Novel NAD+ Metabolomic Technologies and Their Applications to Nicotinamide Riboside Interventions

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Theses and Dissertations

Spring 2016

Novel NAD+ metabolomic technologies and their applications to Nicotinamide Riboside interventions

Samuel A.J. Trammell University of Iowa

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This dissertation is available at Iowa Research Online: https://ir.uiowa.edu/etd/3203

Recommended Citation Trammell, Samuel A.J.. "Novel NAD+ metabolomic technologies and their applications to Nicotinamide Riboside interventions." PhD (Doctor of Philosophy) thesis, University of Iowa, 2016. https://doi.org/10.17077/etd.mk206led

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NOVEL NAD + METABOLOMIC TECHNOLOGIES AND THEIR APPLICATIONS TO NICOTINAMIDE RIBOSIDE INTERVENTIONS

Samuel A.J. Trammell

A thesis submitted in partial fulfillment of the requirements for the Doctor of Philosophy degree in Genetics in the Graduate College of The University of Iowa

May 2016

Thesis Supervisor: Professor Charles Brenner

Samuel A.J. Trammell 2016

Graduate College The University of Iowa Iowa City, Iowa

CERTIFICATE OF APPROVAL

______

PH.D. THESIS

______

This is to certify that the Ph.D. thesis of

Samuel A.J. Trammell has been approved by the Examining Committee for the thesis requirement for the Doctor of Philosophy degree in Genetics at the May 2016 graduation.

Thesis Committee: ______Charles Brenner, Thesis Supervisor

______Diane C. Slusarski

______Mary E. Wilson

______Michael E. Wright

______Robert C. Piper

ACKNOWLEDGEMENTS

I must give the greatest thanks to current and former members of the Brenner laboratory for being a constant supportive yet critical force in my thesis work. My mentor, Dr. Charles

Brenner, was, is, and always will be a voice of optimism and encouragement that propelled my work forward. Dr. Brenner allowed me to work independently but was always there as a much needed critical, centering voice during the whole of my thesis.

Recounting the contributions of all members of the Brenner laboratory is impossible due to my inability to properly measure the extreme aid and friendship each person provided.

However, I would like to specifically thank Dr. Rebecca Fagan for the light, absurd, and humorous conversations had as co-workers and more importantly as friends. I would like to acknowledge former laboratory mates Dr. Szu-Chieh Mei, her husband Dr. Bokuan Wu, and Dr.

Jennifer Bolyston for making the laboratory a fun and interesting place in which to work and for their insightful commentary into my work. To my current co-workers, thank you for continuing to contribute to the special milieu that is the Brenner laboratory.

I could never properly thank Dr. Lynn Teesch and Mr. Vic Parcell enough. Dr. Teesch quickly became an unofficial advisor throughout my time working in the High Resolution Mass

Spectrometry Facility on all things related to the operation and use of mass spectrometry.

Conversations with Mr. Parcell varied from the deeply technical to the most mirthful. Both provided constant expertise and support that continually reinvigorated my passion for science and undoubtedly helped me through the more difficult portions of my time here at the University of Iowa.

I would like to thank Drs. Diane Slusarski, Marry Wilson, Michael Wright, and Rob Piper for first agreeing to serve on my committee and then for the contributions they have made to my growth as a scientist and to my thesis.

Finally, I would like to thank the many friends outside of my field and the University that I have met in Iowa City. I cannot imagine the person I would be today without their company.

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ABSTRACT

Nicotinamide adenine dinucleotide (NAD +) is a cofactor in hydride transfer reactions and

consumed substrate of several classes of glycohydrolyitc enzymes, including sirtuins. NAD +, its

biosynthetic intermediates, breakdown products, and related nucleotides (the NAD metabolome)

is altered in many metabolic disorders, such as aging and obesity. Supplementation with the

novel NAD + precursor, nicotinamide riboside (NR), ameliorates these alterations and opposes

systemic metabolic dysfunctions in rodent models. Based on the hypothesis that perturbations

of the NAD metabolome are both a symptom and cause of metabolic disease, accurate

assessment of the abundance of these metabolites is expected to provide insight into the

biology of diseases and the mechanism of action of NR in promoting metabolic health. Current

quantitative methods, such as HPLC, lack specificity and sensitivity to detect distinct alterations

to the NAD metabolome. In this thesis, I developed novel sensitive, accurate, robust liquid

chromatography mass spectrometry methodologies to quantify the NAD metabolome and

applied these methods to determine the effects of disease states and NR supplementation on

NAD + metabolism. My investigations indicate that NR robustly increases the NAD metabolome,

especially NAD + in a manner kinetically different than any other NAD + precursor. I provide the

first evidence of effective NAD + supplementation from NR in a healthy, 52 year old human male, suggesting the metabolic promoting qualities of NR uncovered in rodent studies are translatable to humans. During my investigation of NR supplementation, my work establishes an unexpected robust, dramatic increase in deamino–NAD +, NAAD, directly from NR, which I argue could serve as an accessible biomarker for efficacious NAD + supplementation and the effect of disease upon the NAD metabolome. Lastly, I further establish NR as a general therapeutic against metabolic disorder by detailing its ability to oppose aspects of chronic alcoholism and diabetes mellitus.

PUBLIC ABSTRACT

A century ago in the United States, a disease known as Pellagra ravaged areas mainly subsisting on maize. This disease was detrimental to quality of life and in some instances proved fatal. At the time, this disease was considered a major public health problem and many grant initiatives were announced to identify the cause of the disease and develop an effective treatment. Through these efforts, Pellagra was shown to be a non-infectious disease caused by a diet of maize and lard. It was cured by drinking milk and eating more animal meat. These efforts essentially eliminated the disease from high income nations. Further investigation identified the B3 vitamins commonly referred to as niacin as the anti-Pellagra components of milk and animal meat. Today, obesity, diabetes, and heart disease are prevalent in the US and areas around the world. These diseases are a new public health crisis resulting in the loss of billions of dollars and a decreased quality of life and lifespan. As it was a hundred years ago, public funds are now directed to identify effective treatments to counter these prevalent and devastating diseases. Work generously funded by the public has identified the most recently discovered B 3 vitamin, nicotinamide riboside, as a health promoting compound that could treat these diseases. The goal of my thesis was to develop improved tools to answer how this vitamin works in times of health and disease. In so doing, my work further establishes this novel B 3 vitamin as a health promoting compound and describes clinically relevant technologies to assess its effectiveness in future human trials.

TABLE OF CONTENTS LIST OF TABLES ...... xi LIST OF FIGURES ...... xiii LIST OF ABBREVIATIONS ...... xv CHAPTER 1 ...... 1 INTRODUCTION ...... 1 1.1 Significance of NAD + and Description of the Need for Improved Technologies for Its Measurement ...... 1 1.2 NAD + Transactions ...... 3 1.3 Thesis Goals ...... 8 1.4 Figure ...... 11 CHAPTER 2 ...... 12 NAD METABOLOME ANALYSIS VIA LIQUID CHROMATOGRAPHY MASS SPECTROMETRY...... 12 2.1 Quantitative NAD + Metabolomics ...... 12 Optimized Extraction ...... 12 Optimized Internal Standards ...... 14 Optimized Liquid Chromatography ...... 17 Mass Spectrometry Optimization ...... 18 Metabolite Measurement Challenges ...... 19 Results in Mammalian Cell Line ...... 19 Conclusions ...... 20 Acknowledgements ...... 20 2.2 Continued Method Development Post-Initial Publication ...... 20 ATP and ADP: The Other Problem Metabolites ...... 20 Addition of MeNam, Me2PY, and Me4PY to the NAD Metabolomic Assay ...... 20 Considerations of Quantitative NAD Metabolomics in Mammalian Tissues ...... 22 Quantification of the Oxidized NAD Metabolome in Liver ...... 24 Quantification of NAD(P)H and Extraction from Liver ...... 25 Quantification of the Oxidized NAD Metabolome in Skeletal Muscle ...... 29 2.3 Tables and Figures ...... 31 CHAPTER 3 ...... 40

NICOTINAMIDE RIBOSIDE IS A MAJOR NAD + PRECURSOR VITAMIN IN BOVINE MILK ...... 40 3.1 Distribution of Work ...... 40 3.2 Abstract ...... 40 3.3 Introduction ...... 41 3.4 Methods ...... 42 Milk Quality and Herd Health Measurements ...... 42 Bovine Milk Sample Acquisition and Preparations ...... 43 NMR Spectroscopy ...... 43 NR Stability Assays ...... 44 Staph a Growth Experiments ...... 44 LC-MS and LC-MS/MS ...... 45 Statistical Analysis ...... 46 3.5 Results ...... 47

NR is a Major Component of the B 3 Vitamin Content in Bovine Milk ...... 47 Staph a Depletes NR and Nam ...... 48 NR Content as a Function of Organic Certification ...... 49 NR is a Bound Metabolite in Bovine Milk ...... 50 3.6 Discussion ...... 50 3.7 Tables and Figures ...... 52 3.8 Supplemental Tables ...... 55 CHAPTER 4 ...... 57 EFFICACY OF NMN AND NR AS EXTRACELLULAR NAD + PRECURSORS ...... 57 4.1 Distribution of Work ...... 57 4.2 Abstract ...... 57 4.3 Introduction ...... 57 4.4 Materials and Methods ...... 59 Compounds ...... 59 Cell Culture Conditions ...... 59 Extraction ...... 59 LC-MS/MS ...... 60 4.5 Results ...... 60

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NMN is Dephosphorylated Extracellularly and Contributes to the Intracellular NAD + Pool slower than NR ...... 60 4.6 Discussion ...... 61 4.7 Figures ...... 63 CHAPTER 5 ...... 65 NICOTINAMIDE RIBOSIDE IS UNIQUELY BIOAVAILABLE IN MOUSE AND MAN ...... 65 5.1 Distribution of Work ...... 65 5.2 Abstract ...... 65 5.3 Introduction ...... 66 5.4 Methods ...... 69 Materials and Reagents ...... 69 Mice ...... 69 N of 1 Human Experiment ...... 70 Clinical Trial ...... 70 Sample Preparation and LC-MS ...... 71 Statistical Analyses ...... 71 5.5 Results ...... 72 Oral NR Increases the Blood NAD Metabolome in a Healthy Adult Male ...... 72 Oral NR, Nam and NA Elevate Hepatic NAD + with Distinctive Kinetics ...... 73 NR Directly Contributes to Murine Liver NAAD ...... 77 NR Increases Blood Cell NAD + Metabolism in Human Subjects ...... 79 5.6. Discussion ...... 81 5.7 Figures and Table for Chapter 5.5-5.6 ...... 84 5.8 Supplemental Materials ...... 90 Clinical Trial ...... 90 Sample Preparation and LC-MS ...... 91 5.9 Supplemental Tables and Figures for Chapter 5.5-5.6 ...... 95 5.10 Perspective on Chapter 5 ...... 97 Introduction ...... 97 Results and Discussion ...... 97 Methods ...... 102 5.11 Figures for 5.10 ...... 103 CHAPTER 6 ...... 107

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NICOTINAMIDE RIBOSIDE PREVENTS ALCOHOL INDUCED FATTY LIVER ...... 107 6.1 Distribution of Work ...... 107 6.2 Abstract ...... 107 6.3 Introduction ...... 108 6.4 Materials and Methods ...... 110 Animal Husbandry and Experimental Design ...... 110 Mitochondrial Isolation ...... 111 Western Blotting ...... 112 Microscopy ...... 112 NAD Metabolomics ...... 112 Acetylomics ...... 113 Statistical Analysis ...... 117 6.5 Results and Discussion ...... 117 6.6 Tables and Figures ...... 123 CHAPTER 7 ...... 130 NICOTINAMIDE RIBOSIDE OPPOSES TYPE 2 DIABETES AND NEUROPATHY IN MICE ...... 130 7.1 Distribution of Work ...... 130 7.2 Abstract ...... 130 7.3 Introduction ...... 131 7.4 Methods ...... 133 Mouse Models ...... 133 NAD Metabolomics ...... 133 Statistics ...... 133 Study Approval ...... 133 7.5 Results and Discussion ...... 133 7.6 Acknowledgments ...... 138 7.7 Figures and Tables for Sections 3-5...... 139 7.8 Supplemental Materials ...... 143 Sample Extraction for NAD + Metabolomics ...... 143 LC-MS/MS Analysis for NAD Metabolomics ...... 143 7.9 Supplemental Figures ...... 145

