Experimental neurology, 321: 113040.

315 References

Martello, Rita, Mario Leutert, Stephanie Jungmichel, Vera Bilan, Sara C Larsen, Clifford Young, Michael O Hottiger, and Michael L Nielsen. 2016. 'Proteome-wide identification of the endogenous ADP-ribosylome of mammalian cells and tissue', Nature communications, 7: 1-13. Martens, Christopher R, Blair A Denman, Melissa R Mazzo, Michael L Armstrong, Nichole Reisdorph, Matthew B McQueen, Michel Chonchol, and Douglas R Seals. 2018. 'Chronic nicotinamide riboside supplementation is well-tolerated and elevates NAD+ in healthy middle-aged and older adults', Nature communications, 9: 1286. Martín-Guerrero, Sandra M, José A Muñoz-Gámez, María-Carmen Carrasco, Javier Salmerón, María Martín-Estebané, Miguel A Cuadros, Julio Navascués, and David Martín-Oliva. 2017. 'Poly (ADP-ribose) polymerases inhibitors prevent early mitochondrial fragmentation and hepatocyte cell death induced by H2O2', PloS one, 12. Martin-Montalvo, Alejandro, Evi M Mercken, Sarah J Mitchell, Hector H Palacios, Patricia L Mote, Morten Scheibye-Knudsen, Ana P Gomes, Theresa M Ward, Robin K Minor, and Marie-José Blouin. 2013. 'Metformin improves healthspan and lifespan in mice', Nature communications, 4: 2192. Martino Carpi, Francesco, Manuela Cortese, Giuseppe Orsomando, Valeria Polzonetti, Silvia Vincenzetti, Benedetta Moreschini, Michael Coleman, Giulio Magni, and Stefania Pucciarelli. 2018. 'Simultaneous quantification of nicotinamide mononucleotide and related pyridine compounds in mouse tissues by UHPLC–MS/MS', Separation Science Plus, 1: 22-30. Marton, Judit, Tamas Fodor, Lilla Nagy, Andras Vida, Greta Kis, Attila Brunyanszki, Miklos Antal, Bernhard Lüscher, and Peter Bai. 2018. 'PARP10 (ARTD10) modulates mitochondrial function', PloS one, 13. Masaoka, Aya, Natalie R Gassman, Padmini S Kedar, Rajendra Prasad, Esther W Hou, Julie K Horton, Michael Bustin, and Samuel H Wilson. 2012. 'HMGN1 protein regulates poly (ADP-ribose) polymerase-1 (PARP-1) self-PARylation in mouse fibroblasts', Journal of Biological Chemistry, 287: 27648-58. Maslowski, Kendle M, Angelica T Vieira, Aylwin Ng, Jan Kranich, Frederic Sierro, Di Yu, Heidi C Schilter, Michael S Rolph, Fabienne Mackay, and David Artis. 2009. 'Regulation of inflammatory responses by gut microbiota and chemoattractant receptor GPR43', Nature, 461: 1282-86. Masson, Murielle, Claude Niedergang, Valérie Schreiber, Sylviane Muller, Josiane Menissier- de Murcia, and Gilbert de Murcia. 1998. 'XRCC1 is specifically associated with poly (ADP-ribose) polymerase and negatively regulates its activity following DNA damage', Molecular and cellular biology, 18: 3563-71. Massudi, Hassina, Ross Grant, Nady Braidy, Jade Guest, Bruce Farnsworth, and Gilles J Guillemin. 2012a. 'Age-associated changes in oxidative stress and NAD+ metabolism in human tissue', PloS one, 7. ———. 2012b. 'Age-associated changes in oxidative stress and NAD+ metabolism in human tissue', PloS one, 7: e42357. Mathewson, Nathan D, Robert Jenq, Anna V Mathew, Mark Koenigsknecht, Alan Hanash, Tomomi Toubai, Katherine Oravecz-Wilson, Shin-Rong Wu, Yaping Sun, and Corinne Rossi. 2016. 'Gut microbiome–derived metabolites modulate intestinal epithelial cell damage and mitigate graft-versus-host disease', Nature immunology, 17: 505. Mathias, Rommel A, Todd M Greco, Adam Oberstein, Hanna G Budayeva, Rumela Chakrabarti, Elizabeth A Rowland, Yibin Kang, Thomas Shenk, and Ileana M Cristea. 2014. 'Sirtuin 4 is a lipoamidase regulating pyruvate dehydrogenase complex activity', Cell, 159: 1615-25.

316 References

Mattison, Julie A, Ricki J Colman, T Mark Beasley, David B Allison, Joseph W Kemnitz, George S Roth, Donald K Ingram, Richard Weindruch, Rafael De Cabo, and Rozalyn M Anderson. 2017. 'Caloric restriction improves health and survival of rhesus monkeys', Nature communications, 8: 14063. McCarthy, Anna R, Marijke CC Sachweh, Maureen Higgins, Johanna Campbell, Catherine J Drummond, Ingeborg MM van Leeuwen, Lisa Pirrie, Marcus JGW Ladds, Nicholas J Westwood, and Sonia Laín. 2013. 'Tenovin-D3, a novel small-molecule inhibitor of sirtuin SirT2, increases p21 (CDKN1A) expression in a p53-independent manner', Molecular cancer therapeutics, 12: 352-60. McCay, CM, MF Crowell, and LA Maynard. 1935. 'The effect of retarded growth upon the length of life span and upon the ultimate body size: one figure', Nutrition, 10: 63-79. McMurdie, Paul J, and Susan Holmes. 2013. 'phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data', PloS one, 8. Mehl, Ryan A, Cynthia Kinsland, and Tadhg P Begley. 2000. 'Identification of the Escherichia coli nicotinic acid mononucleotide adenylyltransferase gene', Journal of bacteriology, 182: 4372-74. Mehr, Ali Poyan, Mei T Tran, Kenneth M Ralto, David E Leaf, Vaughan Washco, Joseph Messmer, Adam Lerner, Ajay Kher, Steven H Kim, and Charbel C Khoury. 2018. 'De novo NAD+ biosynthetic impairment in acute kidney injury in humans', Nature medicine, 24: 1351-59. Mehta, Anuja, and James E Haber. 2014. 'Sources of DNA double-strand breaks and models of recombinational DNA repair', Cold Spring Harbor perspectives in biology, 6: a016428. Mendelsohn, Andrew R, and James W Larrick. 2014. 'Partial reversal of skeletal muscle aging by restoration of normal NAD+ levels', Rejuvenation research, 17: 62-69. Meng, Bing, Dong Wu, Jianhua Gu, Songying Ouyang, Wei Ding, and Zhi‐Jie Liu. 2014. 'Structural and functional analyses of human tryptophan 2, 3‐dioxygenase', Proteins: Structure, Function, and Bioinformatics, 82: 3210-16. Meyer-Ficca, Mirella L, Motomasa Ihara, Jessica J Bader, N Adrian Leu, Sascha Beneke, and Ralph G Meyer. 2015. 'Spermatid head elongation with normal nuclear shaping requires ADP-ribosyltransferase PARP11 (ARTD11) in mice', Biology of reproduction, 92: 80, 1-13. Michishita, Eriko, Ronald A McCord, Elisabeth Berber, Mitomu Kioi, Hesed Padilla-Nash, Mara Damian, Peggie Cheung, Rika Kusumoto, Tiara LA Kawahara, and J Carl Barrett. 2008. 'SIRT6 is a histone H3 lysine 9 deacetylase that modulates telomeric chromatin', Nature, 452: 492-96. Michishita, Eriko, Jean Y Park, Jenna M Burneskis, J Carl Barrett, and Izumi Horikawa. 2005. 'Evolutionarily conserved and nonconserved cellular localizations and functions of human SIRT proteins', Molecular biology of the cell, 16: 4623-35. Mielgo-Ayuso, Juan, Raquel Aparicio-Ugarriza, Josune Olza, Javier Aranceta-Bartrina, Ángel Gil, Rosa M Ortega, Lluis Serra-Majem, Gregorio Varela-Moreiras, and Marcela González-Gross. 2018. 'Dietary Intake and Food Sources of Niacin, Riboflavin, Thiamin and Vitamin B6 in a Representative Sample of the Spanish Population. The ANIBES Study', Nutrients, 10: 846. Mills, Kathryn F, Shohei Yoshida, Liana R Stein, Alessia Grozio, Shunsuke Kubota, Yo Sasaki, Philip Redpath, Marie E Migaud, Rajendra S Apte, and Koji Uchida. 2016. 'Long-term administration of nicotinamide mononucleotide mitigates age-associated physiological decline in mice', Cell metabolism, 24: 795-806.

317 References

Min, Kyung‐Jin, Rochele Yamamoto, Susanne Buch, Michael Pankratz, and Marc Tatar. 2008. 'Drosophila lifespan control by dietary restriction independent of insulin‐like signaling', Aging cell, 7: 199-206. Minhas, Paras S, Ling Liu, Peter K Moon, Amit U Joshi, Christopher Dove, Siddhita Mhatre, Kevin Contrepois, Qian Wang, Brittany A Lee, and Michael Coronado. 2019. 'Macrophage de novo NAD+ synthesis specifies immune function in aging and inflammation', Nature immunology, 20: 50-63. Mitchell, Sarah J, Michel Bernier, Miguel A Aon, Sonia Cortassa, Eun Young Kim, Evandro F Fang, Hector H Palacios, Ahmed Ali, Ignacio Navas-Enamorado, and Andrea Di Francesco. 2018. 'Nicotinamide improves aspects of healthspan, but not lifespan, in mice', Cell metabolism, 27: 667-76. e4. Mitchell, Sarah J, Michel Bernier, Julie A Mattison, Miguel A Aon, Tamzin A Kaiser, R Michael Anson, Yuji Ikeno, Rozalyn M Anderson, Donald K Ingram, and Rafael de Cabo. 2019. 'Daily fasting improves health and survival in male mice independent of diet composition and calories', Cell metabolism, 29: 221-28. e3. Miyo, M, H Yamamoto, M Konno, H Colvin, N Nishida, J Koseki, K Kawamoto, H Ogawa, A Hamabe, and M Uemura. 2015. 'Tumour-suppressive function of SIRT4 in human colorectal cancer', British journal of cancer, 113: 492-99. Mohammed, Altaf, Naveena B Janakiram, Misty Brewer, Rebekah L Ritchie, Anuj Marya, Stan Lightfoot, Vernon E Steele, and Chinthalapally V Rao. 2013. 'Antidiabetic drug metformin prevents progression of pancreatic cancer by targeting in part cancer stem cells and mTOR signaling', Translational oncology, 6: 649-IN7. Montgomery, Magdalene K, CE Fiveash, JP Braude, Brenna Osborne, SHJ Brown, Todd W Mitchell, and Nigel Turner. 2015. 'Disparate metabolic response to fructose feeding between different mouse strains', Scientific reports, 5: 18474. Monti, Daniela, Gianluca Ottolina, Giacomo Carrea, and Sergio Riva. 2011. 'Redox reactions catalyzed by isolated enzymes', Chemical reviews, 111: 4111-40. Moore, J Kent, and James E Haber. 1996. 'Cell cycle and genetic requirements of two pathways of nonhomologous end-joining repair of double-strand breaks in Saccharomyces cerevisiae', Molecular and cellular biology, 16: 2164-73. Moretti, Paolo, Katie Freeman, Lavanya Coodly, and David Shore. 1994. 'Evidence that a complex of SIR proteins interacts with the silencer and telomere-binding protein RAP1', Genes & development, 8: 2257-69. Morgan, Anthony J, Lianne C Davis, Margarida Ruas, and Antony Galione. 2015. "TPC: the NAADP discovery channel?" In.: Portland Press Ltd. Morin-Papunen, Laure, Anni S Rantala, Leila Unkila-Kallio, Aila Tiitinen, Maritta Hippeläinen, Antti Perheentupa, Helena Tinkanen, Risto Bloigu, Katri Puukka, and Aimo Ruokonen. 2012. 'Metformin improves pregnancy and live-birth rates in women with polycystic ovary syndrome (PCOS): a multicenter, double-blind, placebo- controlled randomized trial', The Journal of Clinical Endocrinology, 97: 1492-500. Mortusewicz, Oliver, Jean-Christophe Ame, Valerie Schreiber, and Heinrich Leonhardt. 2007. 'Feedback-regulated poly (ADP-ribosyl) ation by PARP-1 is required for rapid response to DNA damage in living cells', Nucleic acids research, 35: 7665-75. Moser, Jill, Hanneke Kool, Ioannis Giakzidis, Keith Caldecott, Leon HF Mullenders, and Maria I Fousteri. 2007. 'Sealing of chromosomal DNA nicks during nucleotide excision repair requires XRCC1 and DNA ligase IIIα in a cell-cycle-specific manner', Molecular cell, 27: 311-23. Moss, Joel, Sam Garrison, Norman J Oppenheimer, and Stephen H Richardson. 1979. 'NAD- dependent ADP-ribosylation of arginine and proteins by Escherichia coli heat-labile enterotoxin', Journal of Biological Chemistry, 254: 6270-72.