7.10 Perspective on Chapter 7 ...... 147 7.11 Results and Discussion ...... 147 Type 1 Diabetes in Rat Compared to Type 2 Diabetes in Mouse ...... 147 NADH and NADPH Measurement in T2D Murine Liver ...... 151 7.12 Methods ...... 152 7.13 Tables and Figures ...... 154 CHAPTER 8 ...... 158 GENERAL SUMMARY AND FUTURE DIRECTIONS ...... 158 8.1 General Summary ...... 158 8.2 Regulation of the NAD + Metabolome: a Future Avenue of Inquiry ...... 163 8.3 Future Investigations of NR as a Health Promoting Agent ...... 164 APPENDIX A ...... 166 APPENDIX B ...... 172 REFERENCES ...... 174

LIST OF TABLES Table 2.1 Alkaline separation gradient...... 31 Table 2.2 Acidic separation gradient...... 31 Table 2.3 LCMS/MS SRM parameters, sensitivity, and robustness for each metabolite...... 32 Table 2.4 NAD +/nucleotides metabolome of LN428/MPG cell line...... 34 Table 2.5 LCMS/MS SRM parameters, sensitivity, and robustness for methylated nicotinamide metabolites...... 35 Table 2.6 Recovery of murine hepatic oxidized NAD metabolome...... 36 Table 2.7 Gradient for analysis of NAD(P)H...... 39 Table 2.8 Recovery of the oxidized NAD metabolome in murine quadriceps...... 39 Table 3.1 Mean NAD + metabolomes of 18 raw bovine milk samples...... 52

Table 3.2. Correlation coefficients between B 3 vitamin concentrations and milk quality...... 52

Table 3.3. Vitamin B 3 content in store bought bovine milk...... 54 Table 3.4 NAD + precursor concentrations in 19 individual milk samples...... 55 Table 3.5. Individual milk quality assessments and breed...... 56 Table 5.1 PBMC NAD + metabolites (µm) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days...... 85 Table 5.2 Plasma NAD + metabolites (µm) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days...... 95 Table 5.3 Urinary NAD + metabolites (µmol/mmol creatinine) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days...... 95 Table 6.1 Ethanol induced NAD metabolome alterations are opposed by NR...... 123 Table 6.2 Pathways affected by ethanol-induced acetylation...... 125 Table 6.3 Pathways affected by ethanol-induced protein acetylation and responsive to NR treatment...... 126 Table 7.1 The hepatic pool of NADP + and NADPH is depressed by PD and T2D and is partially restored by NR...... 142 Table 7.2 Glycemic control, dyslipidemia, and overall health were not improved by NR in T1D...... 154 Table 7.3 NR opposes T1D neuropathy...... 154 Table 7.4 NR tends to improve STZ induced NAD metabolome defects in sciatic nerve homogenate...... 155

Table 7.5 B 3 vitamins were ineffective in improving glycemic control and overall health...... 155

Table 7.6 Among the B 3 vitamins, NR consistently opposed aspects of T1D neuropathy...... 156

Table 7.7 All three B 3 vitamins tended to alter the NAD metabolome in sciatic nerve...... 156

xii

LIST OF FIGURES Figure 1.1 NAD + biosynthesis in yeast and vertebrates...... 11 Figure 2.1 Chromatograms of all 19 metabolites generated from injection of complex standard solutions using MRM LC-MS...... 33 Figure 2.2 Chromatograms of the methylated nicotinamide species generated from injection of complex standard solutions using MRM LC-MS...... 35

13 Figure 2.3 NADH and its [ C10 ] isotopologue in murine liver sample...... 36 Figure 2.4 Hydrophobic ammonium salts cause intense ion suppression...... 37 Figure 2.5 Carryover of NADPH when TBA is used in the mobile phase...... 37 Figure 2.6 Extracted ion currents for NAD(P)H on a C18 column with TEA in the mobile phase...... 38 Figure 3.1. NR is stable in milk and is degraded by Staph a ...... 53 Figure 3.2. NR-binding to milk demonstrated by NMR...... 54 Figure 4.1 Proposed model for NMN utilization...... 63 Figure 4.2. Extracellular NMN is dephosphorylated extracellular and incorporated into the intracellular NAD + pool at a slower rate compared to extracellular NR...... 64 Figure 5.1 The NAD metabolome...... 84 Figure 5.2. NR elevates hepatic NAD + metabolism distinctly with respect to other vitamins...... 86 Figure 5.3 NR contributes directly to hepatic NAAD...... 87 Figure 5.4 Dose-dependent effects of NR on the NAD Metabolome of human subjects...... 88 Figure 5.5 Hepatic NR, NAR, and Me2PY concentrations after gavage of NR, Nam and NA. ...96 Figure 5.6 IP administration of NR, Nam, and NA produce similar effects on murine liver NAD metabolome...... 103 Figure 5.7 NR directly contributes to hepatic NAAD after IP injection...... 104 Figure 5.8 NR contributes to muscle NAAD following gavage...... 105 Figure 5.9 NR contributes to muscle NAAD following IP...... 106 Figure 6.1 NR tends to oppose ethanol induced hepatic lipid deposition...... 123 Figure 6.2 NR does not oppose ethanol induced global hyperacetylation...... 124 Figure 6.3 Proteins acetylated by ethanol and sensitive to NR...... 127 Figure 6.4 NR opposed increased mortality experienced by ethanol fed mice...... 128 Figure 6.5 NR increased diet consumption in both control and ethanol animals...... 128 Figure 7.1 NR improves metabolic parameters in PD and T2D...... 139 Figure 7.2 NR opposes PDPN and T2DPN...... 140

xiii

Figure 7.3 Activity of NR in DPN can be monitored by corneal confocal microscopy (CCM).... 141 Figure 7.4 Experimental design and weight gain...... 145 Figure 7.5 GTT primary data used for Figure 7.1 i and j...... 146 Figure 7.6 NADP + and NADPH were equally depressed by PD and T2D and improved by NR...... 157

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LIST OF ABBREVIATIONS

AMP Adenosine Monophosphate

ADP Adenosine Diphosphate

ATP Adenosine Triphosphate

ADPR Adenosine diphosphate ribose

CMP Cytosine Monophosphate

IMP Inosine monophosphate

LC-MS Liquid Chromatography Mass Spectrometry

LC-MS/MS Liquid Chromatography Tandem Mass Spectrometry

Me2PY N1-Methyl-2-pyridone-5-carboxamide

Me4PY N1-Methyl-4-pyridone-5-carboxamide

MeNam N1-Methyl-Nicotinamide

NA Nicotinic Acid

NAAD Nicotinamide Adenine Dinucleotide

NAD + Nicotinamide Adenine Dinucleotide

NADH Nicotinamide Adenine Dinucleotide, reduced

NADP + Nicotinamide Adenine Dinucleotide Phosphate

NADPH Nicotinamide Adenine Dinucleotide Phosphate, reduced

NAMN Nicotinic Acid Mononucleotide

NAR Nicotinic acid Riboside

NMN Nicotinamide Mononucleotide

NR Nicotinamide Riboside

Trp Tryptophan

UMP Uridine Monophosphate

CHAPTER 1 INTRODUCTION “Targeted, LCMS-based Metabolomics for Quantitative Measurement of NAD + Metabolites”*

Samuel A.J. Trammell 1,2 and Charles Brenner 1,2

1Department of Biochemistry, 2 Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

*Sections 1.1 and 1.2 and Figure 1.1 of the following chapter are reprinted from a published

article in Computational and Structural Biotechnology Journal volume 4 (1). The copyright of the

article belongs to the authors. The manuscript was written by Samuel Trammell with guidance

from Charles Brenner, PhD.

1.1 Significance of NAD + and Description of the Need for Improved Technologies

for Its Measurement

The essentiality of NAD +-dependent processes in fuel utilization, gene regulation, DNA

repair, protein modification, and cell signaling events makes the analysis of NAD + metabolites central to an understanding of what a tissue is doing. NAD + is the key hydride transfer

coenzyme for a wide variety of oxidoreductases and is also the consumed substrate of sirtuins,

poly(adenosine diphosphate ribose (ADPr)) polymerase, mono ADPr transferases, and cyclic

ADPr synthases (2, 3). Measurement of NAD + and related metabolites including several

nucleosides and nucleotides (hereafter, the NAD + metabolome) serves as a powerful indicator of

the ability of a cell or tissue to perform processes such as glycolysis, gluconeogenesis, fatty

acid oxidation, reactive oxygen species detoxification, among others. Moreover, the state of the

NAD + metabolome can serve as an indication of nutrition, health and disease.

Because NAD + and related metabolites vary in cellular concentration from ~1 µM to ~1

mM, the analytical procedure must be robust, reproducible, and rapid. Liquid chromatography

(LC)-based assays afford the ability to measure multiple metabolites in a timely fashion with the

duration of each run ranging from 10 minutes to an hour. However, quantification through

HPLC-UV-Vis methods is severely compromised based on the complexity of samples. In

complex mixtures, a single peak may contain the metabolite of interest in addition to many other

metabolites of identical retention time. In addition, peak shapes are rarely unaffected by

complexity. Some investigators use a UV-vis signal at a retention time as the primary means for

identification of a metabolite of interest—collected fractions are then subjected to mass

spectrometry to confirm (nonquantitatively) the presence of the metabolite. This process leaves

a great deal of data in the dark. Because every NAD+ metabolite can be converted to one or

more other metabolites, snapshots of the levels of NAD + , nicotinamide (Nam) or any other

NAD + metabolite without assessment of the NAD + metabolome on a common scale has the

potential to be misleading.

Because of its specificity and sensitivity, LC coupled to mass spectrometry (LC-MS) is a

leading analytical method in the measurement of small molecules in complex samples. As with

HPLC-UV-vis methods, LC serves to separate compounds of interest and must be optimized in

the same way as any HPLC method. Because all LC-MS data contain at least two dimensions

of data (retention time plus the mass:charge ratio, termed m/z ), LC-MS increases specificity with

respect to LC-UV-vis methods that report complex absorbance spectra as a function of retention

time or matrix-assisted laser desorption ionization (MALDI)-based methods that report complex

m/z data without retention times. Multidimensional MS, i.e. , LC-MS n, provides further information

because a particular analyte breaks down to component ions at a particular ionization energy.

An ideal LC-MS method identifies an optimal extraction and separation method for all molecules

of interest, detects the compounds in either negative or positive ion mode MS, and has sufficient

LC separation to subject each molecule of interest to MS n analysis. The method is then a series

of selective reaction monitoring (SRM) 1 protocols in which analytes are identified and quantified

by MS as they come off the LC.

Whereas metabolomic discovery projects require high mass resolution instruments,

targeted quantitative LC-MS assays can make use of lower resolution tandem mass

spectrometers such as triple quadrupoles (QQQ). Here, the multidimensional data (retention

time, m/z , and MS 2 transitions) are used to distinguish closely related metabolites, such as

NAD + from NADH. Limits of quantification in optimized targeted, quantitative LC-MS assays are

in the femtomole range.

Though mass spectrometers offer great analytical power for measuring the NAD +

metabolome, they also present technical challenges not encountered in other analytical

techniques. These challenges include development of optimal mass spectrometry conditions,

proper separation of metabolites, and best choice of internal standards. Here we discuss NAD +

metabolism and describe an optimized LC-MS 2 assay of the NAD + metabolome.

1.2 NAD + Transactions

In fungi and vertebrates, NAD + concentration is maintained by either de novo synthesis

from tryptophan (4) or through salvage of nicotinic acid (NA) (5), nicotinamide (Nam) (6), and

the recently identified NAD + precursor vitamin nicotinamide riboside (NR) (7) (Figure 1.1). Some

organisms, such as Candida glabrata , lack de novo synthesis (8). Many vertebrate cell types

turn this pathway off (3). De novo synthesis proceeds from tryptophan in six steps to produce

nicotinic acid mononucleotide (NAMN) and in two additional steps to produce NAD +. When

NAD + is the substrate of an enzyme such as glyceraldehyde phosphate dehydrogenase

(GAPDH), fuel oxidation reactions will reduce NAD + to NADH. In the case of GAPDH, the

reaction is reversible, such that NADH is reoxidized to NAD + in the gluconeogenic direction.