318 References

Mostoslavsky, Raul, Katrin F Chua, David B Lombard, Wendy W Pang, Miriam R Fischer, Lionel Gellon, Pingfang Liu, Gustavo Mostoslavsky, Sonia Franco, and Michael M Murphy. 2006. 'Genomic instability and aging-like phenotype in the absence of mammalian SIRT6', Cell, 124: 315-29. Mouchiroud, Laurent, Riekelt H Houtkooper, and Johan Auwerx. 2013. 'NAD+ metabolism: a therapeutic target for age-related metabolic disease', Critical reviews in biochemistry and molecular biology, 48: 397-408. Mouchiroud, Laurent, Riekelt H Houtkooper, Norman Moullan, Elena Katsyuba, Dongryeol Ryu, Carles Cantó, Adrienne Mottis, Young-Suk Jo, Mohan Viswanathan, and Kristina Schoonjans. 2013. 'The NAD+/sirtuin pathway modulates longevity through activation of mitochondrial UPR and FOXO signaling', Cell, 154: 430-41. Muñoz-Cobo, I, FB Belloch, T Díaz-Perdigón, E Puerta, and RM Tordera. 2017. 'SIRT2 inhibition reverses anhedonia in the VGLUT1+/− depression model', Behavioural brain research, 335: 128-31. Muñoz-Gámez, José Antonio, José Manuel Rodríguez-Vargas, Rosa Quiles-Pérez, Rocío Aguilar-Quesada, David Martín-Oliva, Gilbert de Murcia, Josiane Menissier de Murcia, Antonio Almendros, Mariano Ruiz de Almodóvar, and F Javier Oliver. 2009. 'PARP-1 is involved in autophagy induced by DNA damage', Autophagy, 5: 61-74. Murai, Junko, Shar-Yin N Huang, Amèlie Renaud, Yiping Zhang, Jiuping Ji, Shunichi Takeda, Joel Morris, Beverly Teicher, James H Doroshow, and Yves Pommier. 2014. 'Stereospecific PARP trapping by BMN 673 and comparison with olaparib and rucaparib', Molecular cancer therapeutics, 13: 433-43. Murai, Junko, N Huang Shar-yin, Benu Brata Das, Amelie Renaud, Yiping Zhang, James H Doroshow, Jiuping Ji, Shunichi Takeda, and Yves Pommier. 2012. 'Trapping of PARP1 and PARP2 by clinical PARP inhibitors', Cancer research, 72: 5588-99. Muthurajan, Uma M, Maggie RD Hepler, Aaron R Hieb, Nicholas J Clark, Michael Kramer, Tingting Yao, and Karolin Luger. 2014. 'Automodification switches PARP-1 function from chromatin architectural protein to histone chaperone', Proceedings of the National Academy of Sciences, 111: 12752-57. Nabokina, Svetlana M, Moti L Kashyap, and Hamid M Said. 2005. 'Mechanism and regulation of human intestinal niacin uptake', American Journal of Physiology-Cell Physiology, 289: C97-C103. Nakagawa, Takashi, David J Lomb, Marcia C Haigis, and Leonard Guarente. 2009. 'SIRT5 Deacetylates carbamoyl phosphate synthetase 1 and regulates the urea cycle', Cell, 137: 560-70. Nakamura, Yasuhiko, Masahito Ogura, Kasane Ogura, Daisuke Tanaka, and Nobuya Inagaki. 2012. 'SIRT5 deacetylates and activates urate oxidase in liver mitochondria of mice', FEBS letters, 586: 4076-81. Nasrin, Nargis, Virendar K Kaushik, Eric Fortier, Daniel Wall, Kevin J Pearson, Rafael De Cabo, and Laura Bordone. 2009. 'JNK1 phosphorylates SIRT1 and promotes its enzymatic activity', PloS one, 4. Nasrin, Nargis, Xiaoping Wu, Eric Fortier, Yajun Feng, Olivia Claire Bare, Sumiao Chen, Xianglin Ren, Zhidan Wu, Ryan S Streeper, and Laura Bordone. 2010. 'SIRT4 regulates fatty acid oxidation and mitochondrial gene expression in liver and muscle cells', Journal of Biological Chemistry, 285: 31995-2002. Nasu, S, Frank D Wicks, and RK Gholson. 1982. 'L-Aspartate oxidase, a newly discovered enzyme of Escherichia coli, is the B protein of quinolinate synthetase', Journal of Biological Chemistry, 257: 626-32. Nechifor, Marina Tamara, and Diana Dinu. 2011. 'Ethanol‐induced redox imbalance in rat kidneys', Journal of biochemical and molecular toxicology, 25: 224-30.

319 References

Nemoto, Shino, Maria M Fergusson, and Toren Finkel. 2004. 'Nutrient availability regulates SIRT1 through a forkhead-dependent pathway', Science, 306: 2105-08. Neven, Ellen, Benjamin Vervaet, Kerstin Brand, Ulrike Gottwald-Hostalek, Britt Opdebeeck, Annelies De Maré, Anja Verhulst, Jean-Daniel Lalau, Said Kamel, and Marc E De Broe. 2018. 'Metformin prevents the development of severe chronic kidney disease and its associated mineral and bone disorder', Kidney international, 94: 102-13. Nicolae, Claudia M, Erin R Aho, Katherine N Choe, Daniel Constantin, He-Juan Hu, Deokjae Lee, Kyungjae Myung, and George-Lucian Moldovan. 2015. 'A novel role for the mono-ADP-ribosyltransferase PARP14/ARTD8 in promoting homologous recombination and protecting against replication stress', Nucleic acids research, 43: 3143-53. Nicolae, Claudia M, Erin R Aho, Alexander HS Vlahos, Katherine N Choe, Subhajyoti De, Georgios I Karras, and George-Lucian Moldovan. 2014. 'The ADP-ribosyltransferase PARP10/ARTD10 interacts with proliferating cell nuclear antigen (PCNA) and is required for DNA damage tolerance', Journal of Biological Chemistry, 289: 13627-37. Niedel, James, and LS Dietrich. 1973. 'Nicotinate phosphoribosyltransferase of human erythrocytes Purification and properties', Journal of Biological Chemistry, 248: 3500- 05. Niemann, Bernd, Ying Chen, Hassan Issa, Rolf-Edgar Silber, and Susanne Rohrbach. 2010. 'Caloric restriction delays cardiac ageing in rats: role of mitochondria', Cardiovascular research, 88: 267-76. Niimi, Kimie, Patrick Hardy, Brian Bilecki, and Eiki Takahashi. 2019. 'Rearing and breeding of germ-free mice for over 1 year in a sealed positive pressure cage system', Japanese Journal of Veterinary Research, 67: 119-25. Niimi, Kimie, and Eiki Takahashi. 2019. 'New system to examine the activity and water and food intake of germ-free mice in a sealed positive-pressure cage', Heliyon, 5: e02176. Nijhof, Inger S, Tineke Casneuf, Jeroen Van Velzen, Berris van Kessel, Amy E Axel, Khaja Syed, Richard WJ Groen, Mark van Duin, Pieter Sonneveld, and Monique C Minnema. 2016. 'CD38 expression and complement inhibitors affect response and resistance to daratumumab therapy in myeloma', Blood, The Journal of the American Society of Hematology, 128: 959-70. Nikiforov, Andrey, Christian Dölle, Marc Niere, and Mathias Ziegler. 2011. 'Pathways and subcellular compartmentation of NAD biosynthesis in human cells from entry of extracellular precursors to mitochondrial NAD generation', Journal of Biological Chemistry, 286: 21767-78. Noh, Sang Jae, Myoung Jae Kang, Kyoung Min Kim, Jun Sang Bae, Ho Sung Park, Woo Sung Moon, Myoung Ja Chung, Ho Lee, Dong Geun Lee, and Kyu Yun Jang. 2013. 'Acetylation status of P53 and the expression of DBC1, SIRT1, and androgen receptor are associated with survival in clear cell renal cell carcinoma patients', Pathology, 45: 574-80. Noriega, Lilia G, Jérôme N Feige, Carles Canto, Hiroyasu Yamamoto, Jiujiu Yu, Mark A Herman, Chikage Mataki, Barbara B Kahn, and Johan Auwerx. 2011. 'CREB and ChREBP oppositely regulate SIRT1 expression in response to energy availability', EMBO reports, 12: 1069-76. Oelze, Matthias, Swenja Kröller-Schön, Sebastian Steven, Edith Lubos, Christopher Doppler, Michael Hausding, Silke Tobias, Christoph Brochhausen, Huige Li, and Michael Torzewski. 2014. 'Glutathione peroxidase-1 deficiency potentiates dysregulatory modifications of endothelial nitric oxide synthase and vascular dysfunction in aging', Hypertension, 63: 390-96.

320 References

Ogata, Norio, Kunihiro Ueda, and Osamu Hayaishi. 1980. 'ADP-ribosylation of histone H2B. Identification of glutamic acid residue 2 as the modification site', Journal of Biological Chemistry, 255: 7610-15. Ogura, Yoshio, Munehiro Kitada, Itaru Monno, Ai Watanabe, and Daisuke Koya. 2018. "Renal Mitochondrial Oxidative Stress Induced by NAD+-Dependent Sirt3 Inactivation via Overexpression of CD38 (NAD+ ase) in Diabetic Kidney Disease." In.: Am Diabetes Assoc. Ohashi, Kazuto, Shigeyuki Kawai, and Kousaku Murata. 2012. 'Identification and characterization of a human mitochondrial NAD kinase', Nature communications, 3: 1- 9. Olivera, Baldomero M, and IR Lehman. 1967. 'Diphosphopyridine nucleotide: a cofactor for the polynucleotide-joining enzyme from Escherichia coli', Proceedings of the National Academy of Sciences of the United States of America, 57: 1700. Osterloh, Jeannette M, Jing Yang, Timothy M Rooney, A Nicole Fox, Robert Adalbert, Eric H Powell, Amy E Sheehan, Michelle A Avery, Rachel Hackett, and Mary A Logan. 2012. 'dSarm/Sarm1 is required for activation of an injury-induced axon death pathway', Science, 337: 481-84. Outeiro, Tiago Fleming, Eirene Kontopoulos, Stephen M Altmann, Irina Kufareva, Katherine E Strathearn, Allison M Amore, Catherine B Volk, Michele M Maxwell, Jean- Christophe Rochet, and Pamela J McLean. 2007. 'Sirtuin 2 inhibitors rescue α- synuclein-mediated toxicity in models of Parkinson's disease', Science, 317: 516-19. Pabarcus, Michael K, and John E Casida. 2002. 'Kynurenine formamidase: determination of primary structure and modeling-based prediction of tertiary structure and catalytic triad', Biochimica et Biophysica Acta (BBA)-Protein Structure and Molecular Enzymology, 1596: 201-11. Pal, Rituraj, Tanner O Monroe, Michela Palmieri, Marco Sardiello, and George G Rodney. 2014. 'Rotenone induces neurotoxicity through Rac1-dependent activation of NADPH oxidase in SHSY-5Y cells', FEBS letters, 588: 472-81. Palacios, Orsolya M, Juan J Carmona, Shaday Michan, Ke Yun Chen, Yasuko Manabe, Jack Lee Ward III, Laurie J Goodyear, and Qiang Tong. 2009. 'Diet and exercise signals regulate SIRT3 and activate AMPK and PGC-1α in skeletal muscle', Aging (Albany NY), 1: 771. Palazzo, Luca, Orsolya Leidecker, Evgeniia Prokhorova, Helen Dauben, Ivan Matic, and Ivan Ahel. 2018. 'Serine is the major residue for ADP-ribosylation upon DNA damage', Elife, 7: e34334. Palego, Lionella, Laura Betti, Alessandra Rossi, and Gino Giannaccini. 2016. 'Tryptophan biochemistry: structural, nutritional, metabolic, and medical aspects in humans', Journal of Amino Acids, 2016. Palmieri, Ferdinando, Benjamin Rieder, Angela Ventrella, Emanuela Blanco, Phuc Thi Do, Adriano Nunes-Nesi, A Ulrike Trauth, Giuseppe Fiermonte, Joachim Tjaden, and Gennaro Agrimi. 2009. 'Molecular identification and functional characterization of Arabidopsis thaliana mitochondrial and chloroplastic NAD+ carrier proteins', Journal of Biological Chemistry, 284: 31249-59. Pan, Haihui, and Toren Finkel. 2017. 'Key proteins and pathways that regulate lifespan', Journal of Biological Chemistry, 292: 6452-60. Pandey, Raghav, Julia C Wilmanns, Olivia Hon, Nadia A Rosenthal, Milena B Furtado, and Mauro W Costa. 2019. 'Modulation of Energy Metabolism by Metformin Prevents Diet Induced Cardiac Dysfunction in a Mouse Model of Adult Congenital Heart Disease', Circulation Research, 125: A275-A75.

321 References

Pandithage, Ruwin, Richard Lilischkis, Kai Harting, Alexandra Wolf, Britta Jedamzik, Juliane Lüscher-Firzlaff, Jörg Vervoorts, Edwin Lasonder, Elisabeth Kremmer, and Bernd Knöll. 2008. 'The regulation of SIRT2 function by cyclin-dependent kinases affects cell motility', The Journal of cell biology, 180: 915-29. Pang, Jing, Ju Cui, Huan Gong, Chao Xi, and Tie‐mei Zhang. 2015. 'Effect of NAD on PARP‐ mediated insulin sensitivity in oleic acid treated hepatocytes', Journal of cellular physiology, 230: 1607-13. Panozzo, Cristina, Magdalena Nawara, Catherine Suski, Roza Kucharczyka, Marek Skoneczny, Anne-Marie Bécam, Joanna Rytka, and Christopher J Herbert. 2002. 'Aerobic and anaerobic NAD+ metabolism in Saccharomyces cerevisiae', FEBS letters, 517: 97-102. Papa, Sergio, Pietro Luca Martino, Giuseppe Capitanio, Antonio Gaballo, Domenico De Rasmo, Anna Signorile, and Vittoria Petruzzella. 2012. 'The oxidative phosphorylation system in mammalian mitochondria.' in, Advances in Mitochondrial Medicine (Springer). Park, Jeongsoon, Yue Chen, Daniel X Tishkoff, Chao Peng, Minjia Tan, Lunzhai Dai, Zhongyu Xie, Yi Zhang, Bernadette MM Zwaans, and Mary E Skinner. 2013. 'SIRT5-mediated lysine desuccinylation impacts diverse metabolic pathways', Molecular cell, 50: 919- 30. Park, Ji H, Aaron Long, Katrina Owens, and Tibor Kristian. 2016. 'Nicotinamide mononucleotide inhibits post-ischemic NAD+ degradation and dramatically ameliorates brain damage following global cerebral ischemia', Neurobiology of disease, 95: 102-10. Partida-Sánchez, Santiago, Debra A Cockayne, Simon Monard, Elaine L Jacobson, Norman Oppenheimer, Beth Garvy, Kim Kusser, Stephen Goodrich, Maureen Howard, and Allen Harmsen. 2001. 'Cyclic ADP-ribose production by CD38 regulates intracellular calcium release, extracellular calcium influx and chemotaxis in neutrophils and is required for bacterial clearance in vivo', Nature medicine, 7: 1209-16. Pearse, Barbara MF, and Michael A Rosemeyer. 1974. 'The molecular weight and subunit structure of human erythrocyte 6‐phosphogluconate dehydrogenase', European journal of biochemistry, 42: 225-35. ———. 1975. '[49] 6-Phosphogluconate dehydrogenase from human erythrocytes.' in, Methods in enzymology (Elsevier). Pedersen, Steen Bønløkke, J Ølholm, Søren Kildeberg Paulsen, Marianne Faurholt Bennetzen, and Bjørn Richelsen. 2008. 'Low Sirt1 expression, which is upregulated by fasting, in human adipose tissue from obese women', International Journal of Obesity, 32: 1250- 55. Penfound, Thomas, and John W Foster. 1999. 'NAD-dependent DNA-binding activity of the bifunctional NadR regulator of Salmonella typhimurium', Journal of bacteriology, 181: 648-55. Pereira, Gustavo JS, Hanako Hirata, Gian M Fimia, Lúcia G Do Carmo, Claudia Bincoletto, Sang W Han, Roberta S Stilhano, Rodrigo P Ureshino, Duncan Bloor-Young, and Grant Churchill. 2011. 'Nicotinic acid adenine dinucleotide phosphate (NAADP) regulates autophagy in cultured astrocytes', Journal of Biological Chemistry, 286: 27875-81. Petrack, Barbara, Paul Greengard, Ann Craston, and Helen J Kalinsky. 1963. 'Nicotinamide deamidase in rat liver and the biosynthesis of NAD', Biochemical and biophysical research communications, 13: 472-77. Petrack, Barbara, Paul Greengard, Ann Craston, and Fred Sheppy. 1965. 'Nicotinamide deamidase from mammalian liver', Electrophoresis, 88: 49.