NAD + and NADH can be phosphorylated to NADP + and NADPH. NADP + is required for the

1 Multiple SRMs are referred to as multiple reaction monitoring (MRM).

pentose phosphate pathway (PPP), which produces NADPH. NADPH is required for

detoxification of reactive oxygen species and reductive biosynthesis of lipids and steroids. Just

as glucose-6-phosphate oxidation by the PPP produces NADPH, glutathione reactivation and

reductive biosynthesis reoxidizes NADPH to NADP +.

Beyond serving as a coenzyme in hydride reactions, NAD + is a consumed substrate for

enzymes such as sirtuins, PARPs, and other ADPr transfer enzymes (2, 3, 9, 10). Though CD38

has an activity on NADP +, at least in vitro (11), the typical activity of an NAD +-consuming

enzyme involves NAD + as the substrate, and products that include Nam and an NAD +-derived

ADPr moiety. Thus, to sustain intracellular NAD + levels, actions of NAD +-consuming enzymes must be accompanied by Nam salvage (2, 3). Nam salvage differs between fungi and vertebrates. In fungi, Nam is hydrolyzed by the PNC1 -encoded nicotinamidase to NA (6). NA is

then converted by the first enzyme of the Preiss-Handler pathway, the NPT1 -encoded NA

phosphoribosyltransferase, to form NAMN. The second and third steps of Preiss-Handler

salvage correspond to the final two steps of de novo synthesis, whose last step is glutamine-

dependent NAD + synthetase (12). In vertebrates, Nam produced as a product of NAD +-

consuming enzymes cannot be salvaged as NA intracellularly. However, if Nam goes through

the gut, bacterial nicotinamidases produce NA (13), which circulates and is used via Preiss-

Handler salvage.

Intracellular Nam salvage in vertebrates depends on a Nam phosphoribosyltranferase,

which entered the scientific literature with the names pre-B cell colony enhancing factor (PBEF)

(14) and Visfatin (15). Now termed Nampt, this protein is widely expressed as an intracellular

enzyme and also circulates as an active extracellular molecule (16, 17). First predicted to be

part of a partially extracellular NAD + biosynthetic cycle (2) along with CD73, a homolog of

bacterial NMN 5’-nucleotidase, extracellular Nampt clearly has enzymatic activity (17). However,

extracellular NMN remains controversial in part due to deficiencies in NAD + metabolite

quantification. As a phosphoribosyltransferase, Nampt activity depends on PRPP, an

extracellular source of which has not been demonstrated (18). By an HPLC-UV method, which

may have been distorted by co-eluting analytes, the abundance of extracellular NMN was

reported to be 80 µM (17). However, using LC- MS n, it was reported that PRPP and NMN are

virtually absent and, moreover, are unstable in mouse plasma (18). It stands to reason that

extracellular Nampt may have activity in local environments and developmental/nutritional

conditions in which the substrates, Nam and PRPP, and the ATP activator are at substantial

levels. Systemic NMN at 80 µM appears to be implausible, however.

Nam and NA can also be methylated, which would be predicted to block salvage. In

plants, NA N-methyltransferase produces a compound known as trigonelline by transfer of the methyl group from S-adenosyl-methionine (19, 20). The corresponding Nam N- methyltransferase (NNMT) has been well characterized in vertebrates (21). Increased NNMT expression has been observed in Parkinson’s Disease (22) with a potential role in disease etiology (23, 24). NNMT is also increased in malignancy (25) and plays an apparent role in cell migration (26). Despite the reported roles in disease, N-methyl Nam (NMNam 2) is a natural

metabolite in healthy individuals with reported antithrombotic (27) and vasorelaxant (28)

activities that is increased in plasma and urine after endurance exercise (29). NMNam is

ultimately converted to N1 -Methyl-2-pyridone-5-carboxamide and N1 -Methyl-4-pyridone-5- carboxamide.

Though the primary breakdown product of NAD + is Nam and the complete bacterially digested product is NA, nicotinamide riboside (NR) is an additional salvageable precursor that exists intracellularly and in milk (7, 30, 31). The unique NR salvage pathway is via nicotinamide riboside kinases (7). In addition, NR can be split into a Nam moiety and resynthesized to NAD +

via Nam salvage enzymes (32). Nicotinic acid riboside (NAR) is an alternate substrate of

2 In the future, this metabolite is abbreviated MeNam.

nicotinamide riboside kinases (33) and purine nucleoside phosphorylase (13) that has been

shown to be an intracellular NAD + precursor (30) but has not been reported to circulate.

Whereas NA is the salvageable precursor of NAD + that has been exposed to the most digestive enzymes and Nam is the salvageable precursor that is produced by every cell with

NAD +-consuming enzymes, the main source of dietary NR is probably partial digestion of NAD +.

Depending on one’s nutrition and potentially one’s microbiome, the three vitamin precursors of

NAD + (NA, Nam and NR) and trp should be in circulation (3). The existence of extracellular

enzymes with the potential to produce and consume NMN, and which consume NAD +, suggests

the circulation of pyridine nucleotides (2). Moreover, NMN supplementation of mice on high fat

diet (HFD) increases insulin sensitivity, glucose tolerance, and intracellular NAD + compared to non-treated mice on the same diet (34). Though extracellular NMN was interpreted to function via direct incorporation of the nucleotide into cells (34), careful examination indicates that extracellular NA, Nam, and NR increase intracellular NAD + in yeast and vertebrate cells, whereas NMN requires dephosphorylation to NR (35). Consistent with the prediction that the ectoenzyme CD73 has NMN 5’-nucleotidase activity (2), CD73 has the requisite biochemical activity to catalyze NMN dephosphorylation (36). In the yeast system, NR extends replicative longevity in a manner that depends on conversion to NAD + (32). In mice on high fat diet, NR improves glucose control and insulin sensitivity, while moderating the observed increase in adiposity (37).

In addition to the major difference in Nam salvage between vertebrate and yeast systems, there is a mitochondrial compartmentalization problem in vertebrates. In yeast, transporters Ndt1 and Ndt2 carry NAD + across the mitochondrial inner membrane (38) and the

only mitochondrial NAD + biosynthetic enzyme is NADH kinase, Pos5 (39). However, in

vertebrate cells, the nucleocytoplasm and the mitochondrial matrix constitute distinct pools of

NAD +, NADH, NADP + and NADPH owing to impermeability of the mitochondrial inner

membrane to these compounds. Though systems such as the malate-aspartate shuttle and

nicotinamide nucleotide transhydrogenase transfer reducing equivalents across mitochondrial

membranes, vertebrate mitochondria require a system to import an NAD + precursor into the matrix for conversion to NAD +. On the basis of localization of NAD + biosynthetic enzymes, that precursor is NMN (35) 3. Nmnat3, which converts NMN to NAD +, is localized to the mitochondrial matrix. Nmnat3 is one of three vertebrate NAMN/NMN adenylyltranferases—the other two are localized in the nucleus and on the cytosolic face of Golgi. Though one could argue that the ability of Nmnat3 to convert NAMN to NAAD suggests that NAMN or NMN could be the mitochondrial NAD + precursor, the NAAD product of the NAMN reaction requires glutamine- dependent NAD + synthetase for conversion to NAD +. Glutamine-dependent NAD + synthetase is

not mitochondrially localized (35).

As shown in Figure 1.1, the implication of NMN as the limiting precursor for vertebrate

mitochondrial NAD + biosynthesis is profound. De novo synthesis and NA-dependent Preiss-

Handler synthesis can only supply mitochondria with NAD + by nucleocytosolic conversion to

NAD + followed by the pyrophosphate-dependent conversion of NAD + to NMN in a back reaction

of Nmnat first demonstrated by Arthur Kornberg in 1948 (44) or by conversion of NAD + to Nam

and subsequent conversion of Nam to NMN. In contrast, Nam and NR can be converted directly

to NMN by Nampt and NR kinases, respectively.

In mitochondria that are burning fuel, the redox reactions are largely directional because

fuel oxidation converts NAD + to NADH and complex I of the electron transfer chain reoxidizes

NADH to NAD +. Three vertebrate sirtuins, Sirt3-5, reside in mitochondria, where they consume

NAD + in reactions that either modify proteins or relieve protein modifications (45). For the

3 The origin of mammalian mitochondrial NAD + is controversial. Later investigations revealed Nmnat3 is expressed in erythrocytes, which do not contain mitochondria (40). Further, Nmnat3 deficiency does not alter mitochondrial NAD + (41) and Nmnat3 knockout animals are viable and capable of maintaining in NAD + both fractions (42), suggesting the nucleocytoplasmic and mitochondrial pool are continuous. So far, no mammalian mitochondrial NAD + importer has been identified (43) and the partitioning or lack thereof of NAD + requires further investigation.

mitochondrial sirtuins to work and avoid robbing redox enzymes of NAD +, NMN must be imported from the cytosol.

Completing the major NAD + transactions, yeast possess two cytosolic NAD +/NADH

kinases and the mitochondrial NADH kinase, Pos5 (39). Vertebrate cytosolic NAD +/NADH kinase is related to the yeast enzymes (46), whereas the vertebrate mitochondrial NAD +/NADH kinase was recently identified as a homolog of A. thaliana Nadk3, which can use ATP or polyphosphate as the phosphate donor (47).

1.3 Thesis Goals

The main focus of my thesis research was to develop LC-MS/MS technologies for the

quantitation of NAD + and related metabolites to further our understanding of NR interventions in healthy and diseased states. Previous members of my thesis laboratory focused upon the enzymes related to NAD + and its biosynthesis from NAR and NR (7, 30, 32, 33, 48). In their investigations, the first NAD metabolome assay was developed and included substrates and products of these enzymes and other enzymes related to NAD + metabolism (31). From their

work, NR was established as a bona fide salvageable NAD + precursor that could extend lifespan of yeast (32) and work from other groups revealed NR extends life-span in C. elegans (49) and acts to oppose metabolic (37, 50-54) and neurodegenerative disorders (55, 56) in rodents.

Translating these health promoting effects of NR in the clinic required the improvement and development of novel technologies for accurate and robust quantitation of NAD + and related metabolites.

In my thesis work, I improved upon the previous assay by including internal standards, adding additional NAD + related metabolites, and further optimizing extraction procedures for cell

and tissues. With these improvements, we were able to produce a detailed metabolic image of

the fate of NAD + metabolism in a variety of biological contexts with and without NR

interventions. The technologies described herein allowed for both the confirmation and

generation of hypotheses regarding the effect of NR on NAD + metabolism in the normal and abnormal function of a cell or organism.

In chapter 2, I include a reprint of the remaining publication used to introduce my dissertation in this chapter. Sections are added after section 2.2 Conclusions to describe further method development as necessitated by my thesis work.

Chapters 3, 4, 5, 6, and 7 are demonstrations of the technologies described in chapter 2.

Our work in chapter 3 establishes the true B 3 vitamin content of bovine milk, uncovers that farming practices may influence the vitamin quality of milk, and reveals that B 3 vitamin

fortification of bovine milk may be a future route of delivery of NR to populations at risk for

developing neurological and metabolic disorders. Specifically, we show that NR represents 40%

of the B 3 vitamin content of bovine milk from a herd of Bos taurus and in store procured cow’s

milk. We uncover that organic milk tends to contain less NR than conventional milk and suggest

that, in part, Staphylococcus aureus infection may be responsible. We then uncover that NR is

stable in and binds to milk. This chapter illustrates how the same technology utilized to merely

elucidate the composition of a food product can be utilized to generate and test hypotheses for

how the vitamin content of a food.

In chapter 4, we establish that NR is a superior NAD + precursor compared to NMN using

stable isotope labeling technologies. Work in chapter 3 and 4 were crucial for later work

described in chapter 5 where more complex stable isotope labeled experiments were

performed.