322 References

Pettit, Flora H, Lynn Hamilton, Petr Munk, Genshin Namihira, Michael H Eley, Charles R Willms, and Lester J Reed. 1973. 'α-Keto Acid Dehydrogenase Complexes XIX. Subunit structure of the Escherichia coli α-ketoglutarate dehydrogenase complex', Journal of Biological Chemistry, 248: 5282-90. Pham, Tho X, Minkyung Bae, Mi-Bo Kim, Yoojin Lee, Siqi Hu, Hyunju Kang, Young-Ki Park, and Ji-Young Lee. 2019. 'Nicotinamide riboside, an NAD+ precursor, attenuates the development of liver fibrosis in a diet-induced mouse model of liver fibrosis', Biochimica et Biophysica Acta (BBA)-Molecular Basis of Disease. Picard, Frédéric, Martin Kurtev, Namjin Chung, Acharawan Topark-Ngarm, Thanaset Senawong, Rita Machado de Oliveira, Mark Leid, Michael W McBurney, and Leonard Guarente. 2004. 'Sirt1 promotes fat mobilization in white adipocytes by repressing PPAR-γ', Nature, 429: 771-76. Pillai, Jyothish B, Ayman Isbatan, Shin-ichiro Imai, and Mahesh P Gupta. 2005. 'Poly (ADP- ribose) polymerase-1-dependent cardiac myocyte cell death during heart failure is mediated by NAD+ depletion and reduced Sir2α deacetylase activity', Journal of Biological Chemistry, 280: 43121-30. Pillai, Samyuktha Muralidharan, and David Meredith. 2011. 'SLC36A4 (hPAT4) is a high affinity amino acid transporter when expressed in Xenopus laevis oocytes', Journal of Biological Chemistry, 286: 2455-60. Pinedo, Ignacio, Thomas Ledger, Macarena Greve, and María J Poupin. 2015. 'Burkholderia phytofirmans PsJN induces long-term metabolic and transcriptional changes involved in Arabidopsis thaliana salt tolerance', Frontiers in plant science, 6: 466. Pines, Alex, Mischa G Vrouwe, Jurgen A Marteijn, Dimitris Typas, Martijn S Luijsterburg, Medine Cansoy, Paul Hensbergen, André Deelder, Anton de Groot, and Syota Matsumoto. 2012. 'PARP1 promotes nucleotide excision repair through DDB2 stabilization and recruitment of ALC1', Journal of Cell Biology, 199: 235-49. Pittelli, Maria, Roberta Felici, Vanessa Pitozzi, Lisa Giovannelli, Elisabetta Bigagli, Francesca Cialdai, Giovanni Romano, Flavio Moroni, and Alberto Chiarugi. 2011. 'Pharmacological effects of exogenous NAD on mitochondrial bioenergetics, DNA repair, and apoptosis', Molecular pharmacology, 80: 1136-46. Pollak, Nadine, Marc Niere, and Mathias Ziegler. 2007. 'NAD kinase levels control the NADPH concentration in human cells', Journal of Biological Chemistry, 282: 33562- 71. Polletta, Lucia, Enza Vernucci, Ilaria Carnevale, Tania Arcangeli, Dante Rotili, Silvia Palmerio, Clemens Steegborn, Theresa Nowak, Mike Schutkowski, and Laura Pellegrini. 2015. 'SIRT5 regulation of ammonia-induced autophagy and mitophagy', Autophagy, 11: 253-70. Potaman, Vladimir N, Luda S Shlyakhtenko, Elena A Oussatcheva, Yuri L Lyubchenko, and Viatcheslav A Soldatenkov. 2005. 'Specific binding of poly (ADP-ribose) polymerase- 1 to cruciform hairpins', Journal of molecular biology, 348: 609-15. Preiss, Jack, and Philip Handler. 1957. 'Enzymatic synthesis of nicotinamide mononucleotide', Journal of Biological Chemistry, 225: 759-70. ———. 1958a. 'Biosynthesis of diphosphopyridine nucleotide I. Identification of intermediates', Journal of Biological Chemistry, 233: 488-92. ———. 1958b. 'Biosynthesis of diphosphopyridine nucleotide II. Enzymatic aspects', Journal of Biological Chemistry, 233: 493-500. Pryor, Rosina, Daniel Martinez-Martinez, Leonor Quintaneiro, and Filipe Cabreiro. 2020. 'The role of the microbiome in drug response', Annual review of pharmacology and toxicology, 60: 417-35.

323 References

Puhl, Nathan J, Richard RE Uwiera, L Jay Yanke, L Brent Selinger, and G Douglas Inglis. 2012. 'Antibiotics conspicuously affect community profiles and richness, but not the density of bacterial cells associated with mucosa in the large and small intestines of mice', Anaerobe, 18: 67-75. Pullen, Jeffrey K, Shu-Mei Liang, Milan S Blake, Sharon Mates, and Joseph Y Tai. 1995. 'Production of Haemophilus influenzae type-b porin in Escherichia coli and its folding into the trimeric form', Gene, 152: 85-88. Purohit, Nupur K, Mihaela Robu, Rashmi G Shah, Nicholas E Geacintov, and Girish M Shah. 2016. 'Characterization of the interactions of PARP-1 with UV-damaged DNA in vivo and in vitro', Scientific reports, 6: 1-11. Qi, Guangying, Yasusei Kudo, Bo Tang, Tian Liu, Shengjian Jin, Jing Liu, Xiaoxu Zuo, Sisi Mi, Wenhuan Shao, and Xiaojuan Ma. 2016. 'PARP6 acts as a tumor suppressor via downregulating Survivin expression in colorectal cancer', Oncotarget, 7: 18812. Qin, Junjie, Ruiqiang Li, Jeroen Raes, Manimozhiyan Arumugam, Kristoffer Solvsten Burgdorf, Chaysavanh Manichanh, Trine Nielsen, Nicolas Pons, Florence Levenez, and Takuji Yamada. 2010. 'A human gut microbial gene catalogue established by metagenomic sequencing', Nature, 464: 59-65. Qin, Junjie, Yingrui Li, Zhiming Cai, Shenghui Li, Jianfeng Zhu, Fan Zhang, Suisha Liang, Wenwei Zhang, Yuanlin Guan, and Dongqian Shen. 2012. 'A metagenome-wide association study of gut microbiota in type 2 diabetes', Nature, 490: 55-60. Qin, Yuan-Yuan, Mei Li, Xing Feng, Jian Wang, Lijuan Cao, Xi-Kui Shen, Jieyu Chen, Meiling Sun, Rui Sheng, and Feng Han. 2017. 'Combined NADPH and the NOX inhibitor apocynin provides greater anti-inflammatory and neuroprotective effects in a mouse model of stroke', Free Radical Biology and Medicine, 104: 333-45. Qiu, Longxin, Jianhui Lin, Fangui Xu, Yuehong Gao, Cuilin Zhang, Ying Liu, Yu Luo, and James Y Yang. 2011. 'Inhibition of aldose reductase activates hepatic peroxisome proliferator-activated receptor-α and ameliorates hepatosteatosis in diabetic db/db mice', Experimental diabetes research, 2012. Qiu, Xiaolei, Katharine Brown, Matthew D Hirschey, Eric Verdin, and Danica Chen. 2010. 'Calorie restriction reduces oxidative stress by SIRT3-mediated SOD2 activation', Cell metabolism, 12: 662-67. Quarona, Valeria, Gianluca Zaccarello, Antonella Chillemi, Enrico Brunetti, Vijay Kumar Singh, Enza Ferrero, Ada Funaro, Alberto L Horenstein, and Fabio Malavasi. 2013. 'CD38 and CD157: a long journey from activation markers to multifunctional molecules', Cytometry Part B: Clinical Cytometry, 84: 207-17. Raffaelli, Nadia, Teresa Lorenzi, P Luigi Mariani, Monica Emanuelli, Adolfo Amici, Silverio Ruggieri, and Giulio Magni. 1999. 'The Escherichia coli NadR regulator is endowed with nicotinamide mononucleotide adenylyltransferase activity', Journal of bacteriology, 181: 5509-11. Raffaelli, Nadia, Leonardo Sorci, Adolfo Amici, Monica Emanuelli, Francesca Mazzola, and Giulio Magni. 2002. 'Identification of a novel human nicotinamide mononucleotide adenylyltransferase', Biochemical and biophysical research communications, 297: 835- 40. Rahman, Mahbuba, Mohammad Rubayet Hasan, Takahiro Oba, and Kazuyuki Shimizu. 2006. 'Effect of rpoS gene knockout on the metabolism of Escherichia coli during exponential growth phase and early stationary phase based on gene expressions, enzyme activities and intracellular metabolite concentrations', Biotechnology and bioengineering, 94: 585-95. Raj, Christol James Theoga, Trevor Croft, Padmaja Venkatakrishnan, Benjamin Groth, Gagandeep Dhugga, Timothy Cater, and Su-Ju Lin. 2019. 'The copper-sensing

324 References

transcription factor Mac1, the histone deacetylase Hst1, and nicotinic acid regulate de novo NAD+ biosynthesis in budding yeast', Journal of Biological Chemistry, 294: 5562-75. Rajavel, Mathumathi, Dominique Lalo, Jeffrey W Gross, and Charles Grubmeyer. 1998. 'Conversion of a cosubstrate to an inhibitor: phosphorylation mutants of nicotinic acid phosphoribosyltransferase', Biochemistry, 37: 4181-88. Rajman, Luis, Karolina Chwalek, and David A Sinclair. 2018. 'Therapeutic potential of NAD- boosting molecules: the in vivo evidence', Cell metabolism, 27: 529-47. Ramsey, Kathryn Moynihan, Jun Yoshino, Cynthia S Brace, Dana Abrassart, Yumiko Kobayashi, Biliana Marcheva, Hee-Kyung Hong, Jason L Chong, Ethan D Buhr, and Choogon Lee. 2009. 'Circadian clock feedback cycle through NAMPT-mediated NAD+ biosynthesis', Science, 324: 651-54. Rascón, Brenda, Basil P Hubbard, David A Sinclair, and Gro V Amdam. 2012. 'The lifespan extension effects of resveratrol are conserved in the honey bee and may be driven by a mechanism related to caloric restriction', Aging (Albany NY), 4: 499. Ratajczak, Joanna, Magali Joffraud, Samuel AJ Trammell, Rosa Ras, Núria Canela, Marie Boutant, Sameer S Kulkarni, Marcelo Rodrigues, Philip Redpath, and Marie E Migaud. 2016. 'NRK1 controls nicotinamide mononucleotide and nicotinamide riboside metabolism in mammalian cells', Nature communications, 7: 13103. Reikvam, Dag Henrik, Alexander Erofeev, Anders Sandvik, Vedrana Grcic, Frode Lars Jahnsen, Peter Gaustad, Kathy D McCoy, Andrew J Macpherson, Leonardo A Meza- Zepeda, and Finn-Eirik Johansen. 2011. 'Depletion of murine intestinal microbiota: effects on gut mucosa and epithelial gene expression', PloS one, 6: e17996. Rekdal, Vayu Maini, Elizabeth N Bess, Jordan E Bisanz, Peter J Turnbaugh, and Emily P Balskus. 2019. 'Discovery and inhibition of an interspecies gut bacterial pathway for Levodopa metabolism', Science, 364: eaau6323. Remie, Carlijn ME, Kay HM Roumans, Michiel PB Moonen, Niels J Connell, Bas Havekes, Julian Mevenkamp, Lucas Lindeboom, Vera HW de Wit, Tineke van de Weijer, and Suzanne ABM Aarts. 2020. 'Nicotinamide riboside supplementation alters body composition and skeletal muscle acetylcarnitine concentrations in healthy obese humans', The American Journal of Clinical Nutrition. Revollo, Javier R, Andrew A Grimm, and Shin-ichiro Imai. 2004. 'The NAD biosynthesis pathway mediated by nicotinamide phosphoribosyltransferase regulates Sir2 activity in mammalian cells', Journal of Biological Chemistry, 279: 50754-63. Revollo, Javier R, Antje Körner, Kathryn F Mills, Akiko Satoh, Tao Wang, Antje Garten, Biplab Dasgupta, Yo Sasaki, Cynthia Wolberger, and R Reid Townsend. 2007. 'Nampt/PBEF/visfatin regulates insulin secretion in β cells as a systemic NAD biosynthetic enzyme', Cell metabolism, 6: 363-75. Reynolds, Edward. 2006. 'Vitamin B12, folic acid, and the nervous system', The lancet neurology, 5: 949-60. Rhodes, Katherine A, and Herbert P Schweizer. 2016. 'Antibiotic resistance in Burkholderia species', Drug Resistance Updates, 28: 82-90. Rine, Jasper, Jeffrey N Strathern, James B Hicks, and Ira Herskowitz. 1979. 'A suppressor of mating-type locus mutations in Saccharomyces cerevisiae: evidence for and identification of cryptic mating-type loci', Genetics, 93: 877-901. Robu, Mihaela, Rashmi G Shah, Nancy Petitclerc, Julie Brind’Amour, Febitha Kandan- Kulangara, and Girish M Shah. 2013. 'Role of poly (ADP-ribose) polymerase-1 in the removal of UV-induced DNA lesions by nucleotide excision repair', Proceedings of the National Academy of Sciences, 110: 1658-63.

325 References

Robu, Mihaela, Rashmi G Shah, Nupur K Purohit, Pengbo Zhou, Hanspeter Naegeli, and Girish M Shah. 2017. 'Poly (ADP-ribose) polymerase 1 escorts XPC to UV-induced DNA lesions during nucleotide excision repair', Proceedings of the National Academy of Sciences, 114: E6847-E56. Rodgers, Joseph T, Carlos Lerin, Wilhelm Haas, Steven P Gygi, Bruce M Spiegelman, and Pere Puigserver. 2005. 'Nutrient control of glucose homeostasis through a complex of PGC-1α and SIRT1', Nature, 434: 113-18. Rodionov, Dmitry A, Xiaoqing Li, Irina A Rodionova, Chen Yang, Leonardo Sorci, Etienne Dervyn, Dariusz Martynowski, Hong Zhang, Mikhail S Gelfand, and Andrei L Osterman. 2008. 'Transcriptional regulation of NAD metabolism in bacteria: genomic reconstruction of NiaR (YrxA) regulon', Nucleic acids research, 36: 2032-46. Rogina, Blanka, and Stephen L Helfand. 2004. 'Sir2 mediates longevity in the fly through a pathway related to calorie restriction', Proceedings of the National Academy of Sciences, 101: 15998-6003. Rongvaux, Anthony, Fabienne Andris, Frédéric Van Gool, and Oberdan Leo. 2003. 'Reconstructing eukaryotic NAD metabolism', Bioessays, 25: 683-90. Rosen, Evan D, and Bruce M Spiegelman. 2006. 'Adipocytes as regulators of energy balance and glucose homeostasis', Nature, 444: 847-53. Rossato, Marco, Marnie Granzotto, Veronica Macchi, Andrea Porzionato, Lucia Petrelli, Alessandra Calcagno, Juri Vencato, Diego De Stefani, Valentina Silvestrin, and Rosario Rizzuto. 2014. 'Human white adipocytes express the cold receptor TRPM8 which activation induces UCP1 expression, mitochondrial activation and heat production', Molecular and cellular endocrinology, 383: 137-46. Rothgiesser, Karin M, Süheda Erener, Susanne Waibel, Bernhard Lüscher, and Michael O Hottiger. 2010. 'SIRT2 regulates NF-κB-dependent gene expression through deacetylation of p65 Lys310', Journal of cell science, 123: 4251-58. Rothkamm, Kai, Ines Krüger, Larry H Thompson, and Markus Löbrich. 2003. 'Pathways of DNA double-strand break repair during the mammalian cell cycle', Molecular and cellular biology, 23: 5706-15. Ruas, Margarida, Lianne C Davis, Cheng‐Chang Chen, Anthony J Morgan, Kai‐Ting Chuang, Timothy F Walseth, Christian Grimm, Clive Garnham, Trevor Powell, and Nick Platt. 2015. 'Expression of Ca2+‐permeable two‐pore channels rescues NAADP signalling in TPC‐deficient cells', The EMBO journal, 34: 1743-58. Ruscetti, Tracy, Bruce E Lehnert, James Halbrook, Hai Le Trong, Merl F Hoekstra, David J Chen, and Scott R Peterson. 1998. 'Stimulation of the DNA-dependent protein kinase by poly (ADP-ribose) polymerase', Journal of Biological Chemistry, 273: 14461-67. Ryu, Dongryeol, Hongbo Zhang, Eduardo R Ropelle, Vincenzo Sorrentino, Davi AG Mázala, Laurent Mouchiroud, Philip L Marshall, Matthew D Campbell, Amir Safi Ali, and Gary M Knowels. 2016. 'NAD+ repletion improves muscle function in muscular dystrophy and counters global PARylation', Science translational medicine, 8: 361ra139- 361ra139. Sadhukhan, Sushabhan, Xiaojing Liu, Dongryeol Ryu, Ornella D Nelson, John A Stupinski, Zhi Li, Wei Chen, Sheng Zhang, Robert S Weiss, and Jason W Locasale. 2016. 'Metabolomics-assisted proteomics identifies succinylation and SIRT5 as important regulators of cardiac function', Proceedings of the National Academy of Sciences, 113: 4320-25. Said, Hamid M, Svetlana M Nabokina, Krishnaswamy Balamurugan, Zainab M Mohammed, Cecilia Urbina, and Moti L Kashyap. 2007. 'Mechanism of nicotinic acid transport in human liver cells: experiments with HepG2 cells and primary hepatocytes', American Journal of Physiology-Cell Physiology, 293: C1773-C78.

326 References

Salminen, A, J Ojala, J Huuskonen, A Kauppinen, T Suuronen, and K Kaarniranta. 2008. 'Interaction of aging-associated signaling cascades: Inhibition of NF-κB signaling by longevity factors FoxOs and SIRT1', Cellular and molecular life sciences, 65: 1049- 58. Sánchez-Carrón, Guiomar, Ana Belén Martínez-Moñino, Agustín Sola-Carvajal, Hideto Takami, Francisco García-Carmona, and Álvaro Sánchez-Ferrer. 2013. 'New insights into the phylogeny and molecular classification of nicotinamide mononucleotide deamidases', PloS one, 8: e82705. Sarma, DSR, S Rajalakshmi, and PS Sarma. 1961. 'Deamidation of nicotinamide and NMN', Biochemical and biophysical research communications, 6: 389-93. Sasaki, Tsutomu, Bernhard Maier, Katarzyna D Koclega, Maksymilian Chruszcz, Wendy Gluba, P Todd Stukenberg, Wladek Minor, and Heidi Scrable. 2008. 'Phosphorylation regulates SIRT1 function', PloS one, 3. Sasaki, Yo, Takashi Nakagawa, Xianrong Mao, Aaron DiAntonio, and Jeffrey Milbrandt. 2016. 'NMNAT1 inhibits axon degeneration via blockade of SARM1-mediated NAD+ depletion', Elife, 5: e19749. Sasiak, Krystyna, and Priscilla P Saunders. 1996. 'Purification and properties of a human nicotinamide ribonucleoside kinase', Archives of biochemistry and biophysics, 333: 414-18. Satchell, Margaret A, Xiaopeng Zhang, Patrick M Kochanek, C Edward Dixon, Larry W Jenkins, John Melick, Csaba Szabó, and Robert SB Clark. 2003. 'A dual role for poly‐ ADP‐ribosylation in spatial memory acquisition after traumatic brain injury in mice involving NAD+ depletion and ribosylation of 14‐3‐3γ', Journal of neurochemistry, 85: 697-708. Satoh, Masahiko S, and Tomas Lindahl. 1992. 'Role of poly (ADP-ribose) formation in DNA repair', Nature, 356: 356-58. Savage, Dwayne C. 1977. 'Microbial ecology of the gastrointestinal tract', Annual review of microbiology, 31: 107-33. ———. 1986. 'Gastrointestinal microflora in mammalian nutrition', Annual review of nutrition, 6: 155-78. Scheibye-Knudsen, Morten, Sarah J Mitchell, Evandro F Fang, Teruaki Iyama, Theresa Ward, James Wang, Christopher A Dunn, Nagendra Singh, Sebastian Veith, and Md Mahdi Hasan-Olive. 2014. 'A high-fat diet and NAD+ activate Sirt1 to rescue premature aging in cockayne syndrome', Cell metabolism, 20: 840-55. Schleicher, Emily M, Adri M Galvan, Yuka Imamura-Kawasawa, George-Lucian Moldovan, and Claudia M Nicolae. 2018. 'PARP10 promotes cellular proliferation and tumorigenesis by alleviating replication stress', Nucleic acids research, 46: 8908-16. Schlenk, F, and H v Euler. 1936. 'Cozymase', Naturwissenschaften, 24: 794-95. Schlicker, Christine, Melanie Gertz, Panagiotis Papatheodorou, Barbara Kachholz, Christian FW Becker, and Clemens Steegborn. 2008. 'Substrates and regulation mechanisms for the human mitochondrial sirtuins Sirt3 and Sirt5', Journal of molecular biology, 382: 790-801. Schmidt, Mark S, and Charles Brenner. 2019. 'Absence of evidence that Slc12a8 encodes a nicotinamide mononucleotide transporter', Nature metabolism, 1: 660-61. Schreiber, Valérie, Jean-Christophe Amé, Pascal Dollé, Inès Schultz, Bruno Rinaldi, Valérie Fraulob, Josiane Ménissier-de Murcia, and Gilbert de Murcia. 2002. 'Poly (ADP-ribose) polymerase-2 (PARP-2) is required for efficient base excision DNA repair in association with PARP-1 and XRCC1', Journal of Biological Chemistry, 277: 23028- 36.

327 References

Schuette, SALLY A, and RICHARD C Rose. 1983. 'Nicotinamide uptake and metabolism by chick intestine', American Journal of Physiology-Gastrointestinal and Liver Physiology, 245: G531-G38. Schultz, Michael B, and David A Sinclair. 2016. 'Why NAD+ declines during aging: It’s destroyed', Cell metabolism, 23: 965-66. Schultz, Niklas, Elena Lopez, Nasrollah Saleh‐Gohari, and Thomas Helleday. 2003. 'Poly (ADP‐ribose) polymerase (PARP‐1) has a controlling role in homologous recombination', Nucleic acids research, 31: 4959-64. Schwager, Stephan, Kirsty Agnoli, Manuela Köthe, Friederike Feldmann, Michael Givskov, Aurelien Carlier, and Leo Eberl. 2013. 'Identification of Burkholderia cenocepacia strain H111 virulence factors using nonmammalian infection hosts', Infection and immunity, 81: 143-53. Schweiger, Manfred, Klaus Hennig, Felicitas Lerner, Marc Niere, Monica Hirsch-Kauffmann, Thomas Specht, Christoph Weise, Shiao Li Oei, and Mathias Ziegler. 2001. 'Characterization of recombinant human nicotinamide mononucleotide adenylyl transferase (NMNAT), a nuclear enzyme essential for NAD synthesis', FEBS letters, 492: 95-100. Seelig, Gail Foure, and Roberta F Colman. 1978. 'Characterization of the physicochemical and catalytic properties of human heart NADP-dependent isocitrate dehydrogenase', Archives of biochemistry and biophysics, 188: 394-409. Segata, Nicola, Susan Kinder Haake, Peter Mannon, Katherine P Lemon, Levi Waldron, Dirk Gevers, Curtis Huttenhower, and Jacques Izard. 2012. 'Composition of the adult digestive tract bacterial microbiome based on seven mouth surfaces, tonsils, throat and stool samples', Genome biology, 13: R42. Seifert, Jochen, Kunz Norbert, Ralf Flachmann, Albrecht Läufer, Jany Klaus-Dieter, and Hans Günter Gassen. 1990. 'Expression of the E. coli nadB gene and characterization of the gene product L-aspartate oxidase', Biological chemistry Hoppe-Seyler, 371: 239-48. Sermswan, Rasana W, Phairat Royros, Nittaya Khakhum, Surasakdi Wongratanacheewin, and Apichai Tuanyok. 2015. 'Direct detection of Burkholderia pseudomallei and biological factors in soil', Transactions of the Royal Society of Tropical Medicine and Hygiene, 109: 462-68. Shah, Talmeez Zeb, Abdul Basit Ali, Saghir Ahmad Jafri, and MH Qazi. 2013. 'Effect of Nicotinic Acid (Vitamin B3 or Niacin) on the lipid profile of diabetic and non–diabetic rats', Pakistan journal of medical sciences, 29: 1259. Shahrour, Maher Awni, Claudia M Nicolae, Simon Edvardson, Motee Ashhab, Adri M Galvan, Daniel Constantin, Bassam Abu-Libdeh, George-Lucian Moldovan, and Orly Elpeleg. 2016. 'PARP10 deficiency manifests by severe developmental delay and DNA repair defect', neurogenetics, 17: 227-32. Shankaraiah, Ram C, Elisa Callegari, Paola Guerriero, Alessandro Rimessi, Paolo Pinton, Laura Gramantieri, Enrico M Silini, Silvia Sabbioni, and Massimo Negrini. 2019. 'Metformin prevents liver tumourigenesis by attenuating fibrosis in a transgenic mouse model of hepatocellular carcinoma', Oncogene, 38: 7035-45. Shao, Genze, Jeffrey Patterson-Fortin, Troy E Messick, Dan Feng, Niraj Shanbhag, Yingqun Wang, and Roger A Greenberg. 2009. 'MERIT40 controls BRCA1–Rap80 complex integrity and recruitment to DNA double-strand breaks', Genes & development, 23: 740-54. Shats, Igor, Jason G Williams, Juan Liu, Mikhail V Makarov, Xiaoyue Wu, Fred B Lih, Leesa J Deterding, Chaemin Lim, Xiaojiang Xu, and Thomas A Randall. 2020. 'Bacteria boost mammalian host NAD metabolism by engaging the deamidated biosynthesis pathway', Cell metabolism, 31: 564-79. e7.

328 References

She, David T, Lap Jack Wong, Sang-Ha Baik, and Thiruma V Arumugam. 2018. 'SIRT2 inhibition confers neuroprotection by downregulation of FOXO3a and MAPK signaling pathways in ischemic stroke', Molecular neurobiology, 55: 9188-203. Sheibani-Tezerji, Raheleh, Thomas Rattei, Angela Sessitsch, Friederike Trognitz, and Birgit Mitter. 2015. 'Transcriptome profiling of the endophyte Burkholderia phytofirmans PsJN indicates sensing of the plant environment and drought stress', MBio, 6: e00621- 15. Shen, Jiamei, Mitchell Huber, Ethan Y Zhao, Changya Peng, Fengwu Li, Xiaorong Li, Xiaokun Geng, and Yuchuan Ding. 2016. 'Early rehabilitation aggravates brain damage after stroke via enhanced activation of nicotinamide adenine dinucleotide phosphate oxidase (NOX)', Brain research, 1648: 266-76. Shi, Tong, Fei Wang, Emily Stieren, and Qiang Tong. 2005. 'SIRT3, a mitochondrial sirtuin deacetylase, regulates mitochondrial function and thermogenesis in brown adipocytes', Journal of Biological Chemistry, 280: 13560-67. Shibata, Katsumi, Takashi Hayakawa, and Kazuo Iwai. 1986. 'Tissue distribution of the enzymes concerned with the biosynthesis of NAD in rats', Agricultural and biological chemistry, 50: 3037-41. Shimazu, Tadahiro, Matthew D Hirschey, Lan Hua, Kristin E Dittenhafer-Reed, Bjoern Schwer, David B Lombard, Yu Li, Jakob Bunkenborg, Frederick W Alt, and John M Denu. 2010. 'SIRT3 deacetylates mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase 2 and regulates ketone body production', Cell metabolism, 12: 654-61. Shimoyama, Makoto, Yoshinori Tanigawa, Toichi Ito, Ryuzo Murashima, Iwao Ueda, and Tsunesuke Tomoda. 1971. 'Nicotinamide deamidation by microorganisms in rat stomach', Journal of bacteriology, 108: 191-95. Shin, Jiyung, Ming He, Yufei Liu, Silvana Paredes, Lidia Villanova, Katharine Brown, Xiaolei Qiu, Noushin Nabavi, Mary Mohrin, and Kathleen Wojnoonski. 2013. 'SIRT7 represses Myc activity to suppress ER stress and prevent fatty liver disease', Cell reports, 5: 654- 65. Shin, Jongoh, Sooin Lee, Min-Jeong Go, Sang Yup Lee, Sun Chang Kim, Chul-Ho Lee, and Byung-Kwan Cho. 2016. 'Analysis of the mouse gut microbiome using full-length 16S rRNA amplicon sequencing', Scientific reports, 6: 29681. Shore, David, Mike Squire, and Kim A Nasmyth. 1984. 'Characterization of two genes required for the position‐effect control of yeast mating‐type genes', The EMBO journal, 3: 2817- 23. Shrimp, Jonathan H, Jing Hu, Min Dong, Brian S Wang, Robert MacDonald, Hong Jiang, Quan Hao, Andrew Yen, and Hening Lin. 2014. 'Revealing CD38 cellular localization using a cell permeable, mechanism-based fluorescent small-molecule probe', Journal of the American Chemical Society, 136: 5656-63. Shu, Bingyan, Ye Feng, Yuan Gui, Qingmiao Lu, Wei Wei, Xian Xue, Xiaoli Sun, Weichun He, Junwei Yang, and Chunsun Dai. 2018. 'Blockade of CD38 diminishes lipopolysaccharide-induced macrophage classical activation and acute kidney injury involving NF-κB signaling suppression', Cellular signalling, 42: 249-58. Sims, Carrie A, Yuxia Guan, Sarmistha Mukherjee, Khushboo Singh, Paul Botolin, Antonio Davila Jr, and Joseph A Baur. 2018. 'Nicotinamide mononucleotide preserves mitochondrial function and increases survival in hemorrhagic shock', JCI insight, 3. Sinclair, David A, and Leonard Guarente. 1997. 'Extrachromosomal rDNA circles—a cause of aging in yeast', Cell, 91: 1033-42. Singh, S Kumar, Oleg V Kurnasov, Baozhi Chen, Howard Robinson, Nick V Grishin, Andrei L Osterman, and Hong Zhang. 2002. 'Crystal structure of Haemophilus influenzae