The work described in chapter 5 is the culmination of my thesis work. This chapter is my

perspective and narration of a work that was written by my advisor Dr. Charles Brenner and

includes data generated using methods pioneered by me but performed by both myself and Dr.

Mark Schmidt, a current staff member of the Brenner laboratory. In this work, we quantified the

NAD metabolome in a healthy middle-aged human subject after initial and subsequent

supplementation of NR and uncover that NAAD is a potential, non-obvious, accessible

biomarker for NR supplementation. NAAD is a non-obvious product of NR since there is

currently no known mammalian deamidating NAD + pathway (Figure 1.1). We then compare the effect of NA, Nam, and NR on the murine hepatic NAD metabolome. All three precursors increase NAD + as expected. However, both Nam and NR increase NAAD. Additionally, we

report for the first time that NR is a far superior effector in NAD + metabolism, increasing both hepatic NAD + and NAAD to a greater extent compared to NA and Nam. Further, we tested

whether and confirmed that NR directly contributes to NAAD using stable isotope technologies.

In Dr. Schmidt’s work, we confirm that NAAD positively correlates with NR dosage in a group of

healthy human subjects. Together, these works performed in human and murine systems prove

+ NR is superior to other B 3 vitamins effecting the NAD metabolome and increasing NAD in

particular and uncover that NAAD may be a future, clinical biomarker for the effect of NR on

NAD + metabolism.

Chapters 6 and 7 are essentially the phenotypic effects of NR supplementation on

metabolic syndromes. In chapter 6, we tested the hypothesis that NR supplementation would

prevent alcohol-induced fatty liver disease by increasing hepatic NAD + and consequently

reversing the metabolic damage of alcohol on mitochondrial metabolism. In Chapter 7, we

tested the hypothesis that the alteration of the NAD metabolome in diabetic animals is involved

in the etiology of diabetic peripheral neuropathy and that supplementation with NR could

prevent this devastating complication of diabetes. In this chapter, I present our work with a Type

I diabetic animal model and NR and my perspective on my work with a Type II diabetic animal

model and its context alongside the work performed by Dr. Mark Yorek and coworkers detailed

in Chapter 7.1-4 written by Dr. Charles Brenner, thesis advisor.

1.4 Figure

Figure 1.1 NAD + biosynthesis in yeast and vertebrates. Intracellular NAD+ is derived from either de novo synthesis from tryptophan or from salvage of NA, Nam, or NR. In yeast, Nam is converted to NA by nicotinamidase Pnc1p (dotted line). In yeast and vertebrates, NA is phosphoribosylated to NAMN, an intermediate in de novo synthesis, and converted to NAD+ by way of NAAD in a step catalyzed by glutamine-dependent NAD+ synthetase (12). In vertebrates, Nam conversion to NMN is catalyzed by Nampt (16). The other source of NMN in yeast and vertebrates is phosphorylation of NR by NR kinases. NR and NAR can be split to the corresponding pyridine bases. NAR phosphorylation yields NAMN. NMN is converted to NAD+ by NMN adenylyltransferase activity, which is reversible. As shown, in vertebrates, NMN must be imported into mitochondria for conversion to NAD+. Enzymatic NAD+ and NADP+ consumption releases the Nam moiety and produces ADPr products. Finally, Nam and NA can be converted to non-salvageable products.

CHAPTER 2 NAD METABOLOME ANALYSIS VIA LIQUID CHROMATOGRAPHY MASS SPECTROMETRY “Targeted, LCMS-based Metabolomics for Quantitative Measurement of NAD + Metabolites”*

Samuel A.J. Trammell 1,2 and Charles Brenner 1,2

1Department of Biochemistry, 2 Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

*The following chapter is a description of the general liquid chromatography and mass spectrometry methodologies employed in all subsequent chapters. Sections 1 – 2 are reprints of the rest of the publication included in Chapter 1.1-1.2 (1) which was written by myself with guidance and editing by CB.

2.1 Quantitative NAD + Metabolomics

The NAD + metabolome 4, as defined here, includes dinucleotides, nucleotides,

nucleosides, nucleobases and related compounds (Table 2.3). The masses of many of the

analytes differ by a single Dalton, necessitating optimal separation and careful MS. The current

method is an improvement over methods, which measured only select metabolites (17, 57), and

more recent methods, which embraced a more complete set of metabolites, but which lacked

resolution of several compounds (31, 58). Here we review optimization of all parameters and a

solution to the ionization suppression problem that plagued previous methods.

Optimized Extraction

Methods that do not inactivate enzymatic activities upon cell lysis (58) are clearly flawed

and, based upon the amount of time of sample autolysis, cellular NAD + can be degraded to ~1%

of expected values (~10 µM) with elevation of apparent NR concentration to ~100 times

4 After publication of this document, we have referred to the NAD + metabolome interchangeably with the NAD metabolome.

expected values (1 mM) (59). The preferred method of extraction is to use boiled, buffered ethanol (60), which is well validated for NAD + metabolites (30, 31).

For yeast samples, an ideal cell number is 2 to 4.5 x 10 7, the midrange of which can be obtained by harvesting 25 ml of cells at an OD 600 nm of 0.7. For mammalian cell culture, we typically use 4 to 20 x 10 6 cells, depending upon the cell type. Yeast cell pellets are extracted directly. Mammalian cell pellets are washed once in ice-cold potassium buffered saline. Cells are resuspended in 300 µL of a 75% ethanol/25% 10 mM HEPES, pH 7.1 v/v (buffered ethanol) solution, preheated to 80 °C. Samples are shaken at 1000 rpm in an 80 °C block for three minutes. Soluble metabolites are separated from particulate by refrigerated microcentrifugation

(10 min, 16k g). Though the ethanol-soluble extract contains all the metabolites of interest, the weight of the particulate can be used to determine the optimized resuspension volume for dried metabolites. Thus, both the particulate and soluble metabolites are dried by speed vacuum at

40 °C.

Empirically, we determined that 3.6 mg of yeast or mammalian cell-derived particulate corresponds to a metabolite pellet, which can be resuspended in a 100 µl volume and produce the desired absorbance and LC-MS signals. Thus, the dry weight of each pellet is recorded, divided by 3.6 mg, and multiplied by 100 µl to obtain the initial resuspension volume. Extracts are resuspended in 1% (v/v) acetic acid adjusted to pH 9 with ammonium hydroxide (ammonium acetate buffer). These conditions were chosen to preserve NADH and NADPH prior to analysis 5

(61).

Resuspended metabolites (2 µl) are checked in a Nanodrop (ThermoFisher) to determine the OD 260 nm , which is typically greater than or equal to 14. The remaining volume is diluted to an OD 260 nm of 14 in ammonium acetate buffer to obtain the final resuspension volume.

5 In Chapter 2.4, I describe problems in analysis uncovered after publication which required alterations to the re-suspension solvent and to the metabolites included in the metabolomic assay and the way with which they were dealt.

For LC-MS, this material is diluted two-fold into two different metabolite standards and 2.5 µl of

the resulting material is injected and analyzed. Because these dilutions convert the total

intracellular volume into a known volume of which an effective volume of 1.25 µl is analyzed, it

is straightforward to calculate the intracellular volume of cells under analysis.

The calculation of intracellular volume is as follows. For yeast cells, the intracellular

volume of a single cell is taken as 70 fl (62). Thus, the calculated intracellular volume is

obtained as 70 fl times the cell number. For mammalian cells, we use 2.5 pl as volume of a

HeLa cell (63) and calculate the total extracted intracellular volume in the same way as for yeast

cells. For example, an extraction of 3 x 10 7 yeast cells has a calculated intracellular volume of

2.1 µl. If this sample were resuspended into 100 µl and require no further adjustment after checking on the Nanodrop, the 1.25 µl of cell extract in a 2.5 µl injection would represent 1.25% of 2.1 µl = 26 nl. Because the internal standards permit metabolites to be quantified on a mol scale, intracellular metabolite concentrations are determined, in this example, as mol of metabolite divided by 2.6 x 10 -8 l.

Optimized Internal Standards

Ionization suppression is the tendency for sample components to dampen the ionization

and detectability of particular analytes. Thus, one cannot reliably depend on the peak height or

area of a metabolite in a standard curve of purified metabolites to be on the same scale as its

peak size in a complex mixture. In the most advanced previous quantification method for NAD +

metabolites, ionization suppression was a problem for NAD +, inosine and NA (31).

Two internal standard sets are employed. One set is used to quantify 16 metabolites (all analytes except NR, Nam, and NA) and is used in an alkaline separation. The other set is used to quantify NR, Nam, and NA in an acidic separation.

For analytes in the alkaline separation, an extract of Fleischmann’s yeast metabolites is prepared from cells grown in 99% uniformly labeled 13 C glucose (Icon Isotopes, Summit, New

Jersey). In the course of this culture, the PPP converts 13 C glucose to 13 C ribose-5-phosphate,

such that all of the cells’ nucleic acids, mononucleotides and dinucleotides incorporate 13 C.

Mononucleotides incorporate 5 additional Da from one ribosyl moiety, whereas dinucleotides incorporate 10 additional Da from two ribosyl moieties 6. Complete incorporation is achieved by

growing two serial starter cultures in 13 C glucose synthetic dextrose complete media, followed

13 by inoculation of a 250 ml volume of C glucose media at a starting OD 600 of 0.2 and growth to

OD 600 of 0.8. Cells in 50 ml aliquots are then pelleted and stored at -80 °C prior to extraction

with 300 µl buffered ethanol solution. Metabolites are resuspended in 100 µl of the ammonium

acetate buffer and have a typical OD 260 nM of 100. For LC-MS, Fleischmann’s extract is diluted

1:40 into ammonium acetate buffer. This diluted Fleischmann’s extract is further diluted 1:1 with

experimental samples. In the LC-MS analysis, 2.5 µl of the fully diluted material is injected. This

material represents 1.25 µl of the experimental extract and an appropriate amount of

Fleischmann’s extract for metabolite quantification.

Because the vitamins Nam and NA do not contain a carbohydrate group, they are not

labeled by heavy labeled glucose and require the second set of internal standards for accurate

quantification. To each sample or standard solution, heavy labeled Nam and NR are added

such that the final concentration is 1.5 µM. 18 O labeled Nam is prepared by

18 tetramethylguanidine- catalyzed hydrolysis with 3-cyano-pyridine and H 2 O, as described (64).

Though heavy NR is present in the Fleischmann’s yeast extract standard, NR is best separated

and quantified in an acidic separation with Nam and NA. Heavy labeled NR is made as

described (65).

We prepare stock solutions (typically at 10 mM) of each metabolite to be quantified and

then prepare a set of standard solutions containing the whole set of metabolites to be quantified

6 This process creates isotopologues, which are compounds that differ in isomer composition + 13 13 + (e.g. the NAD produced from yeast supplemented with [ C6]-glucose produce [ C10 ]-NAD , the isotopologue to 12-carbon NADH. Isotopologues should not be confused with isotopomers, which are molecules that contain the same isotopic composition but differ in the location of the isotope.

at a range of concentrations (0, 0.1, 0.2, 0.6, 2, 6, 20, 60, and 200 µM). For the alkaline

separation, these standards are mixed 1:1 with the 1:40 dilution of Fleischmann’s extract so that

each quantifiable metabolite in the extract can be set to a pmol amount. For example, if a

particular metabolite in the Fleischmann’s sample were to interpolate precisely between the 2

µM and 6 µM standards (1 µM and 3 µM) in the 2.5 µl injected volume, we would calculate there

to be 5 pmol of that metabolite in the standard amount of Fleischmann’s extract that will be used

for all subsequent samples. This determination is made in technical replicates. Importantly, we

cannot expect the 5 pmol peak area of that metabolite to remain constant when Fleischmann’s

extract is mixed with experimental yeast extracts because peak shapes are often distorted, and

ionization is suppressed due to sample complexity. However, because the 13 C metabolites in

Fleischmann’s extract will have the same degree of ionization suppression as the 12 C metabolites in the experimental extract, the peak area ratios of 12 C to 13 C metabolites and the known mol amounts of the Fleischmann’s metabolites allow calculation of the amounts of the experimental metabolites. Molar calculation of the Fleischmann’s metabolites are not performed.