329 References

NadR protein a bifunctional enzyme endowed with NMN adenylyltransferase and ribosylnicotinamide kinase activities', Journal of Biological Chemistry, 277: 33291-99. Smith, Aidan C, Ryan C Holden, Sherry M Rasmussen, Michael R Hoane, and Michael J Hylin. 2019. 'Effects of nicotinamide on spatial memory and inflammation after juvenile traumatic brain injury', Behavioural brain research, 364: 123-32. Smith, Karen, Kathy D McCoy, and Andrew J Macpherson. 2007. "Use of axenic animals in studying the adaptation of mammals to their commensal intestinal microbiota." In Seminars in immunology, 59-69. Elsevier. Smith, Susan, Izabela Giriat, Anja Schmitt, and Titia De Lange. 1998. 'Tankyrase, a poly (ADP-ribose) polymerase at human telomeres', Science, 282: 1484-87. Snow, Anson, Biehuoy Shieh, Kun-Che Chang, Arttatrana Pal, Patricia Lenhart, David Ammar, Philip Ruzycki, Suryanarayana Palla, G Bhanuprakesh Reddy, and J Mark Petrash. 2015. 'Aldose reductase expression as a risk factor for cataract', Chemico- biological interactions, 234: 247-53. Soares, Sandra, Michael Thompson, Thomas White, Amir Isbell, Michiko Yamasaki, Yodeta Prakash, Frances E Lund, Antony Galione, and Eduardo N Chini. 2007. 'NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells', American Journal of Physiology-Cell Physiology, 292: C227-C39. Sobol, Robert W, Julie K Horton, Ralf Kühn, Hua Gu, Rakesh K Singhal, Rajendra Prasad, Klaus Rajewsky, and Samuel H Wilson. 1996. 'Requirement of mammalian DNA polymerase-β in base-excision repair', Nature, 379: 183-86. Sofue, M, Y Yoshimura, M Nishida, and J Kawada. 1991. 'Uptake of nicotinamide by rat pancreatic β cells with regard to streptozotocin action', Journal of endocrinology, 131: 135-38. Someya, Shinichi, Wei Yu, William C Hallows, Jinze Xu, James M Vann, Christiaan Leeuwenburgh, Masaru Tanokura, John M Denu, and Tomas A Prolla. 2010. 'Sirt3 mediates reduction of oxidative damage and prevention of age-related hearing loss under caloric restriction', Cell, 143: 802-12. Son, Myung Jin, Youjeong Kwon, Taekwon Son, and Yee Sook Cho. 2016. 'Restoration of mitochondrial NAD+ levels delays stem cell senescence and facilitates reprogramming of aged somatic cells', Stem Cells, 34: 2840-51. Son, Ni-Huiping, Radha Ananthakrishnan, Shuiqing Yu, Raffay S Khan, Hongfeng Jiang, Ruiping Ji, Hirokazu Akashi, Qing Li, Karen O'Shea, and Shunichi Homma. 2012. 'Cardiomyocyte aldose reductase causes heart failure and impairs recovery from ischemia', PloS one, 7. Sonowal, Himangshu, Pabitra B Pal, Jian-Jun Wen, Sanjay Awasthi, Kota V Ramana, and Satish K Srivastava. 2017. 'Aldose reductase inhibitor increases doxorubicin-sensitivity of colon cancer cells and decreases cardiotoxicity', Scientific reports, 7: 1-14. Sorci, Leonardo, Lucia Brunetti, Lucia Cialabrini, Francesca Mazzola, Marat D Kazanov, Sabato D’Auria, Silverio Ruggieri, and Nadia Raffaelli. 2014. 'Characterization of bacterial NMN deamidase as a Ser/Lys hydrolase expands diversity of serine amidohydrolases', FEBS letters, 588: 1016-23. Soriano-Garcia, M, R Parthasarathy, B Paul, and ARP Paterson. 1984. 'Structure and conformation of 6-(4-nitrobenzyl) thioinosine, C17H17N5O6S, a potent inhibitor of nucleoside transport', Acta Crystallographica Section C: Crystal Structure Communications, 40: 1897-901. Sousa, Tiago, Ronnie Paterson, Vanessa Moore, Anders Carlsson, Bertil Abrahamsson, and Abdul W Basit. 2008. 'The gastrointestinal microbiota as a site for the biotransformation of drugs', International journal of pharmaceutics, 363: 1-25.

330 References

Spagnolo, Laura, Jody Barbeau, Nicola J Curtin, Edward P Morris, and Laurence H Pearl. 2012. 'Visualization of a DNA-PK/PARP1 complex', Nucleic acids research, 40: 4168- 77. Spanogiannopoulos, Peter, Elizabeth N Bess, Rachel N Carmody, and Peter J Turnbaugh. 2016. 'The microbial pharmacists within us: a metagenomic view of xenobiotic metabolism', Nature Reviews Microbiology, 14: 273. Speakman, John R, and Sharon E Mitchell. 2011. 'Caloric restriction', Molecular aspects of medicine, 32: 159-221. Spencer, Richard L, and Jack Preiss. 1967. 'Biosynthesis of diphosphopyridine nucleotide the purification and the properties of diphosphopyridine nucleotide synthetase from Escherichia coli', Journal of Biological Chemistry, 242: 385-92. Spies, Tom D, Clark Cooper, and MA Blankenhorn. 1938. 'The use of nicotinic acid in the treatment of pellagra', Journal of the American Medical Association, 110: 622-27. Spinelli, Jessica B, Haejin Yoon, Alison E Ringel, Sarah Jeanfavre, Clary B Clish, and Marcia C Haigis. 2017. 'Metabolic recycling of ammonia via glutamate dehydrogenase supports breast cancer biomass', Science, 358: 941-46. Sporty, Jennifer, Su‐Ju Lin, Michiko Kato, Ted Ognibene, Benjamin Stewart, Ken Turteltaub, and Graham Bench. 2009. 'Quantitation of NAD+ biosynthesis from the salvage pathway in Saccharomyces cerevisiae', Yeast, 26: 363-69. Srivastava, Satish K, Umesh CS Yadav, Aramati BM Reddy, Ashish Saxena, Ravinder Tammali, Mohammad Shoeb, Naseem H Ansari, Aruni Bhatnagar, Mark J Petrash, and Sanjay Srivastava. 2011. 'Aldose reductase inhibition suppresses oxidative stress- induced inflammatory disorders', Chemico-biological interactions, 191: 330-38. Srivastava, Shireesh, Ulrich Baxa, Gang Niu, Xiaoyuan Chen, and Richard L. Veech. 2013. 'A ketogenic diet increases brown adipose tissue mitochondrial proteins and UCP1 levels in mice', IUBMB life, 65: 58-66. Stanicka, Joanna, Eileen G Russell, John F Woolley, and Thomas G Cotter. 2015. 'NADPH oxidase-generated hydrogen peroxide induces DNA damage in mutant FLT3- expressing leukemia cells', Journal of Biological Chemistry, 290: 9348-61. Steen, Mareike, Tanja Kirchberger, and Andreas H Guse. 2007. 'NAADP mobilizes calcium from the endoplasmic reticular Ca2+ store in T-lymphocytes', Journal of Biological Chemistry, 282: 18864-71. Stefka, Andrew T, Taylor Feehley, Prabhanshu Tripathi, Ju Qiu, Kathy McCoy, Sarkis K Mazmanian, Melissa Y Tjota, Goo-Young Seo, Severine Cao, and Betty R Theriault. 2014. 'Commensal bacteria protect against food allergen sensitization', Proceedings of the National Academy of Sciences, 111: 13145-50. Stein, Liana R, and Shin‐ichiro Imai. 2014. 'Specific ablation of Nampt in adult neural stem cells recapitulates their functional defects during aging', The EMBO journal, 33: 1321- 40. Stewart, Grant S, Bin Wang, Colin R Bignell, A Malcolm R Taylor, and Stephen J Elledge. 2003. 'MDC1 is a mediator of the mammalian DNA damage checkpoint', Nature, 421: 961-66. Stokvis, Ellen, Hilde Rosing, and Jos H Beijnen. 2005. 'Stable isotopically labeled internal standards in quantitative bioanalysis using liquid chromatography/mass spectrometry: necessity or not?', Rapid Communications in Mass Spectrometry: An International Journal Devoted to the Rapid Dissemination of Up‐to‐the‐Minute Research in Mass Spectrometry, 19: 401-07. Stopnisek, Nejc, Natacha Bodenhausen, Beat Frey, Noah Fierer, Leo Eberl, and Laure Weisskopf. 2014. 'Genus‐wide acid tolerance accounts for the biogeographical

331 References

distribution of soil Burkholderia populations', Environmental microbiology, 16: 1503- 12. Strahl-Bolsinger, Sabine, Andreas Hecht, Kunheng Luo, and Michael Grunstein. 1997. 'SIR2 and SIR4 interactions differ in core and extended telomeric heterochromatin in yeast', Genes & development, 11: 83-93. Stromsdorfer, Kelly L, Shintaro Yamaguchi, Myeong Jin Yoon, Anna C Moseley, Michael P Franczyk, Shannon C Kelly, Nathan Qi, Shin-ichiro Imai, and Jun Yoshino. 2016. 'NAMPT-mediated NAD+ biosynthesis in adipocytes regulates adipose tissue function and multi-organ insulin sensitivity in mice', Cell reports, 16: 1851-60. Stubbs, Marion, RL Veech, and HA Krebs. 1972. 'Control of the redox state of the nicotinamide–adenine dinucleotide couple in rat liver cytoplasm', Biochemical Journal, 126: 59-65. Stucki, Manuel, Julie A Clapperton, Duaa Mohammad, Michael B Yaffe, Stephen J Smerdon, and Stephen P Jackson. 2005. 'MDC1 directly binds phosphorylated histone H2AX to regulate cellular responses to DNA double-strand breaks', Cell, 123: 1213-26. Su, Fan, Cédric Jacquard, Sandra Villaume, Jean Michel, Fanja Rabenoelina, Christophe Clément, Essaid A Barka, Sandrine Dhondt-Cordelier, and Nathalie Vaillant-Gaveau. 2015. 'Burkholderia phytofirmans PsJN reduces impact of freezing temperatures on photosynthesis in Arabidopsis thaliana', Frontiers in plant science, 6: 810. Suda, Yasuyuki, Hiroyuki Tachikawa, Ayako Yokota, Hideki Nakanishi, Nobuhiko Yamashita, Yutaka Miura, and Nobuhiro Takahashi. 2003. 'Saccharomyces cerevisiae QNS1 codes for NAD+ synthetase that is functionally conserved in mammals', Yeast, 20: 995-1005. Sukhanova, Maria V, Sanae Abrakhi, Vandana Joshi, David Pastre, Mikhail M Kutuzov, Rashid O Anarbaev, Patrick A Curmi, Loic Hamon, and Olga I Lavrik. 2016. 'Single molecule detection of PARP1 and PARP2 interaction with DNA strand breaks and their poly (ADP-ribosyl) ation using high-resolution AFM imaging', Nucleic acids research, 44: e60-e60. Sultani, G, AF Samsudeen, B Osborne, and N Turner. 2017. 'NAD+: a key metabolic regulator with great therapeutic potential', Journal of neuroendocrinology, 29: e12508. Summers, Daniel W, Daniel A Gibson, Aaron DiAntonio, and Jeffrey Milbrandt. 2016. 'SARM1-specific motifs in the TIR domain enable NAD+ loss and regulate injury- induced SARM1 activation', Proceedings of the National Academy of Sciences, 113: E6271-E80. Sun, Chaonan, Ling Chen, and Zhu Shen. 2019. 'Mechanisms of gastrointestinal microflora on drug metabolism in clinical practice', Saudi Pharmaceutical Journal. Takanaga, Hitomi, Hiroshi Maeda, Hikaru Yabuuchi, Ikumi Tamai, Haruhiro Higashida, and Akira Tsuji. 1996. 'Nicotinic acid transport mediated by pH‐dependent anion antiporter and proton cotransporter in rabbit intestinal brush‐border membrane', Journal of pharmacy and pharmacology, 48: 1073-77. Tang, Wai Ho, Jeremiah Stitham, Yu Jin, Renjing Liu, Seung Hee Lee, Jing Du, Gourg Atteya, Scott Gleim, Geralyn Spollett, and Kathleen Martin. 2014. 'Aldose Reductase– Mediated Phosphorylation of p53 Leads to Mitochondrial Dysfunction and Damage in Diabetic Platelets', Circulation, 129: 1598-609. Tang, Xinghong, Hongying Zhang, Yan Long, Hui Hua, Yangfu Jiang, and Jing Jing. 2018. 'PARP9 is overexpressed in human breast cancer and promotes cancer cell migration', Oncology letters, 16: 4073-77. Tanigawa, Yoshinori, Makoto Shimoyama, Juro Asamizu, and Iwao Ueda. 1971. 'Subcellular localization of nicotinamide deamidase in rat liver', Biochimica et Biophysica Acta (BBA)-General Subjects, 230: 510-17.

332 References

Tanner, Kirk G, Joseph Landry, Rolf Sternglanz, and John M Denu. 2000. 'Silent information regulator 2 family of NAD-dependent histone/protein deacetylases generates a unique product, 1-O-acetyl-ADP-ribose', Proceedings of the National Academy of Sciences, 97: 14178-82. Tanno, Masaya, Jun Sakamoto, Tetsuji Miura, Kazuaki Shimamoto, and Yoshiyuki Horio. 2007. 'Nucleocytoplasmic shuttling of the NAD+-dependent histone deacetylase SIRT1', Journal of Biological Chemistry, 282: 6823-32. Tanny, Jason C, and Danesh Moazed. 2001. 'Coupling of histone deacetylation to NAD breakdown by the yeast silencing protein Sir2: Evidence for acetyl transfer from substrate to an NAD breakdown product', Proceedings of the National Academy of Sciences, 98: 415-20. Tao, Randa, Mitchell C Coleman, J Daniel Pennington, Ozkan Ozden, Seong-Hoon Park, Haiyan Jiang, Hyun-Seok Kim, Charles Robb Flynn, Salisha Hill, and W Hayes McDonald. 2010. 'Sirt3-mediated deacetylation of evolutionarily conserved lysine 122 regulates MnSOD activity in response to stress', Molecular cell, 40: 893-904. Tarantini, Stefano, Marta Noa Valcarcel-Ares, Peter Toth, Andriy Yabluchanskiy, Zsuzsanna Tucsek, Tamas Kiss, Peter Hertelendy, Michael Kinter, Praveen Ballabh, and Zoltán Süle. 2019. 'Nicotinamide mononucleotide (NMN) supplementation rescues cerebromicrovascular endothelial function and neurovascular coupling responses and improves cognitive function in aged mice', Redox biology, 24: 101192. Tarragó, Mariana G, Claudia CS Chini, Karina S Kanamori, Gina M Warner, Ariel Caride, Guilherme C de Oliveira, Micaela Rud, Adrienne Samani, Kyaw Z Hein, and Runqing Huang. 2018. 'A potent and specific CD38 inhibitor ameliorates age-related metabolic dysfunction by reversing tissue NAD+ decline', Cell metabolism, 27: 1081-95. e10. Tateishi, Kensuke, Fumi Higuchi, Julie J Miller, Mara VA Koerner, Nina Lelic, Ganesh M Shankar, Shota Tanaka, David E Fisher, Tracy T Batchelor, and A John Iafrate. 2017. 'The alkylating chemotherapeutic temozolomide induces metabolic stress in IDH1- mutant cancers and potentiates NAD+ depletion–mediated cytotoxicity', Cancer research, 77: 4102-15. Teeuwisse, Wouter M, Ralph L Widya, Marit Paulides, Hildo J Lamb, Johannes WA Smit, Albert de Roos, Mark A Van Buchem, Hanno Pijl, and Jeroen van der Grond. 2012. 'Short-term caloric restriction normalizes hypothalamic neuronal responsiveness to glucose ingestion in patients with type 2 diabetes', Diabetes, 61: 3255-59. Tempel, Wolfram, Wael M Rabeh, Katrina L Bogan, Peter Belenky, Marzena Wojcik, Heather F Seidle, Lyudmila Nedyalkova, Tianle Yang, Anthony A Sauve, and Hee-Won Park. 2007. 'Nicotinamide riboside kinase structures reveal new pathways to NAD+', PLoS biology, 5. Thomas, David C. 2017. 'The phagocyte respiratory burst: historical perspectives and recent advances', Immunology Letters, 192: 88-96. Thwaites, David T, and Catriona MH Anderson. 2011. 'The SLC36 family of proton‐coupled amino acid transporters and their potential role in drug transport', British journal of pharmacology, 164: 1802-16. Tilg, Herbert, and Arthur Kaser. 2011. 'Gut microbiome, obesity, and metabolic dysfunction', The Journal of clinical investigation, 121: 2126-32. Todisco, Simona, Gennaro Agrimi, Alessandra Castegna, and Ferdinando Palmieri. 2006. 'Identification of the mitochondrial NAD+ transporter in Saccharomyces cerevisiae', Journal of Biological Chemistry, 281: 1524-31. Tolner, B, B Poolman, B Wallace, and WN Konings. 1992. 'Revised nucleotide sequence of the gltP gene, which encodes the proton-glutamate-aspartate transport protein of Escherichia coli K-12', Journal of bacteriology, 174: 2391-93.

333 References

Tong, Lei, and John M Denu. 2010. 'Function and metabolism of sirtuin metabolite O-acetyl- ADP-ribose', Biochimica et Biophysica Acta (BBA)-Proteins and Proteomics, 1804: 1617-25. Trammell, Samuel AJ, and Charles Brenner. 2013. 'Targeted, LCMS-based metabolomics for quantitative measurement of NAD+ metabolites', Computational and structural biotechnology journal, 4: e201301012. Trammell, Samuel AJ, Mark S Schmidt, Benjamin J Weidemann, Philip Redpath, Frank Jaksch, Ryan W Dellinger, Zhonggang Li, E Dale Abel, Marie E Migaud, and Charles Brenner. 2016a. 'Nicotinamide riboside is uniquely and orally bioavailable in mice and humans', Nature communications, 7: 12948. ———. 2016b. 'Nicotinamide riboside is uniquely and orally bioavailable in mice and humans', Nature communications, 7: 1-14. Trammell, Samuel AJ, Benjamin J Weidemann, Ankita Chadda, Matthew S Yorek, Amey Holmes, Lawrence J Coppey, Alexander Obrosov, Randy H Kardon, Mark A Yorek, and Charles Brenner. 2016c. 'Nicotinamide riboside opposes type 2 diabetes and neuropathy in mice', Scientific reports, 6: 26933. Trammell, Samuel AJ, Liping Yu, Philip Redpath, Marie E Migaud, and Charles Brenner. 2016d. 'Nicotinamide riboside is a major NAD+ precursor vitamin in cow milk', The Journal of Nutrition, 146: 957-63. Trefely, Sophie, Joyce Liu, Katharina Huber, Mary T Doan, Helen Jiang, Jay Singh, Eliana von Krusenstiern, Anna Bostwick, Peining Xu, and Juliane G Bogner-Strauss. 2019. 'Subcellular metabolic pathway kinetics are revealed by correcting for artifactual post harvest metabolism', Molecular metabolism, 30: 61-71. Tseng, Anne H-H, Li-Hong Wu, Shyan-Shu Shieh, and Danny Ling Wang. 2014. 'SIRT3 interactions with FOXO3 acetylation, phosphorylation and ubiquitinylation mediate endothelial cell responses to hypoxia', Biochemical Journal, 464: 157-68. Tubbs, Anthony, and André Nussenzweig. 2017. 'Endogenous DNA damage as a source of genomic instability in cancer', Cell, 168: 644-56. Tunaru, Sorin, Jukka Kero, Annette Schaub, Christian Wufka, Andree Blaukat, Klaus Pfeffer, and Stefan Offermanns. 2003. 'PUMA-G and HM74 are receptors for nicotinic acid and mediate its anti-lipolytic effect', Nature medicine, 9: 352. Tuncel, Handan, Shinji Tanaka, Shiro Oka, Shiro Nakai, Ryuichiro Fukutomi, Mayumi Okamoto, Takahide Ota, Hiroshi Kaneko, Masaaki Tatsuka, and Fumio Shimamoto. 2012. 'PARP6, a mono (ADP-ribosyl) transferase and a negative regulator of cell proliferation, is involved in colorectal cancer development', International journal of oncology, 41: 2079-86. Turkiew, Elliot, Debbie Falconer, Nicole Reed, and Ahmet Höke. 2017. 'Deletion of Sarm1 gene is neuroprotective in two models of peripheral neuropathy', Journal of the Peripheral Nervous System, 22: 162-71. Ubeda, Carles, Ying Taur, Robert R Jenq, Michele J Equinda, Tammy Son, Miriam Samstein, Agnes Viale, Nicholas D Socci, Marcel RM Van Den Brink, and Mini Kamboj. 2010. 'Vancomycin-resistant Enterococcus domination of intestinal microbiota is enabled by antibiotic treatment in mice and precedes bloodstream invasion in humans', The Journal of clinical investigation, 120: 4332-41. Uchida, YKTT, K Izai, T Orii, and T Hashimoto. 1992. 'Novel fatty acid beta-oxidation enzymes in rat liver mitochondria. II. Purification and properties of enoyl-coenzyme A (CoA) hydratase/3-hydroxyacyl-CoA dehydrogenase/3-ketoacyl-CoA thiolase trifunctional protein', Journal of Biological Chemistry, 267: 1034-41. Uddin, Golam Mezbah, Neil A Youngson, Bronte M Doyle, David A Sinclair, and Margaret J Morris. 2017. 'Nicotinamide mononucleotide (NMN) supplementation ameliorates the

334 References

impact of maternal obesity in mice: comparison with exercise', Scientific reports, 7: 15063. Ueda, Kunihiro, and Osamu Hayaishi. 1985. 'ADP-ribosylation', Annual review of biochemistry, 54: 73-100. Uehlinger, Susanne, Stephan Schwager, Steve P Bernier, Kathrin Riedel, David T Nguyen, Pamela A Sokol, and Leo Eberl. 2009. 'Identification of specific and universal virulence factors in Burkholderia cenocepacia strains by using multiple infection hosts', Infection and immunity, 77: 4102-10. Umapathy, Nagavedi Siddaramappa, Joyce Gonzales, Sadanand Fulzele, Kyung-mi Kim, Rudolf Lucas, and Alexander Dimitrievich Verin. 2012. 'β-Nicotinamide adenine dinucleotide attenuates lipopolysaccharide-induced inflammatory effects in a murine model of acute lung injury', Experimental lung research, 38: 223-32. Ummarino, Simone, Massimo Mozzon, Federica Zamporlini, Adolfo Amici, Francesca Mazzola, Giuseppe Orsomando, Silverio Ruggieri, and Nadia Raffaelli. 2017. 'Simultaneous quantitation of nicotinamide riboside, nicotinamide mononucleotide and nicotinamide adenine dinucleotide in milk by a novel enzyme-coupled assay', Food chemistry, 221: 161-68. Vachharajani, Vidula T, Tiefu Liu, Xianfeng Wang, Jason J Hoth, Barbara K Yoza, and Charles E McCall. 2016. 'Sirtuins link inflammation and metabolism', Journal of immunology research, 2016. Vakhrusheva, Olesya, Christian Smolka, Praveen Gajawada, Sawa Kostin, Thomas Boettger, Thomas Kubin, Thomas Braun, and Eva Bober. 2008. 'Sirt7 increases stress resistance of cardiomyocytes and prevents apoptosis and inflammatory cardiomyopathy in mice', Circulation Research, 102: 703-10. Van Meter, Michael, Matthew Simon, Gregory Tombline, Alfred May, Timothy D Morello, Basil P Hubbard, Katie Bredbenner, Rosa Park, David A Sinclair, and Vilhelm A Bohr. 2016. 'JNK phosphorylates SIRT6 to stimulate DNA double-strand break repair in response to oxidative stress by recruiting PARP1 to DNA breaks', Cell reports, 16: 2641-50. Vander Heiden, Matthew G, Jason W Locasale, Kenneth D Swanson, Hadar Sharfi, Greg J Heffron, Daniel Amador-Noguez, Heather R Christofk, Gerhard Wagner, Joshua D Rabinowitz, and John M Asara. 2010. 'Evidence for an alternative glycolytic pathway in rapidly proliferating cells', Science, 329: 1492-99. Vassilopoulos, Athanassios, J Daniel Pennington, Thorkell Andresson, David M Rees, Allen D Bosley, Ian M Fearnley, Amy Ham, Charles Robb Flynn, Salisha Hill, and Kristie Lindsey Rose. 2014. 'SIRT3 deacetylates ATP synthase F1 complex proteins in response to nutrient-and exercise-induced stress', Antioxidants & redox signaling, 21: 551-64. Vaziri, Homayoun, Scott K Dessain, Elinor Ng Eaton, Shin-Ichiro Imai, Roy A Frye, Tej K Pandita, Leonard Guarente, and Robert A Weinberg. 2001. 'hSIR2SIRT1 functions as an NAD-dependent p53 deacetylase', Cell, 107: 149-59. Vinogradov, Andrei D, and Vera G Grivennikova. 2016. 'Oxidation of NADH and ROS production by respiratory complex I', Biochimica et Biophysica Acta (BBA)- Bioenergetics, 1857: 863-71. Vyas, Sejal, Melissa Chesarone-Cataldo, Tanya Todorova, Yun-Han Huang, and Paul Chang. 2013. 'A systematic analysis of the PARP protein family identifies new functions critical for cell physiology', Nature communications, 4: 2240. Vyas, Sejal, Ivan Matic, Lilen Uchima, Jenny Rood, Roko Zaja, Ronald T Hay, Ivan Ahel, and Paul Chang. 2014. 'Family-wide analysis of poly (ADP-ribose) polymerase activity', Nature communications, 5: 1-13.

335 References

Wang, Ban, Youjun Zhang, Wei Cao, Xunbing Wei, James Chen, and Weihai Ying. 2016a. 'SIRT2 plays significant roles in lipopolysaccharides-induced neuroinflammation and brain injury in mice', Neurochemical research, 41: 2490-500. Wang, F, CH Chan, K Chen, X Guan, Hui-Kuan Lin, and Q Tong. 2012. 'Deacetylation of FOXO3 by SIRT1 or SIRT2 leads to Skp2-mediated FOXO3 ubiquitination and degradation', Oncogene, 31: 1546-57. Wang, Fei, Margaret Nguyen, F Xiao‐Feng Qin, and Qiang Tong. 2007. 'SIRT2 deacetylates FOXO3a in response to oxidative stress and caloric restriction', Aging cell, 6: 505-14. Wang, Fei, and Qiang Tong. 2009. 'SIRT2 suppresses adipocyte differentiation by deacetylating FOXO1 and enhancing FOXO1's repressive interaction with PPARγ', Molecular biology of the cell, 20: 801-08. Wang, Jinyi, Zhibo Hong, Chao Zeng, Qiujun Yu, and Haichang Wang. 2014a. 'NADPH oxidase 4 promotes cardiac microvascular angiogenesis after hypoxia/reoxygenation in vitro', Free Radical Biology and Medicine, 69: 278-88. Wang, Juanjuan, Chenggang Zhu, Dan Song, Ruiqi Xia, Wenbo Yu, Yongjun Dang, Yiyan Fei, Long Yu, and Jiaxue Wu. 2017a. 'Epigallocatechin-3-gallate enhances ER stress- induced cancer cell apoptosis by directly targeting PARP16 activity', Cell death discovery, 3: 1-9. Wang, Lei, Yongjin J Zhou, Debin Ji, Xinping Lin, Yuxue Liu, Yixin Zhang, Wujun Liu, and Zongbao K Zhao. 2014b. 'Identification of UshA as a major enzyme for NAD degradation in Escherichia coli', Enzyme and microbial technology, 58: 75-79. Wang, Minli, Weizhong Wu, Wenqi Wu, Bustanur Rosidi, Lihua Zhang, Huichen Wang, and George Iliakis. 2006a. 'PARP-1 and Ku compete for repair of DNA double strand breaks by distinct NHEJ pathways', Nucleic acids research, 34: 6170-82. Wang, Shengjun, Xue Yang, Youting Lin, Xiaoxue Qiu, Hui Li, Xiuhe Zhao, Lili Cao, Xuewu Liu, Yuejiu Pang, and Xuping Wang. 2013. 'Cellular NAD depletion and decline of SIRT1 activity play critical roles in PARP-1-mediated acute epileptic neuronal death in vitro', Brain research, 1535: 14-23. Wang, Sufan, Ting Wan, Mingtong Ye, Yun Qiu, Lei Pei, Rui Jiang, Nengzhi Pang, Yuanling Huang, Baoxia Liang, and Wenhua Ling. 2018. 'Nicotinamide riboside attenuates alcohol induced liver injuries via activation of SirT1/PGC-1α/mitochondrial biosynthesis pathway', Redox biology, 17: 89-98. Wang, Suping, Zili Xing, Peter S Vosler, Hannah Yin, Wenjin Li, Feng Zhang, Armando P Signore, R Anne Stetler, Yanqin Gao, and Jun Chen. 2008. 'Cellular NAD replenishment confers marked neuroprotection against ischemic cell death: role of enhanced DNA repair', Stroke, 39: 2587-95. Wang, Tao, Xiangbin Zhang, Poonam Bheda, Javier R Revollo, Shin-ichiro Imai, and Cynthia Wolberger. 2006b. 'Structure of Nampt/PBEF/visfatin, a mammalian NAD+ biosynthetic enzyme', nature structural & molecular biology, 13: 661-62. Wang, Xiaonan, Xuejun Hu, Yang Yang, Toshihiro Takata, and Takashi Sakurai. 2016b. 'Nicotinamide mononucleotide protects against β-amyloid oligomer-induced cognitive impairment and neuronal death', Brain research, 1643: 1-9. Wang, Xiaowan, Qiao Zhang, Ruisi Bao, Nannan Zhang, Yingzhen Wang, Luis Polo-Parada, Andrew Tarim, Aidan Alemifar, Xianlin Han, and Heather M Wilkins. 2017b. 'Deletion of nampt in projection neurons of adult mice leads to motor dysfunction, neurodegeneration, and death', Cell reports, 20: 2184-200. Wang, Yan, Yu Mu, Xiaorui Zhou, Huaixue Ji, Xing Gao, Wen Wen Cai, Qiuhua Guan, and Tie Xu. 2017c. 'SIRT2-mediated FOXO3a deacetylation drives its nuclear translocation triggering FasL-induced cell apoptosis during renal ischemia reperfusion', Apoptosis, 22: 519-30.