Mol amounts of metabolites in the experimental samples are converted to molar using the calculation of intracellular volume described above. Because the IMP peak in Fleischmann’s extract is quite small, we use relative peak areas of IMP and NMN in the standard solutions to derive a correction factor that allows IMP in experimental samples to be quantified against the

Fleischmann’s NMN peak 7. For each metabolite, the peak area used for quantification is that of the MS 2 transition.

In theory, inclusion of 1.5 µM 18 O labeled Nam and 18 O labeled NR should be sufficient

to quantify nonlabeled Nam and NR in the experimental extracts. In practice, because label

7 Later analysis revealed that UMP, ADPR, NADP + internal standards are also of poor signal batch-to-batch. The yeast produced internal standard for NAAD is also found to be weak in signal and sometimes co-eluting with what is believed to be the alpha isomer of NAD + internal standard. When signal is too poor, NAD + internal standard is utilized for ADPR, NAAD, and NADP +. CMP internal standard is used for UMP quantitation.

incorporation may vary and there is greater accuracy in preparation of 10 mM standards than

1.5 µM radiolabeled standards, we quantify the 18 O peaks of Nam and NR against a standard curve of NA, Nam and NR. These analyses result in a mol amount of heavy Nam and NR determined from the light standards. Because the same amount of heavy Nam and NR will be in all experimental samples, relative peak areas allow conversion of experimental Nam and NR peaks to mol and molar. The peak area ratio between heavy Nam and NA in the standard solutions is used to derive a correction factor that allows the heavy Nam peak to calculate the amount of NA in experimental samples.

Optimized Liquid Chromatography

Earlier, we described an assay of the NAD + metabolome based on hydrophilic interaction liquid chromatography (31). Encouraged by its track record for separation of nucleosides and nucleotides (66-68), we have since developed an improved separation with the porous graphitic carbon reversed phase material, Hypercarb (Thermo). Resolution of all compounds is done with two different mobile phases on two Hypercarb columns, each used solely for one separation.

In the alkaline separation, solvent A is 7.5 mM ammonium acetate with 0.05% (v/v) ammonium hydroxide and solvent B is 0.1% (v/v) ammonium hydroxide in acetonitrile. The optimized gradient is described in Table 2.1 with a flow rate of 0.08 ml/min and a column temperature of 60 °C. As shown in Table 2.1, the complete run takes 32.2 min on a 1 mm x 100 mm Hypercarb column and must be equilibrated for 20 min prior to first injection.

In the acid separation, solvent A is 10 mM ammonium acetate with 0.1% formic acid and solvent B is 0.1% formic acid in acetonitrile. The optimized gradient is described in Table 2.2 with a flow rate of 0.2 mL/min and a column temperature of 60 °C. As shown in Table 2.2, the complete run takes 23.4 min on a 2.1 mm x 100 mm Hypercarb column. Extracted ion currents for each resolved metabolite are provided in Figure 2.

Mass Spectrometry Optimization

The power of triple quadrupole mass spectrometers is the ability to perform multiple

SRM protocols in a single run, i.e. multiple reaction monitoring (MRM). Modern QQQs are equipped with automatic optimization software to detect transitions and optimize ionization. The software can be the best friend and greatest enemy in method development. Since many of the metabolites are of similar structure and mass, specific diagnostic fragments must be identified and optimized. The carboxylic acid versus carboxamide metabolites and the oxidized versus reduced metabolites differ by only one Dalton. The 13 C peaks produced from metabolites such as NAD + and NADP + would produce crosstalk with NADH and NADPH, respectively (Figure

2.1A). However, all four compounds produce diagnostic fragments, allowing for specific quantification. Current automatic optimization software identifies fragments that are most easily produced and not necessarily those that are diagnostic for the metabolite in the context of structural similarities in the NAD + metabolome. Online searchable libraries such as Metlin

(http://metlin.scripps.edu/ ) and Massbank ( http://www.massbank.jp/?lang=en ) provide MS/MS

spectra for many metabolites with identified fragment structures (69, 70). Specific fragments for

NADH and NADPH not identified by the automatic software were chosen based on these

searches. The transitions were optimized manually. Transitions and optimized conditions are

detailed in Table 2.3.

The cone voltage must be optimized when measuring NAD + especially for NR, NAR,

NAMN, and NMN to reduce on-source fragmentation. NR and NMN readily produce Nam signal, whereas NAR and NAMN produce NA signal (Figure 2.1B, Nam extracted ion current).

Optimization of cone voltage decreases but does not completely remove crosstalk. This unavoidable crosstalk greatly illustrates the need for robust LC separation. After development, overall robustness was determined based on the capacity factors ( k’ ), quantitative range, linear goodness of fit (R 2), replicative standard deviation (RSD) of the method, and RSD of the system. Capacity factors were above 2 for all analytes with the exception of CMP ( k’ = 1) (Table

1). Standard curves were linear from 0.125 picomoles to 250 picomoles with R 2 values falling above 0.99 for all but NAR (0.948), inosine (0.974), NR (0.981) and NADH (0.988). RSD of the method was measured with six separate standard solutions at 10 µM concentrations. RSD of the system was measured with four injections of the same standard solution. Method RSDs were below 10% for all but cytidine, NAR, inosine, NAMN, ATP, NAAD, NADH, and NADP.

System RSDs were below 10% for all but cytidine and NAR. Limits of quantification (LOQ) were measured repeatedly empirically and defined as the concentration producing a signal-to-noise ratio of 10. LOQ were below 100 fmol for all but NMN (1 pmol), ATP (1 pmol), NA (2.5 pmol), and uridine (3.1 pmol). Moreover, the LOQ using this method was at least 3-fold lower for seven metabolites than the previously used method. The previous method is 3-fold more sensitive for one metabolite (Table 1.3) (31).

Metabolite Measurement Challenges

In our hands, AMP and NADPH cannot be reliably quantified by these methods. ATP

and ADP fragment on source to AMP, similar to NR fragmentation to Nam (Figure 2.1B Nam

extracted ion current). Given poor resolution of AMP from both metabolites, detected AMP

signal would represent biological AMP as well as that derived from ATP and ADP. Further, the

NADP peak may represent the sum of cellular NADP and a portion of cellular NADPH, which

has become oxidized. Thus, care should be taken in interpreting the NADP peak. Nam is

strikingly membrane permeable. Nam in cells can be easily lost into post-cellular supernatants

and we suspect that Nam in organelles can be easily lost into post-organelle supernatants.

Results in Mammalian Cell Line

To test our method on a real sample, we analyzed a glioma cell line, LN428/MPG (a gift

of Dr. Robert Sobol, University of Pittsburgh), which had been grown in MEMalpha (10% FBS

HI, gentamycin, geneticin) media in triplicate 150 mm dishes (2 x 10 7 cells per dish by CASY

cell count). The results of the analysis are reported in Table 2.4. As expected, nucleotides, such

as ATP, ADP, UMP, and NAD +, are high in abundance compared to NAD + precursors and biosynthetic intermediates. The NAD +:NADH ratio is ~39 and the NAD +:NADP ratio is 4.6. As expected for cells grown in a type of Dulbecco’s modified Eagle’s media, Nam, but not other vitamins, is detectable. The concentration of NMN is low when compared with yeast, suggesting the NMN pool is converted rapidly to NAD + (31).

Conclusions

Here, an improved LC-MS method has been developed to quantify the NAD +

metabolome. Its principle features are resolution and quantification of 16 metabolites in an

alkaline separation, and resolution and separation of 3 metabolites in an acidic separation, both

on a porous graphitic carbon stationary phase. The problem of ionization suppression that

plagued earlier methods has been eliminated. Preservation and quantification of NADPH

remains a challenge.

Acknowledgements

Work was supported, in part, by grant MCB-0822581 from the National Science Foundation and

by NIH Pre-Doctoral Training Program in Genetics T32 grant GM008629.

2.2 Continued Method Development Post-Initial Publication

ATP and ADP: The Other Problem Metabolites

ATP and ADP were found to be labile in the buffered boiled extraction protocol. Since

ATP degrades to ADP, this complicates analysis of both metabolites. Further, ATP and ADP are

thought to be at 10:1 ratio but this ratio was not observed in LN428 cells utilizing our method

(Table 2.4), suggesting improper metabolic quenching. For this reason, ATP and ADP were not

routinely included in future experiments.

Addition of MeNam, Me2PY, and Me4PY to the NAD Metabolomic Assay

In mammals, Nam serves as a precursor to NAD + and to MeNam (Chapter 1.2). MeNam

is further oxidized by aldehyde oxidase 1 (EC: 1.2.3.1) to produce either N-methyl-2-pyridone-5-

carboxamide (Me2PY) or N-methyl-4-pyridone-5-carboxamide (Me4PY). As stated in Chapter 1,

Section 2, MeNam, Me2PY, and Me4PY have biological effects. Since the writing of that

document, we now know that expression of NNMT, the enzyme methylating Nam to MeNam,

positively correlates with adiposity in adipocytes (71) and negatively correlates in liver (72) and

appears to regulate lipid metabolism in a manner that is tissue dependent and relying upon

either methyl metabolism or the MeNam itself (73). MeNam is produced with concomitant

demethylation of the primary methyl-donor, S-adenosyl-methionine (SAM), which is also the

source of nuclear histone methylation. NNMT activity has been shown to deplete SAM and

cause epigenetic changes and contributes to cancer development (74) but also stem cell

maturation (75). MeNam itself increases murine hepatic Sirt1 activity by either directly or

indirectly stabilizing its protein abundance and has inhibitory effects on cholesterol synthesis

(72). Me2PY and Me4PY have also been implicated in calorie mediated increases in C. elegans life-span (76) by increasing catalase activity and promoting resistance of reactive oxygen species damage. Beyond the numerous biological effects of these metabolites or the enzymatic activity synthesizing them, these metabolites are not thought to be NAD + precursors as there is no known demethylase nor oxidoreductase. In this way, these metabolites can be thought of as an indication of NAD + precursor wasting as an increased occurrence of MeNam and oxidized

derivatives represent a diversion of flux from its synthesis. For these reasons, these metabolites

were added to the NAD metabolome.

MeNam, Me2PY, and Me4PY SRM conditions are displayed in Table 2.5 and were

produced as described above in Chapter 2.1. These metabolites are readily resolvable (Figure

2.2) on the Thermo Scientific Hypercarb™ column with separation as described for the acid

separation in Chapter 2.1. Though Me2PY and Me4PY are isomers, little cross-talk is observed

(Figure 2.2). Retention times and precision for each metabolite are displayed in Table 2.5.

Systematic and analytical variability are below 20% for all metabolites.

18 Internal standard for MeNam was produced by methylating O Nam with D 3

iodomethane. Briefly, 125 mg of labeled Nam was dissolved in 0.5 ml of ACS grade methanol.

96 µl of labeled iodomethane was added slowly to the solution. The solution was vortexed, then

allowed to react at 25 °C with constant shaking at 300 rpm for 24 hours. The reaction produced

a yellow needle shaped precipitate and was dried down via speed vacuum for three hours. The product was analyzed using a Waters Premier Q-TOF operated in positive ion mode and the product at m/z 142 was observed alongside smaller amounts of 18 O Nam. Any non-methylated

labeled Nam was not removed since it would contribute to the internal standard signal and not

interfere with analysis of the NAD metabolome. 18 O Nam was utilized as an internal standard for

Me2PY and Me4PY. MeNam is linear from 0.1 to 100 µM. Me2PY is linear from 0.1 to 30 µM

and Me4PY is linear from 0.1 to 100 µM.

Considerations of Quantitative NAD Metabolomics in Mammalian Tissues

Metabolomic analysis within any tissue requires optimization of extraction from that

tissue prior to the final experiment. Tissues vary in type and abundance of proteins and

metabolites. If the metabolite of interest readily binds to a specific protein, its recovery may be

more affected in a tissue that expresses that protein in abundance compared to another.

Additionally, some metabolites may be isobaric 8 with an analyte and interfere with quantitation if

co-eluting and abundant. The results of these differences mean every tissue presents its own

unique challenges for the same set of metabolites.

Though the extraction methods may differ, aspects of extraction procedures for proper

metabolomic analysis are universal. The extraction process must simultaneously quench

metabolism and separate the metabolite(s) from interfering compounds (77). Turnover rates for

bioenergetic metabolites such as ATP can be in the 100s of µmol per second range (78) and

are greatly influenced by metabolic and genetic interventions. NAD + turnover is similarly

8 Isobaric metabolites are metabolites of the same mass and/or m/z.

affected. Hence, accurate measurement requires rapid and effective quenching of all chemical

and enzymatic reactions occurring in the cell, tissue, or fluid.

Early in my thesis work, tissues were prepared as homogenates and no care was taken

to quench enzymatic activity. This naïve choice led to clear changes in the NAD metabolome

(Table 6.1) exemplified with an extremely high ratio of Nam to NAD +. As described in Chapter

2.1, this high ratio is almost certainly a result of non-quenching of NAD + consuming activities

causing a decrease in NAD + with simultaneous synthesis of Nam. To this end, we employed freeze clamping 9 upon tissue collection at the time of sacrifice to quench enzymatic activity and

preserve the NAD metabolome. Unless otherwise stated, all tissues were collected this way

prior to storage at -80 °C.

Prior to extraction, tissues were pulverized to a fine powder using a Bessman pulverizer

cooled to liquid nitrogen temperatures. The powdering of the tissue was performed to allow for

aliquoting of tissue for the complete NAD metabolome analysis and to increase the surface area

exposed to the extraction solvent. To continue to slow enzymatic turnover of the metabolites,

tissue aliquots were kept at dry ice temperature or lower at all times prior to addition of

extraction solvent. Normally, extractions are performed to precipitate protein and other

macromolecules and release the metabolites of interest into the soluble fraction. Metabolite

extractions are often rely on adjusting the pH and often combined with addition of organic

solvents (commonly methanol or acetonitrile) at various temperatures. It is imperative that

tissues are lysed as quickly and efficiently to ensure inhibition of enzymatic activity and robust

recovery of all metabolites from the sample. Unlike mammalian cell culture samples which

easily lyse with repeated pipetting, tissues require mechanical disruption such as bead beating,

9 Freeze clamping of tissue is performed by squeezing tissue between two liquid nitrogen cooled plates for at least 10 seconds. This methodology ensures all enzymatic activity halts due to extreme low temperature.

homogenization, or sonication. We found in certain instances that heating of the suspended

extract maximized recovery of some metabolites, especially NADP. This effect has been

observed by others (79, 80) and has been attributed to greater disruption of protein binding.

Protein precipitation is imperative for accessing metabolites, but further and possibly

more importantly, for preserving the life of the analytical column, in our case the Hypercarb

column. Loaded proteins can precipitate on column leading to blockage or can adhere to the

sorbent 10 and interfere with separation of metabolites. But the protein precipitation method can

produce a soluble fraction that is incompatible with the liquid chromatograph used for

separation. High organic, low aqueous samples disrupt the peak shape and retention time of

metabolites eluting in separations such as described in Chapter 2.1. In some instances, the

soluble extract is diluted with an appropriate amount of pure or buffered water to ensure proper

separation. However, this method obviously dilutes the sample, causing loss of signal for lower

abundant metabolites. Here, we employed drying of the sample and re-suspension in an

appropriate aqueous solvent. Drying methods themselves can impact recovery of a metabolite

(80) and should always be considered when developing a method. These considerations and

others were investigated in developing sample extractions protocols.

Quantification of the Oxidized NAD Metabolome in Liver

Liver is the largest mammalian internal organ and potentially the most metabolically

versatile and active. The complete suite of NAD + biosynthetic routes are expressed in the liver

(3, 81), making the liver very responsive to B 3 vitamins. We employ the same extraction buffer as for cells and extract 5 – 20 mg of frozen pulverized liver in the following manner. Samples are extracted by addition of 0.1 ml of buffered ethanol (3 volumes ethanol: 1 volume 10 mM

HEPES, pH 7.1) at 80 °C. Samples are vortexed vigorously until thawed, sonicated in a bath

10 Sorbents are materials used to absorb and adsorb compounds. It is the material packed into the column that is responsible for separation of the analytes.

sonicator (10 sec followed by 15 sec on ice, repeated twice), vortexed, then placed into a

Thermomixer® (Eppendorf, Hamburg, Germany) set to 80 °C and shaken at 1050 rpm for five

min. Samples are centrifuged (16.1k g, 10 minutes, 4 °C). Clarified supernatants are transferred

to fresh 1.5 ml tubes to dry via speed vacuum for two hours. Prior to LC-MS/MS analysis,

samples were reconstituted in 40 µl of 10 mM ammonium acetate (>99% pure) in LCMS grade

water. Analytes are separated and analyzed as described in Chapter 2.1 unless otherwise

stated. Recovery was measured based on internal standard area counts in sample compared to

its area counts in reconstitution solvent (Table 2.6). With the exception of NR, all metabolites

had ≥ 50% recoveries. NR recovery had only 18%. This may appear to be a problem in

quantitation given the importance of NR as a metabolic activator; however, NR is an obligate

cation and as consequence is very detectable (LOQ = 10 fmol) (Table 2.3). Further, the ~five-

fold deficit in NR recovery should equally affect its internal standard 18 O NR. Since quantitation

is based upon the ratio of NR to its isotopologue, NR quantitative values are accurate as long as

both analyte and internal standard are detected.

Quantification of NAD(P)H and Extraction from Liver

In the published method (Chapter 2.1), NADPH was immediately removed from routine

analysis due to its instability in the ideal conditions for quantitation of the oxidized NAD

metabolome. NADH was included since yeast fed labeled glucose produce its isotopologue,

which could serve as its internal standard and control for its stability and extraction efficiency.

13 However, over time it was found that the [ C10 ]-NADH in yeast extract varied in concentration and was difficult to robustly produce. Further, future method development with differing liquid chromatography separations indicated the presence of closely eluting and sometimes co-eluting unknown compounds that produced signal for the NADH internal standard in sample (Figure

2.3). The origin of the multiple internal standard is likely from the yeast extract itself, but this point was never proven since it was unusable in liver. These complications warranted a complete re-working for the analysis of NADH and led to its removal from the routine NAD

metabolome assay. Because NADH was removed, considerations for its stability, which

negatively impacted the oxidized NAD metabolome, were no longer relevant, the re-suspension

solvent for the oxidized NAD metabolome was altered to a more acidic condition to preserve

metabolite stability.

Though NADH and NADPH quantitation is difficult, the ratio of these metabolites to their

oxidized forms are often reported as indicators for a cell or tissue to perform glycolysis versus

gluconeogenesis and/or as a capacity for ROS detoxification (Chapter 1.1-1.2). Further, the

action of sirtuins, often thought of as metabolic regulators (82, 83), is inhibited by NADH at

physiologically obtainable concentrations (84). For these reasons, we developed and

implemented an LC-MS method for the quantitation of these reduced dinucleotides in liver. In so

doing, we uncovered a carryover problem with a common liquid chromatography separation for

NADH and NADPH (collectively referred hereafter as NAD(P)H) and offer an alternative liquid

chromatography separation.

Since NADH and NADPH carry a -2 and -4 negative charge, it was clear at the onset of

the liquid chromatography method development that an ion pairing agent was required. Ion

pairing agents are additives to the mobile phase 11 that interact with the analytes of interest and produce better retention and peak shape. Hydrophobic Ammonium salts carrying alkane chains are often employed for the separation of negatively charged metabolites and have been utilized in other assays for organic acid and NADPH (85). Since ammonium salts can cause ion suppression in positive ion mode (Figure 2.4), negative ion mode was utilized. Single ion monitoring (SIM) mode was selected in lieu of SRM mode to increase sensitivity. In this mode, the mass spectrometer scans only for the intact m/z of the compound of interest at any one time and does not produce diagnostic fragments for confirmation of the identity of the compound.

Confirmation of the metabolite of interest in a sample in this method relies upon standard

11 The mobile phase is a liquid or gas that flows through a chromatographic system, carrying compounds at different rates over a stationary phase.

addition to the sample and observation of an increase in signal at the suspected retention time.

Though SRM mode increases selectivity and confidence within a measurement, NAD(P)H

weakly fragments, and, consequently, its signal was greatly diminished in this scanning method.

Initially, we utilized10 mM tributylamine (TBA) as the ion pairing agent in the mobile phase and

a Waters Acquity BEH C18 column (inner diameter x length: 2.1 x 100 mm) as the stationary

phase 12 (86). At first, NAD(P)H eluted sharply and were well resolved from each other (Figure

2.4), but over time, the metabolites became increasingly retained on the column and variably

eluted, indicating carryover (Figure 2.5). The same was observed using the Phenomenex

Synergi Hydro-RP sorbent employed by Fan et al. (2014) (86). The carryover continued despite

decreasing the concentration. This carryover could have resulted from either retention of the

metabolites in the injector system or on the column. With this separation appearing incompatible

with robust quantitation of NAD(P)H, this mobile phase was abandoned without further

investigation into the cause of the carryover/increased retention. However, it appears that TBA

is too retentive of negatively charged metabolites and should be used cautiously in the analysis

of other organic acids.

We hypothesized that triethylammonium (TEA) acetate could replace TBA since it would

be charged in the pH of the mobile phase and is less hydrophobic. Indeed, utilization of TEAA at

10 mM decreased retention times compared to TBA (Figure 2.6 versus 2.5) but produced sharp

peak shapes and did not suffer the carryover problems observed with TBA. Hence, TEAA was

utilized in the mobile phase. To decrease analysis time, flow rate was increased to 0.4 ml/min

from 0.2 ml/min and the gradient optimized for separation of NAD(P)H in 13.5 minutes total run

time. Gradient and mobile phase conditions are described in Table 2.7. NADPH and NADH

eluted at 3.34 and 3.69 minutes, respectively. NADH and NADPH are linear from 9 to 60 µM at

12 A stationary phase is the non-mobile portion of a chromatographic system that interacts with compounds carried in the mobile phase and causes the rate of their flow through the system to vary, thus separating the compounds from a complex mixture.

an injection volume of 10 µl and reproducible with a method RSD of 5 and 15%, respectively,

indicating the separation is suitably sensitive and reproducible for quantitation of NAD(P)H.

Extraction of these metabolites required the optimization of a different procedure from

that described for the oxidized NAD metabolome. First, deoxygenating of the extraction solvent

with nitrogen was essential. Second, the pH of the extraction solvent is adjusted to 9 and the

extraction solvent is kept at dry ice temperature throughout extraction to increase stability (61).

Third, nitrogen drying at ambient temperature is employed rather than drying with speed

vacuum. Fourth, multiple rounds of extraction are performed. Inclusion of these changes allows

for 100% recovery of the standards in the absence of tissue.

To extract NAD(P)H from liver, ~20 mg of frozen pulverized liver is sonicated (Branson

Sonifer 450, output control = 4, intensity = 40%, 10 seconds) in 0.5 ml of -80 °C methanol/25

mM ammonium acetate pH 9 (5:1) in a -4 °C acetone/water bath, then rested on dry ice. The

extract is heated at 60 °C using Thermomixer® (Eppendorf, Hamburg, Germany) for three

minutes with constant shaking at 1050 rpm then centrifuged (16.1k g, 10 minutes, 4 °C). The supernatant is transferred to a fresh tube and the pellet re-extracted two more times. The supernatants from each round of extraction are combined. Extracts are dried at ambient temperature using nitrogen gas and reconstituted in 50 mM ammonium acetate (>99%) and

0.05% ammonium hydroxide (>99%) in LCMS grade water immediately prior to analysis. The metabolites remain labile in this solvent and must be analyzed within 8 hours post reconstitution. Recovery of NAD(P)H in murine liver was estimated by spiking either water or

1000 pmol of both analytes into an aliquot of liver and calculated with the following equation:

X X 100 where K is the ratio of the weight of the liver aliquot dosed before processing to the weight of the liver aliquot dosed after processing. NADH and NADPH were recovered at 67 and 77%, respectively. To estimate signal suppression from the sample, the added signal of analyte in the

dosed after processing sample was compared to signal in fresh standard. Signal recovery was approximately 83 and 93% for NADH and NADPH, respectively. Since no reliable internal standard could be produced for NADH (Figure 2.3) and NADPH at the time of method development, a standard addition method was utilized whereby liver extracts were pooled, then aliquoted equally, and finally dosed with varying concentrations of standard. The pooled liver was aliquoted such that approximately 0.5 mg of liver was loaded at 10 µl injection. This amount of tissue is the same amount loaded from samples at 2.5 µl. This method allowed for control of ion suppression.