336 References

Wang, Yong-Xiao, Yun-Min Zheng, Qi-Bing Mei, Qinq-Song Wang, Mei Lin Collier, Sidney Fleischer, Hong-Bo Xin, and Michael I Kotlikoff. 2004. 'FKBP12. 6 and cADPR regulation of Ca2+ release in smooth muscle cells', American Journal of Physiology- Cell Physiology, 286: C538-C46. Wang, Yuanyuan, Changsen Bai, Yuxia Ruan, Miao Liu, Qiaoyun Chu, Li Qiu, Chuanzhen Yang, and Binghui Li. 2019. 'Coordinative metabolism of glutamine carbon and nitrogen in proliferating cancer cells under hypoxia', Nature communications, 10: 1-14. Warburg, O, and Walter Christian. 1936. 'Pyridin, the hydrogen-transferring component of the fermentation enzymes (pyridine nucleotide)', Biochem. Z, 287: 291-328. Warburg, Otto. 1956. 'On the origin of cancer cells', Science, 123: 309-14. Warren, Martin J, Evelyne Raux, Heidi L Schubert, and Jorge C Escalante-Semerena. 2002. 'The biosynthesis of adenosylcobalamin (vitamin B12)', Natural product reports, 19: 390-412. Wątroba, Mateusz, and Dariusz Szukiewicz. 2016. 'The role of sirtuins in aging and age-related diseases', Advances in medical sciences, 61: 52-62. Wei, Chun‐Chun, Yuan‐Yuan Kong, Xia Hua, Guo‐Qiang Li, Si‐Li Zheng, Ming‐He Cheng, Pei Wang, and Chao‐Yu Miao. 2017. 'NAD replenishment with nicotinamide mononucleotide protects blood–brain barrier integrity and attenuates delayed tissue plasminogen activator‐induced haemorrhagic transformation after cerebral ischaemia', British journal of pharmacology, 174: 3823-36. Wells, J Mitchell, and Scott A McLuckey. 2005. 'Collision‐induced dissociation (CID) of peptides and proteins', Methods in enzymology, 402: 148-85. Wermuth, Bendicht, Hanspeter Bürgisser, Kurt Bohren, and Jean‐Pierre Von Wartburg. 1982. 'Purification and characterization of human‐brain aldose reductase', European journal of biochemistry, 127: 279-84. West, RE, Joel Moss, M Vaughan, T Liu, and T-Yung Liu. 1985. 'Pertussis toxin-catalyzed ADP-ribosylation of transducin. Cysteine 347 is the ADP-ribose acceptor site', Journal of Biological Chemistry, 260: 14428-30. White, Thomas A, Mathur S Kannan, and Timothy F Walseth. 2003. 'Intracellular calcium signaling through the cADPR pathway is agonist specific in porcine airway smooth muscle', The FASEB Journal, 17: 482-84. WHO. 2015. World report on ageing and health (World Health Organization). Wieland, Otto H. 1983. 'The mammalian pyruvate dehydrogenase complex: structure and regulation.' in, Reviews of Physiology, Biochemistry and Pharmacology, Volume 96 (Springer). Wiley, Christopher D, Michael C Velarde, Pacome Lecot, Su Liu, Ethan A Sarnoski, Adam Freund, Kotaro Shirakawa, Hyung W Lim, Sonnet S Davis, and Arvind Ramanathan. 2016. 'Mitochondrial dysfunction induces senescence with a distinct secretory phenotype', Cell metabolism, 23: 303-14. Wilk, Anna, Faisal Hayat, Richard Cunningham, Jianfeng Li, Silvia Garavaglia, Leila Zamani, Davide M Ferraris, Peter Sykora, Joel Andrews, and Jennifer Clark. 2020. 'Extracellular NAD+ enhances PARP-dependent DNA repair capacity independently of CD73 activity', Scientific reports, 10: 1-21. Williams, Pete A, Jeffrey M Harder, Nicole E Foxworth, Kelly E Cochran, Vivek M Philip, Vittorio Porciatti, Oliver Smithies, and Simon WM John. 2017. 'Vitamin B3 modulates mitochondrial vulnerability and prevents glaucoma in aged mice', Science, 355: 756- 60. Willison, John C, and Ghislaine Tissot. 1994. 'The Escherichia coli efg gene and the Rhodobacter capsulatus adgA gene code for NH3-dependent NAD synthetase', Journal of bacteriology, 176: 3400-02.

337 References

Wilson, Ian D, and Jeremy K Nicholson. 2017. 'Gut microbiome interactions with drug metabolism, efficacy, and toxicity', Translational Research, 179: 204-22. Witte, AV, M Fobker, R Gellner, S Knecht, and A Flöel. 2009. 'Caloric restriction improves memory in elderly humans', Proceedings of the National Academy of Sciences, 106: 1255-60. Wogulis, Mark, Erin R Chew, Paul D Donohoue, and David K Wilson. 2008. 'Identification of formyl kynurenine formamidase and kynurenine aminotransferase from Saccharomyces cerevisiae using crystallographic, bioinformatic and biochemical evidence', Biochemistry, 47: 1608-21. Wu, Jinzi, Zhen Jin, and Liang-Jun Yan. 2017. 'Redox imbalance and mitochondrial abnormalities in the diabetic lung', Redox biology, 11: 51-59. Wu, Jinzi, Xiaoting Luo, Nopporn Thangthaeng, Nathalie Sumien, Zhenglan Chen, Margaret A Rutledge, Siqun Jing, Michael J Forster, and Liang-Jun Yan. 2017. 'Pancreatic mitochondrial complex I exhibits aberrant hyperactivity in diabetes', Biochemistry and biophysics reports, 11: 119-29. Wu, Lindsay, David A Sinclair, and Kyle Meetze. 2019. "Enzymatic systems and methods for synthesizing nicotinamide mononucleotide and nicotinic acid mononucleotide." In.: Google Patents. Wu, Xuefang, Feng Luo, Jinbang Li, Xueyun Zhong, and Kunping Liu. 2016. 'Tankyrase 1 inhibitior XAV939 increases chemosensitivity in colon cancer cell lines via inhibition of the Wnt signaling pathway', International journal of oncology, 48: 1333-40. Wubbolts, Marcel G, Peter Terpstra, Jan B van Beilen, Jaap Kingma, HA Meesters, and Bernard Witholt. 1990. 'Variation of cofactor levels in Escherichia coli. Sequence analysis and expression of the pncB gene encoding nicotinic acid phosphoribosyltransferase', Journal of Biological Chemistry, 265: 17665-72. Xiao, Wusheng, Rui-Sheng Wang, Diane E Handy, and Joseph Loscalzo. 2018. 'NAD (H) and NADP (H) redox couples and cellular energy metabolism', Antioxidants & redox signaling, 28: 251-72. Xu, Chengming, Banjun Ruan, Yinghao Jiang, Ting Xue, Zhenyu Wang, Huanyu Lu, Ming Wei, Shan Wang, Zicheng Ye, and Dongsheng Zhai. 2017. 'Antibiotics-induced gut microbiota dysbiosis promotes tumor initiation via affecting APC-Th1 development in mice', Biochemical and biophysical research communications, 488: 418-24. Xu, Dejun, Lin Wu, Xiaohan Jiang, Li Yang, Jianyong Cheng, Huali Chen, Rongmao Hua, Guoxia Geng, Lulu Yang, and Qingwang Li. 2019. 'SIRT2 inhibition results in meiotic arrest, mitochondrial dysfunction, and disturbance of redox homeostasis during bovine oocyte maturation', International journal of molecular sciences, 20: 1365. Xu, Feng, Qiongyi Zhang, Kangling Zhang, Wei Xie, and Michael Grunstein. 2007. 'Sir2 deacetylates histone H3 lysine 56 to regulate telomeric heterochromatin structure in yeast', Molecular cell, 27: 890-900. Xu, Ming, Yang Zhang, Min Xia, Xiao-Xue Li, Joseph K Ritter, Fan Zhang, and Pin-Lan Li. 2012. 'NAD (P) H oxidase-dependent intracellular and extracellular O2•− production in coronary arterial myocytes from CD38 knockout mice', Free Radical Biology and Medicine, 52: 357-65. Xu, Zhu, Lei Zhang, Wenjun Zhang, Du Meng, Hongxia Zhang, Ying Jiang, Xiaojun Xu, Michael Van Meter, Andrei Seluanov, and Vera Gorbunova. 2015. 'SIRT6 rescues the age related decline in base excision repair in a PARP1-dependent manner', Cell cycle, 14: 269-76. Yaku, Keisuke, Keisuke Okabe, and Takashi Nakagawa. 2018a. 'NAD metabolism: Implications in aging and longevity', Ageing research reviews, 47: 1-17.

338 References

———. 2018b. 'Simultaneous measurement of NAD metabolome in aged mice tissue using liquid chromatography tandem‐mass spectrometry', Biomedical Chromatography, 32: e4205. Yamada, Kazuo, Nobumasa Hara, Tomoko Shibata, Harumi Osago, and Mikako Tsuchiya. 2006. 'The simultaneous measurement of nicotinamide adenine dinucleotide and related compounds by liquid chromatography/electrospray ionization tandem mass spectrometry', Analytical biochemistry, 352: 282-85. Yamakuchi, Munekazu, and Charles J Lowenstein. 2009. 'MiR-34, SIRT1, and p53: the feedback loop', Cell cycle, 8: 712-15. Yamamoto, Takanobu, Jaemin Byun, Peiyong Zhai, Yoshiyuki Ikeda, Shinichi Oka, and Junichi Sadoshima. 2014a. 'Nicotinamide mononucleotide, an intermediate of NAD+ synthesis, protects the heart from ischemia and reperfusion', PloS one, 9. ———. 2014b. 'Nicotinamide mononucleotide, an intermediate of NAD+ synthesis, protects the heart from ischemia and reperfusion', PloS one, 9: e98972. Yang, Chun-Song, Kasey Jividen, Adam Spencer, Natalia Dworak, Li Ni, Luke T Oostdyk, Mandovi Chatterjee, Beata Kuśmider, Brian Reon, and Mahmut Parlak. 2017a. 'Ubiquitin modification by the E3 ligase/ADP-ribosyltransferase Dtx3L/Parp9', Molecular cell, 66: 503-16. e5. Yang, Fengying, Xiaolei Chu, Miaomiao Yin, Xiaolei Liu, Hairui Yuan, Yanmei Niu, and Li Fu. 2014. 'mTOR and autophagy in normal brain aging and caloric restriction ameliorating age-related cognition deficits', Behavioural brain research, 264: 82-90. Yang, Hongying, Tianle Yang, Joseph A Baur, Evelyn Perez, Takashi Matsui, Juan J Carmona, Dudley W Lamming, Nadja C Souza-Pinto, Vilhelm A Bohr, and Anthony Rosenzweig. 2007a. 'Nutrient-sensitive mitochondrial NAD+ levels dictate cell survival', Cell, 130: 1095-107. Yang, Lu, Luxi Sun, Yaqun Teng, Hao Chen, Ying Gao, Arthur S Levine, Satoshi Nakajima, and Li Lan. 2017b. 'Tankyrase1-mediated poly (ADP-ribosyl) ation of TRF1 maintains cell survival after telomeric DNA damage', Nucleic acids research, 45: 3906-21. Yang, Yonghua, Wei Fu, Jiandong Chen, Nancy Olashaw, Xiaohong Zhang, Santo V Nicosia, Kapil Bhalla, and Wenlong Bai. 2007b. 'SIRT1 sumoylation regulates its deacetylase activity and cellular response to genotoxic stress', Nature cell biology, 9: 1253-62. Yang, Yue, Farheen Sultana Mohammed, Ning Zhang, and Anthony A Sauve. 2019. 'Dihydronicotinamide riboside is a potent NAD+ concentration enhancer in vitro and in vivo', Journal of Biological Chemistry, 294: 9295-307. Yang, Yue, Ning Zhang, Guoan Zhang, and Anthony A Sauve. 2020. 'NRH salvage and conversion to NAD+ requires NRH kinase activity by adenosine kinase', Nature metabolism, 2: 364-79. Yanofsky, CHARLES, VIRGINIA Horn, and PAUL Gollnick. 1991. 'Physiological studies of tryptophan transport and tryptophanase operon induction in Escherichia coli', Journal of bacteriology, 173: 6009-17. Yao, Kang, Jun Fang, YL Yin, Ze-Meng Feng, Zhi-Ru Tang, and Guoyao Wu. 2011. 'Tryptophan metabolism in animals: important roles in nutrition and health', Front Biosci (Schol Ed), 3: 286-97. Yao, Zhiwen, Wenhao Yang, Zhiqiang Gao, and Peng Jia. 2017. 'Nicotinamide mononucleotide inhibits JNK activation to reverse Alzheimer disease', Neuroscience letters, 647: 133-40. Yatsunenko, Tanya, Federico E Rey, Mark J Manary, Indi Trehan, Maria Gloria Dominguez- Bello, Monica Contreras, Magda Magris, Glida Hidalgo, Robert N Baldassano, and Andrey P Anokhin. 2012. 'Human gut microbiome viewed across age and geography', Nature, 486: 222-27.