Though this method could be improved with the synthesis of internal standards, the work described in this section represents a robust and sensitive framework for future quantitative

NAD(P)H assays. Further, through method development, we discovered that TBA, an ion- pairing agent in well-known separations for organic acids (85, 86), causes extreme carryover of

NADPH that could not easily be overcome through reduction of the ion-pairing agent concentration in the mobile phase. The applicability of this effect to other metabolites should be explored. In cases where carryover is experienced, TEA may be an adequate substitution.

Quantification of the Oxidized NAD Metabolome in Skeletal Muscle

Muscle depends upon Nam and NR for NAD + biosynthesis (87). NAD + increase as a function of NR opposes myopathy (50, 88). Unlike liver, muscle is a dense tissue containing a tough actinomyosyin network. As a consequence, extracting the oxidized NAD metabolome from this tissue requires greater mechanical disruption and, like with NAD(P)H, requires heating to increase release of the metabolites. To extract quadriceps, ~20 mg of frozen pulverized tissue is sonicated as described for extraction of NAD(P)H in the presence of 0.5 ml ice cold methanol/water (4:5). The extract is heated for 5 minutes at 85 °C using a Thermomixer®

(Eppendorf, Hamburg, Germany) with constant shaking at 1050 rpm then centrifuged (16.1k g,

10 minutes, 4 °C). The supernatant is dried using a speed vacuum and reconstituted in 40 µl of

10 mM ammonium acetate (>99%) in LCMS grade water. LC-MS/MS conditions are as

described in Chapter 2.1. Recoveries were determined by comparing internal standard area counts in samples in reconstitution solvent (Table 2.8). All recoveries are above 60% except

CMP and NAR which are 38 and 31%, respectively.

2.3 Tables and Figures

Table 2.1 Alkaline separation gradient.13 Time (min) Solvent B (%) Column Volumes 0 5 - 1.8 5 1.8 14 54 12.4 14.1 90 - 17.1 90 3 17.2 5 - 32.2 5 15.3

Table 2.2 Acidic separation gradient.12 Time (min) Solvent B (%) Column Volumes 0 5 - 1.8 5 1 11.2 35.9 5.4 11.3 90 - 13.3 90 1.2 13.4 5 - 23.4 5 5.8

13 Table is a reprint from Trammell and Brenner (2013) (1).

Table 2.3 LCMS/MS SRM parameters, sensitivity, and robustness for each metabolite.14 a 2 d d Metab - Transition CE Cone RT k’ LOQ LOQ R RSD RSD olite (m/z) Volt. (min) this Evans Method System study (pmol) c (pmol) b Nam 123 > 96 16 32 9.82 6 0.6 0.45 0.996 2 1 NA 124 > 53 26 32 8.35 5 2.5 1.20 0.999 2 1 Cytidine 244 > 112 18 18 11.14 7.0 .01 0.1 0.995 22 16 Uridine 245 > 113 16 18 11.5 7.0 3.1 1.20 0.990 7 3 NR 255 > 123 12 14 8.98 5.4 0.01 0.2 0.998 4 2 NAR 256 > 124 13 14 10.33 6.4 0.1 0.06 .948 11 12 Inosine 269 > 137 12 12 12.88 8.2 0.03 0.07 0.974 10 5 CMP 324 > 112 22 16 2.97 1.1 0.09 0.68 1 9 1 UMP 325 > 97 12 20 4.11 1.9 0.06 0.21 0.990 6 4 NMN 335 > 123 12 16 8.92 5.4 1.0 0.5 1 8 3 NAMN 336 > 124 12 18 4.2 2 0.06 0.18 0.999 10 2 IMP 349 > 137 22 14 9.65 5.9 0.1 0.13 0.995 6 3 ADP 428 > 136 26 30 10.43 6.5 .03 NIR f 0.999 8 3 ATP 508 > 410 16 30 10.51 6.5 1.0 NIR f 0.995 11 3 ADPr 560 > 348 16 26 11.08 6.9 .02 NIR f 0.991 3 4 + NAD 664 > 428 26 26 13.64 8.7 0.19 0.17 0.999 8 2 NAAD 665 > 428 24 24 11.89 7.5 0.02 0.26 0.998 12 2 NADH 666 > 649 20 26 12.98 12 0.19 0.06 0.988 10 3 NADP 744 > 604 18 26 12.01 7.6 0.06 0.87 0.996 13 3 NADPH 745 > 729 48 28 acollision energy bLOQ of method described in this paper cLOQ of method in (31) eRSD expressed as percentage of the mean fNot included in report (NIR)

14 See footnote 12 on page 31.

Figure 2.1 Chromatograms of all 19 metabolites generated from injection of complex standard solutions using MRM LC-MS. 15 All compounds were detected in positive ion mode in alkaline (A) and acidic (B) separations. Multiple peaks observed in Uridine and NA illustrate cross talk from other metabolites in mixture. In the case of Uridine, the early eluting peak is the result of the 13 C peak of Cytidine. NA later eluting peaks are the result of 13 C peaks from Nam produced either from the Nam standard or on-source fragmentation of NR to Nam.

15 Figure is a reprint from Trammell and Brenner (2013) (1).

Table 2.4 NAD +/nucleotides metabolome of LN428/MPG cell line.16 Metabolite LN428/MPG Cells (µM) ATP 1010 ± 380 ADP 890 ± 150 UMP 370 ± 80 NAD + 260 ± 40 Inosine 250 ± 150 Uridine 210 ± 80 CMP 170 ± 70 IMP 98 ± 26 NADP 57 ± 10 Nam 39 ± 2 Cytidine 6.7 ± 4.4 NADH 6.7 ± 2.3 ADPr 6.7 ± 2.2 NMN 1.3 ± 0.3 NA <4.0 NR <0.016 NAMN <0.68 NAAD <0.24 NAR <1.1 NAD +/NADH 39

16 See footnote 12 on page 31.

Table 2.5 LCMS/MS SRM parameters, sensitivity, and robustness for methylated nicotinamide metabolites. a 2 d d Metabolite Transition CE Cone RT k’ LOQ LOQ R RSD RSD (m/z) Volt. (min) this Evans Method System study (pmol) c (pmol) b MeNam 137 > 94 20 8 3.47 1.5 0.0625 NIR 1 10 30 Me2PY 153 > 107 22 44 11.18 7.0 2.5 NIR 0.990 10 13 Me4PY 153 > 136 14 24 10.95 6.8 0.375 NIR 0.998 13 5 acollision energy bRSD expressed as percentage of the mean cNot included in report (NIR)

Figure 2.2 Chromatograms of the methylated nicotinamide species generated from injection of complex standard solutions using MRM LC-MS. All three compounds were detected in positive ion mode in the acidic separation described in Table 2.2.

Table 2.6 Recovery of murine hepatic oxidized NAD metabolome. ADPR, NAAD, NADP +, Me2PY, Me4PY, IMP, UMP were not determined due to a lack of internal standard. Metabolite Recovery (%) Cytidine 95 CMP 95 Inosine 58 MeNam 92 NA 53 NAD + 58 Nam 62 NAMN 69 NAR 70 NMN 50 NR 18 Uridine 89

13 Figure 2.3 NADH and its [ C10 ] isotopologue in murine liver sample. 13 NADH (300 pmol) and [ C10 ]-NADH produced from yeast extract (1:80 final dilution) were added to a murine liver sample, extracted as described in Chapter 2.2: Quantification of NAD(P)H and Extraction from Liver. Metabolites were separated using a Waters Acquity BEH C18 (2.1 x 100 mm) column with 10 mM TBA and 15 mM acetic acid in mobile phase A and methanol in mobile phase B and detected in SIM mode on a Waters TQD operated in negative 13 ion mode. Extracted ion currents for NADH (black line) and [ C10 ]-NADH (red line) are plotted on the left and right y-axis, respectively. RT: retention time

Figure 2.4 Hydrophobic ammonium salts cause intense ion suppression. NADH (125 pmol) was injected and separated using a Waters Acquity BEH C18 (2.1 x 100 mm) column with a gradient between mobile phase containing 10 mM TEA and 15 mM acetic acid as described in Table 2.7. Analytes were detected using a Waters Premier Q-TOF operated in positive ion mode with scanning from m/z 120 – 800. The spectra above were averaged over the retention time of NADH and are displayed in arbitrary units. We see that NADH ([M+H] = m/z: 666) forms an intense ion species with TEA (101 Da) to produce a peak with an m/z of 767. If scanning for the protonated m/z of NADH, the signal would be dampened by the adduct formed from the mobile phase. Negative ion mode is hence used in lieu of positive ion mode when including TEA or other hydrophobic ammonium salts in the mobile phase. TEA: triethylamine

Figure 2.5 Carryover of NADPH when TBA is used in the mobile phase. Solvent blanks (50 mM Tris pH 8) were injected after 30 (blue line), 100 (red line), and 300 pmol (black line) was injected on a Waters Acquity BEH C18 (2.1 x 100 mm) column with 10 mM TBA and 15 mM acetic acid in the mobile phase. NADPH was detected in negative ion, SIM mode on a Waters TQD. The extracted ion current for NADPH is displayed above. Carryover was noticed with increasing amounts of NADPH loaded, rendering this method unusable for quantitation of NADPH. RT: retention time; TBA: tributylamine

Figure 2.6 Extracted ion currents for NAD(P)H on a C18 column with TEA in the mobile phase. The chromatogram was produced after loading 100 pmol of NAD(P)H onto a Waters Acquity BEH C18 column (2.1 x 100 mm) and separated with 10 mM TEA and 15 mM acetic acid in the mobile phase with the gradient and conditions described in Table 2.7. Analytes were detected with a Waters TQD operated in negative ion, MRM mode (NADH: m/z 664>159 (solid line); NADPH: m/z 744>159 (dotted line)). Though MRM is less sensitive than SIM, sensitivity was non-limiting for detection of the standards in this experiment. RT: retention time; TEA: triethylamine

Table 2.7 Gradient for analysis of NAD(P)H. NAD(P)H is separated on a Waters Acquity BEH C18 column (2.1 x 100 mm) at a flow rate of 0.4 ml/min with column held at 25 °C with a gradient starting at 100% A (10 mM TEA and 15 mM acetic acid) and 0% B (LCMS grade methanol). Time (min) %B Column Volumes 0 0 - 1.25 0 1.4 2.5 20 1.4 3.75 20 1.4 6.5 55 3.2 7.75 95 1.4 9.25 95 1.7 9.3 0 0.06 13.5 0 4.8

Table 2.8 Recovery of the oxidized NAD metabolome in murine quadriceps. ADPR, NAAD, NADP +, Me2PY, Me4PY, IMP, Inosine, and UMP were not determined due to a lack of internal standard. Metabolite Recovery (%) CMP 38 Cytidine 84 MeNam 60 NA 77 NAD + 74 NADP + 92 NR 76 Nam 67 NAR 31 NAMN 83 NMN 89 Uridine 101

CHAPTER 3 NICOTINAMIDE RIBOSIDE IS A MAJOR NAD + PRECURSOR VITAMIN IN BOVINE MILK Samuel A.J. Trammell 1,2 , Liping Yu 1,3 , Philip Redpath 4, Marie E. Migaud 1,4 , and Charles Brenner 1,2

1 Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA

2 Interdisciplinary Graduate Program in Genetics, University of Iowa, Iowa City, IA

3 Nuclear Magnetic Resonance Facility, Carver College of Medicine, University of Iowa, Iowa

City, IA

4 Queen's University Belfast, School of Pharmacy, Belfast, Northern Ireland, UK

3.1 Distribution of Work

CB and I designed the research, analyzed the results and wrote the manuscript. LY performed the NMR measurements. I performed the LC-MS and LC-MS/MS with heavy standards synthesized by myself or by PR and MEM.