339 References

Ying, Weihai, Philippe Garnier, and Raymond A Swanson. 2003. 'NAD+ repletion prevents PARP-1-induced glycolytic blockade and cell death in cultured mouse astrocytes', Biochemical and biophysical research communications, 308: 809-13. Ying, Weihai, Guangwei Wei, Dongmin Wang, Qing Wang, Xiannan Tang, Jian Shi, Peng Zhang, and Huafei Lu. 2007. 'Intranasal administration with NAD+ profoundly decreases brain injury in a rat model of transient focal ischemia', Front Biosci, 12: 2728- 34. Yoshida, Akira. 1966. 'Glucose 6-phosphate dehydrogenase of human erythrocytes I. Purification and characterization of normal (B+) enzyme', Journal of Biological Chemistry, 241: 4966-76. Yoshino, Jun, Joseph A Baur, and Shin-ichiro Imai. 2018. 'NAD+ intermediates: the biology and therapeutic potential of NMN and NR', Cell metabolism, 27: 513-28. Yoshino, Jun, Kathryn F Mills, Myeong Jin Yoon, and Shin-ichiro Imai. 2011. 'Nicotinamide mononucleotide, a key NAD+ intermediate, treats the pathophysiology of diet-and age- induced diabetes in mice', Cell metabolism, 14: 528-36. Young, Genevieve S, Elena Choleris, Frances E Lund, and James B Kirkland. 2006. 'Decreased cADPR and increased NAD+ in the Cd38−/− mouse', Biochemical and biophysical research communications, 346: 188-92. Young, JD, SYM Yao, L Sun, CE Cass, and SA Baldwin. 2008. 'Human equilibrative nucleoside transporter (ENT) family of nucleoside and nucleobase transporter proteins', Xenobiotica, 38: 995-1021. Yu, Haiyan, Patrick Chaimbault, Igor Clarot, Zilin Chen, and Pierre Leroy. 2019. 'Labeling nitrogen species with the stable isotope 15N for their measurement by separative methods coupled with mass spectrometry: A review', Talanta, 191: 491-503. Yu, Mei, Sabine Schreek, Christa Cerni, Chantal Schamberger, Krzysztof Lesniewicz, Elzbieta Poreba, Jörg Vervoorts, Gesa Walsemann, Joachim Grötzinger, and Elisabeth Kremmer. 2005. 'PARP-10, a novel Myc-interacting protein with poly (ADP-ribose) polymerase activity, inhibits transformation', Oncogene, 24: 1982-93. Yu, Seong-Woon, Hongmin Wang, Marc F Poitras, Carmen Coombs, William J Bowers, Howard J Federoff, Guy G Poirier, Ted M Dawson, and Valina L Dawson. 2002. 'Mediation of poly (ADP-ribose) polymerase-1-dependent cell death by apoptosis- inducing factor', Science, 297: 259-63. Yu, Wei, Kristin E Dittenhafer-Reed, and John M Denu. 2012. 'SIRT3 protein deacetylates isocitrate dehydrogenase 2 (IDH2) and regulates mitochondrial redox status', Journal of Biological Chemistry, 287: 14078-86. Yuan, Fang, Zhi‐Ming Xu, Li‐Yan Lu, Hui Nie, Jun Ding, Wei‐Hai Ying, and Heng‐Li Tian. 2016. 'SIRT 2 inhibition exacerbates neuroinflammation and blood–brain barrier disruption in experimental traumatic brain injury by enhancing NF‐κB p65 acetylation and activation', Journal of neurochemistry, 136: 581-93. Yuan, Min, Susanne B Breitkopf, Xuemei Yang, and John M Asara. 2012. 'A positive/negative ion–switching, targeted mass spectrometry–based metabolomics platform for bodily fluids, cells, and fresh and fixed tissue', Nature protocols, 7: 872. Yuan, Zhigang, and Edward Seto. 2007. 'A functional link between SIRT1 deacetylase and NBS1 in DNA damage response', Cell cycle, 6: 2869-71. Zaarur, Nava, Kathleen Desevin, James Mackenzie, Avery Lord, Alla Grishok, and Konstantin V Kandror. 2019. 'ATGL-1 mediates the effect of dietary restriction and the insulin/IGF-1 signaling pathway on longevity in C. elegans', Molecular metabolism, 27: 75-82. Zarse, Kim, Sebastian Schmeisser, Marco Groth, Steffen Priebe, Gregor Beuster, Doreen Kuhlow, Reinhard Guthke, Matthias Platzer, C Ronald Kahn, and Michael Ristow.

340 References

2012. 'Impaired insulin/IGF1 signaling extends life span by promoting mitochondrial L-proline catabolism to induce a transient ROS signal', Cell metabolism, 15: 451-65. Zhan, Yu, Po-Ju Chen, William D Sadler, Fuyuan Wang, Sara Poe, Gabriel Núñez, Kathryn A Eaton, and Grace Y Chen. 2013. 'Gut microbiota protects against gastrointestinal tumorigenesis caused by epithelial injury', Cancer research, 73: 7199-210. Zhang, Fengjiao, Ruiye Xie, Frances M Munoz, Serrine S Lau, and Terrence J Monks. 2014a. 'PARP-1 hyperactivation and reciprocal elevations in intracellular Ca2+ during ROS- induced nonapoptotic cell death', Toxicological Sciences, 140: 118-34. Zhang, Hao, Mingxu Shao, He Huang, Shujuan Wang, Lili Ma, Huining Wang, Liping Hu, Kai Wei, and Ruiliang Zhu. 2018a. 'The dynamic distribution of small-tail han sheep microbiota across different intestinal segments', Frontiers in microbiology, 9: 32. Zhang, Hong, Tianjun Zhou, Oleg Kurnasov, Sara Cheek, Nick V Grishin, and Andrei Osterman. 2002. 'Crystal structures of E. coli nicotinate mononucleotide adenylyltransferase and its complex with deamido-NAD', Structure, 10: 69-79. Zhang, Hongbo, Dongryeol Ryu, Yibo Wu, Karim Gariani, Xu Wang, Peiling Luan, Davide D’Amico, Eduardo R Ropelle, Matthias P Lutolf, and Ruedi Aebersold. 2016a. 'NAD+ repletion improves mitochondrial and stem cell function and enhances life span in mice', Science, 352: 1436-43. Zhang, Kezhong, Hyunbae Kim, Zhiyao Fu, Yining Qiu, Zhao Yang, Jiemei Wang, Deqiang Zhang, Xin Tong, Lei Yin, and Jing Li. 2018b. 'Deficiency of the mitochondrial NAD kinase causes stress-induced hepatic steatosis in mice', Gastroenterology, 154: 224-37. Zhang, Qinghong, Su-Yan Wang, Capucine Fleuriel, Dominique Leprince, Jonathan V Rocheleau, David W Piston, and Richard H Goodman. 2007. 'Metabolic regulation of SIRT1 transcription via a HIC1: CtBP corepressor complex', Proceedings of the National Academy of Sciences, 104: 829-33. Zhang, Rongli, Yuyan Shen, Lin Zhou, Panjamaporn Sangwung, Hisashi Fujioka, Lilei Zhang, and Xudong Liao. 2017. 'Short-term administration of nicotinamide mononucleotide preserves cardiac mitochondrial homeostasis and prevents heart failure', Journal of molecular and cellular cardiology, 112: 64-73. Zhang, Xuejun, Oleg V Kurnasov, Subramanian Karthikeyan, Nick V Grishin, Andrei L Osterman, and Hong Zhang. 2003. 'Structural characterization of a human cytosolic NMN/NaMN adenylyltransferase and implication in human NAD biosynthesis', Journal of Biological Chemistry, 278: 13503-11. Zhang, Yajie, Jianqi Wang, Ming Ding, and Yonghao Yu. 2013. 'Site-specific characterization of the Asp-and Glu-ADP-ribosylated proteome', Nature methods, 10: 981. Zhang, Yang, Ming Xu, Min Xia, Xiang Li, Krishna M Boini, Mi Wang, Erich Gulbins, Paul H Ratz, and Pin-Lan Li. 2014b. 'Defective autophagosome trafficking contributes to impaired autophagic flux in coronary arterial myocytes lacking CD38 gene', Cardiovascular research, 102: 68-78. Zhang, Youjun, Ban Wang, Xingli Fu, Shaofeng Guan, Wenzheng Han, Jie Zhang, Qian Gan, Weiyi Fang, Weihai Ying, and Xinkai Qu. 2016b. 'Exogenous NAD+ administration significantly protects against myocardial ischemia/reperfusion injury in rat model', American journal of translational research, 8: 3342. Zhang, Yuan, Yang Xie, Eric D Berglund, Katie Colbert Coate, Tian Teng He, Takeshi Katafuchi, Guanghua Xiao, Matthew J Potthoff, Wei Wei, and Yihong Wan. 2012. 'The starvation hormone, fibroblast growth factor-21, extends lifespan in mice', Elife, 1: e00065. Zhao, Kehao, Robyn Harshaw, Xiaomei Chai, and Ronen Marmorstein. 2004. 'Structural basis for nicotinamide cleavage and ADP-ribose transfer by NAD+-dependent Sir2

341 References

histone/protein deacetylases', Proceedings of the National Academy of Sciences, 101: 8563-68. Zhao, Weixing, Justin B Steinfeld, Fengshan Liang, Xiaoyong Chen, David G Maranon, Chu Jian Ma, Youngho Kwon, Timsi Rao, Weibin Wang, and Chen Sheng. 2017. 'BRCA1– BARD1 promotes RAD51-mediated homologous DNA pairing', Nature, 550: 360-65. Zhao, Wenhui, Jan-Philipp Kruse, Yi Tang, Sung Yun Jung, Jun Qin, and Wei Gu. 2008. 'Negative regulation of the deacetylase SIRT1 by DBC1', Nature, 451: 587-90. Zhao, Wenjing, Yapeng Wang, Shuyun Liu, Jiaojiao Huang, Zhengxiao Zhai, Chuan He, Jinmei Ding, Jun Wang, Huijuan Wang, and Weibing Fan. 2015. 'The dynamic distribution of porcine microbiota across different ages and gastrointestinal tract segments', PloS one, 10. Zhao, Yahui, Xiaoding Hu, Li Wei, Dan Song, Juanjuan Wang, Lifang You, Hexige Saiyin, Zhaojie Li, Wenbo Yu, and Long Yu. 2018. 'PARP10 suppresses tumor metastasis through regulation of Aurora A activity', Oncogene, 37: 2921-35. Zhao, Yong Juan, Connie Mo Ching Lam, and Hon Cheung Lee. 2012. 'The membrane-bound enzyme CD38 exists in two opposing orientations', Sci. Signal., 5: ra67-ra67. Zhao, Zhi Ying, Xu Jie Xie, Wan Hua Li, Jun Liu, Zhe Chen, Ben Zhang, Ting Li, Song Lu Li, Jun Gang Lu, and Liangren Zhang. 2019. 'A cell-permeant mimetic of NMN activates SARM1 to produce cyclic ADP-ribose and induce non-apoptotic cell death', IScience, 15: 452-66. Zheng, Chaobo, Jin Han, Weiliang Xia, Shengtao Shi, Jianrong Liu, and Weihai Ying. 2012. 'NAD+ administration decreases ischemic brain damage partially by blocking autophagy in a mouse model of brain ischemia', Neuroscience letters, 512: 67-71. Zheng, Meiling, Juan Cai, Zhiwen Liu, Shaoqun Shu, Ying Wang, Chengyuan Tang, and Zheng Dong. 2019. 'Nicotinamide reduces renal interstitial fibrosis by suppressing tubular injury and inflammation', Journal of cellular and molecular medicine, 23: 3995- 4004. Zhernakova, Alexandra, Alexander Kurilshikov, Marc Jan Bonder, Ettje F Tigchelaar, Melanie Schirmer, Tommi Vatanen, Zlatan Mujagic, Arnau Vich Vila, Gwen Falony, and Sara Vieira-Silva. 2016. 'Population-based metagenomics analysis reveals markers for gut microbiome composition and diversity', Science, 352: 565-69. Zhong, Lei, Agustina D'Urso, Debra Toiber, Carlos Sebastian, Ryan E Henry, Douangsone D Vadysirisack, Alexander Guimaraes, Brett Marinelli, Jakob D Wikstrom, and Tomer Nir. 2010. 'The histone deacetylase Sirt6 regulates glucose homeostasis via Hif1α', Cell, 140: 280-93. Zhou, Can‐Can, Xi Yang, Xia Hua, Jian Liu, Mao‐Bing Fan, Guo‐Qiang Li, Jie Song, Tian‐ Ying Xu, Zhi‐Yong Li, and Yun‐Feng Guan. 2016. 'Hepatic NAD+ deficiency as a therapeutic target for non‐alcoholic fatty liver disease in ageing', British journal of pharmacology, 173: 2352-68. Zhou, Hui-Zhong, Raymond A Swanson, Ursula Simonis, Xiaokui Ma, Gary Cecchini, and Mary O Gray. 2006. 'Poly (ADP-ribose) polymerase-1 hyperactivation and impairment of mitochondrial respiratory chain complex I function in reperfused mouse hearts', American Journal of Physiology-Heart and Circulatory Physiology, 291: H714-H23. Zhou, Minghai, Gregory Ottenberg, Gian Franco Sferrazza, Christopher Hubbs, Mohammad Fallahi, Gavin Rumbaugh, Alicia F Brantley, and Corinne I Lasmezas. 2015. 'Neuronal death induced by misfolded prion protein is due to NAD+ depletion and can be relieved in vitro and in vivo by NAD+ replenishment', Brain, 138: 992-1008. Zhu, Chen-Tseh, and David M Rand. 2012. 'A hydrazine coupled cycling assay validates the decrease in redox ratio under starvation in Drosophila', PloS one, 7.

342 References

Zhu, Xiao-Hong, Ming Lu, Byeong-Yeul Lee, Kamil Ugurbil, and Wei Chen. 2015. 'In vivo NAD assay reveals the intracellular NAD contents and redox state in healthy human brain and their age dependences', Proceedings of the National Academy of Sciences, 112: 2876-81. Zhu, Ying, Ke-ke Zhao, Yao Tong, Ya-li Zhou, Yi-xiao Wang, Pei-quan Zhao, and Zhao-yang Wang. 2016. 'Exogenous NAD+ decreases oxidative stress and protects H 2 O 2-treated RPE cells against necrotic death through the up-regulation of autophagy', Scientific reports, 6: 26322. Zimmerman, Steven B, and Carol K Oshinsky. 1969. 'Enzymatic joining of deoxyribonucleic acid strands iii. Further purification of the deoxyribonucleic acid ligase from escherichia coli and multiple forms of the purified enzyme', Journal of Biological Chemistry, 244: 4689-95. Zlosnik, James EA, Guohai Zhou, Rollin Brant, Deborah A Henry, Trevor J Hird, Eshwar Mahenthiralingam, Mark A Chilvers, Pearce Wilcox, and David P Speert. 2015. 'Burkholderia species infections in patients with cystic fibrosis in British Columbia, Canada. 30 years’ experience', Annals of the American Thoracic Society, 12: 70-78. Zong, Xiangang, Michael Schieder, Hartmut Cuny, Stefanie Fenske, Christian Gruner, Katrin Rötzer, Oliver Griesbeck, Hartmann Harz, Martin Biel, and Christian Wahl-Schott. 2009. 'The two-pore channel TPCN2 mediates NAADP-dependent Ca 2+-release from lysosomal stores', Pflügers Archiv-European Journal of Physiology, 458: 891-99. Zou, Xiao‑Dong, Shao‑Qing Guo, Zhi‑Wei Hu, and Wei‑Lang Li. 2016. 'NAMPT protects against 6-hydroxydopamine-induced neurotoxicity in PC12 cells through modulating SIRT1 activity', Molecular medicine reports, 13: 4058-64. Zschoernig, Barbara, and Ulrich Mahlknecht. 2009. 'Carboxy-terminal phosphorylation of SIRT1 by protein kinase CK2', Biochemical and biophysical research communications, 381: 372-77.

343