3.2 Abstract

Background: Nicotinamide riboside (NR) is a recently discovered nicotinamide adenine dinucleotide (NAD +) precursor vitamin with a unique biosynthetic pathway. Though the presence of NR in bovine milk has been known for more than a decade, the concentration of NR with respect to the other NAD + precursor vitamins was unknown.

Objective: We aimed to determine NAD + precursor vitamin content in raw samples of milk from individual cows and from commercially available bovine milk.

Methods: Liquid chromatography tandem mass spectrometry (LC-MS/MS) and isotope dilution technologies were used to quantify NAD + precursor vitamin content and to measure NR stability in raw and commercial milk. Nuclear magnetic resonance (NMR) spectroscopy was utilized to test for NR binding to substances in milk.

Results: Bovine milk typically contained ~12 m M NAD + precursor vitamins, of which 60% was

present as nicotinamide (Nam) and 40% was present as NR. Nicotinic acid and other NAD +

metabolites were below the limits of detection. Milk from samples testing positive for

Staphylococcus aureus contained lower levels of NR and Nam (Spearman R = -0.58 and -0.43,

respectively) and NR was degraded by Staphylococcus aureus . Conventional milk contained

more NR than milk sold as organic. Nonetheless, NR was stable in organic milk and exhibited an

NMR spectrum consistent with association with a protein fraction in skim milk.

Conclusions: The pellagra-preventive activity of bovine milk is likely a function of NR and

nicotinamide. Control of Staphylococcus aureus may be important to preserve the B 3 vitamin content of milk.

3.3 Introduction

One hundred years ago, pellagra was common in the rural American South. One of the

early treatments for pellagra was consumption of a pint and a half to two pints of bovine milk (89).

In 1937, nicotinamide (Nam) and nicotinic acid (NA) were identified as pellagra-preventive (PP)

factors (90, 91) and tryptophan (trp) was subsequently discovered as a molecule with PP activity

(92). Nam and NA, which are collectively termed niacin, contain a pyridine ring that can be

salvaged to form NAD + in two or three enzymatic steps, whereas trp is the de novo precursor of

NAD +, requiring 7 enzymatic steps (3). Largely because trp can be incorporated into protein,

oxidized as a fuel, and converted to many other metabolites such as serotonin, 50-60 mg of trp is

considered the niacin equivalent of 1 mg of Nam or NA. In addition, much of the niacin equivalent

in food is, in fact, NAD+ (93).

NAD + and its phosphorylated and oxidized derivatives, NADP +, NADH and NADPH are

essential hydride transfer cofactors in hundreds of oxidoreductase reactions and consumed

substrates of several classes of enzymes with activities required for DNA repair, gene expression,

regulation of energy metabolism, and calcium mobilization (2). NAD + is one of the most abundant

metabolites in the human body and is turned over at a rapid pace (87), requiring near constant replenishment. Hence, though pellagra is described as niacin deficiency, at a cellular level, pellagra is a disease of NAD + depletion as a result of diets deficient in NAD + precursors.

It has long been known that the NAD + precursors in milk include Nam (94) and Trp (95).

More recently, it has been discovered that milk also contains NR, another salvageable NAD +

precursor vitamin (7). Boosting NAD + levels with NR extends lifespan in yeast (32) and has been shown to prevent and treat metabolic (37, 50, 53, 54) and neurodegenerative (55, 56) conditions in mouse models. Though these studies suggest that dairy products as a source of NR could be beneficial to many aspects of human health, the amount of NR in milk has not been established.

In this work, we determine the complete B 3 vitamin content in individual and pooled commercial samples of bovine milk using a liquid chromatography tandem mass spectrometry (LC-MS-MS)- based method (1). Our results show that milk from Staphylococcus aureus (Staph a )-positive samples contained lower levels of NR and Nam, and that milk sold as organic milk contained lower levels of NR than conventionally sourced milk. Moreover, we show that NR is stable in milk, is bound by substances in milk, and that approximately 40% of the NAD + precursor vitamin content of bovine milk is present as NR.

3.4 Methods

Milk Quality and Herd Health Measurements

Milk flows and representative samples were obtained from 19 conventionally raised cows

using a Dairy Herd Improvement Association (DHIA) testing meter. Samples were dispensed into

2 ounce snap cap DHI vials containing liquid bronopol for analysis by Dairy Lab Services

(Dubuque, IA) of fat, protein, lactose, other solids, milk urea nitrogen and somatic cells (FOSS,

Denmark). Additional aseptic individual milk samples were obtained for bacterial analysis after

teats were sterilized with 70% ethanol prior to collecting 3 ml of milk from each teat into 12 x 75

mm culture tubes. All samples were frozen before further analysis. Blood agar culture plates were

inoculated with sample, then incubated at 37 °C, and evaluated for bacterial growth at 24 and 48 h. Bacterial growth was characterized by morphology and samples were subjected to confirmatory tests to identify genus and species.

Bovine Milk Sample Acquisition and Preparations

Nineteen milk samples from individual cows plus 8 skim milk samples (4 organic and 4

conventional) purchased in the Iowa City area were analyzed by LC-MS-MS. Two 50 µl aliquots

were extracted from each milk sample. Each aliquot was dosed with either solution A (18.75 pmol

18 18 18 of [ O1]-NR, 18.75 pmol of [ O1]-Nam, 18.75 pmol of [D 3, O1]-MeNam and 150 pmoles [D 4]-

NA) or solution B, a 13 C-labeled yeast extract at 1:50 dilution. Aliquots were extracted with 0.5 ml of 1.5% formic acid at room temperature. Each aliquot was vortexed for 10 s and then sonicated for 10 min in a bath sonicator. Aliquots were then centrifuged at a speed of 16,100 x g for 10 min at room temperature. Extracts were transferred to fresh 1.5 ml centrifuge tubes and dried overnight via speed vacuum. Recovery was greater than 90% for all metabolites of interest.

NMR Spectroscopy

NR 1H resonances were assigned with 1H/ 13 C two-dimensional heteronuclear multiple

quantum coherence (HMQC) and heteronuclear multiple bond coherence (HMBC) experiments.

NR binding to skim milk was analyzed using water-ligand observed via gradient spectroscopy

(WaterLOGSY) (96, 97). To analyze fractions of milk for NR-binding activity, 2 ml of total milk

was centrifuged for 1 h at 4 oC at 16,100 x g. The supernatant was termed the soluble fraction, while the pellet resuspended in 2 ml 50 mM sodium phosphate pH 7 was termed the particulate fraction. NMR samples were prepared by adding 150 µl skim milk, skim milk soluble fraction, or resuspended skim milk particulate fraction to 352.2 µl buffer that contained 300 µl 50 mM sodium phosphate (pH 7), 50 µl D 2O, and 2.2 µl NR stock, giving a final NR concentration in the

NMR samples of 0.3 mM. For the WaterLOGSY experiment, a T 2 relaxation filter of 100 ms was

used just before data acquisition to suppress signals derived from macromolecules, and a water

Nuclear Overhauser Effect (NOE) mixing time of 1 s was used in the experiment. All NMR data were acquired using a Bruker Avance II 800 MHz NMR spectrometer equipped with a sensitive cryoprobe and recorded at 25 oC. The 1H chemical shifts were referenced to 2,2-dimethyl-2- silapentane-5-sulfonate (DSS). NMR spectra were processed using NMRPipe package software

(98) and analyzed using NMRView (99).

NR Stability Assays

18 13 [ O]-labelled and [ C1,D 1]-NR were synthesized as described (65)(Trammell, et. al. in preparation). [ 18 O]-NR was suspended in conventional and organic milk brands or in water at pH

5, 7, or 11 at a concentration of 10 µM and allowed to sit at room temperature. Twenty µl aliquots

were collected at 0, 0.5, 1, 2, 4, and 8 h and extracted as described above.

Staph a Growth Experiments

Strain RN3170 was a kind gift of Patrick Schlievert (University of Iowa) (100). Bacteria

were streaked onto Todd-Hewitt (Becton & Dickinson) 2% agar plates, incubated overnight at

37 °C and then stored at 4 °C. Staph a was then inoculated into Todd-Hewitt media containing 50

13 mM Bis-Tris pH 6.7 and 10 µM of [ C1,D 1]-NR at a starting OD 600 nm of 0.1. Non-inoculated

medium was used as control for NR stability. All cultures were incubated at 37 °C with constant

shaking at 220 rpm. Fifteen ml aliquots were collected at 0, 1, 2, 4, 6, and 8 h. OD 600 and pH

values were recorded at each time point. Aliquots were centrifuged at 2,060 x g for 30 min at 4 °C

at which time, 1 ml of culture medium was collected and snap-frozen in liquid nitrogen. The

remainder of culture medium was aspirated and cell pellets were washed with 1 ml of ice-cold

PBS and recentrifuged at 16,100 x g for 10 min at 4 °C. PBS was aspirated and pellets were flash

frozen. Fifty µl of media were analysed using LC-MS/MS as described below. Cells were extracted

using buffered ethanol (3 parts ethanol to 1 part 10 mM HEPES, pH 7.1) heated to 80 °C for 3

min with constant shaking at 1,050 rpm. Extracts were clarified by centrifugation (16,100 x g, 10

min, 4°C). Pellets were extracted again following the same procedure as above. Supernatants

from both rounds of extraction were combined and dried via speed vacuum. Extracts were analysed using LC-MS as described below.

LC-MS and LC-MS/MS

Media samples were diluted 1:1 with double distilled H 2O. Standard solutions in double distilled H 2O were diluted 1:1 with non-inoculated Todd-Hewitt media containing 50 mM Bis-Tris

13 and [ C1, D 1]-NR producing a standard curve with the final concentrations of 0, 0.1, 0.3, 0.5, 1,

3, 5, and 10 µM. Quality control samples at a final concentration of 0.75 and 7.5 µM were also prepared by diluting standard 1:1 with media. Ten µl of media samples, quality controls, and standards were injected and quantified using a Waters TQD mass spectrometer using the acid separation chromatographic conditions described previously (1). Media were quantified using raw peak areas and converted to µM using background-subtracted standard curves.

For bovine milk, standards (final concentrations of 0.08, 0.24, 0.8, 2.4, 8, 24, 80, 120 µM) and two quality control samples (final concentration of 2.5 and 25 µM) were treated in the same manner as the samples and as described above. Five µl of samples, quality controls, and standards containing solution A or 10 µl of those containing solution B were loaded onto the column and quantified using a Waters TQD mass spectrometer according to the procedures previously described (1). Newly quantified metabolites in the acidic separation, MeNam, Me2PY, and Me4PY were assayed with the following transitions: MeNam (137 > 94 m/z), Me2PY (153 >

107 m/z), and Me4PY (153 > 136 m/z). Analyte peak areas were normalized to internal standard peak areas and converted to µM using the standard curve. Staph a cell pellets were suspended

in 50 µl of 10 mM ammonium acetate in LCMS grade water. A 260 nm values for each sample were

measured using a Thermo Scientific 2000c Nanodrop spectrophotometer operated in nucleic acid

mode. Samples at 0 and 1 h time points were diluted 1:1 with either solution A or solution B.

Samples at all remaining time points were diluted to a final A 260 nm value of 14 then diluted 1:1 with solution A or B. All samples were analyzed according to the chromatography protocols previously described (1) and detected and quantified using a Waters Premier QTOF operated in positive full

scan mode. The alkaline separation was altered by increasing the flow rate to 0.55 ml/min and shortening the run time to 11.6 min. Separation was performed using a modified gradient with initial equilibration at 3% B, a 0.9 min hold, a gradient to 50% B over 6.3 min, followed by a 1 min wash at 90% B, and a 3 min re-equilibration at 3% B. When performing the alkaline separation, the scanning window was set to m/z = 120 – 800 with a scan rate of 0.1 and an interscan rate of

0.01. When performing the acid separation, the scanning window was set to m/z = 120 – 600 with a scan rate of 0.5 and an interscan delay of 0.05. In both cases, leucine enkephalin was infused and utilized for mass accuracy correction. Analyte peak areas were normalized to internal standard peak areas and converted to µM using the standard curve. Nam concentrations were

12 13 18 corrected for the contribution of [ C1]-Nam and [ C1]-Nam to the [ O1]-Nam internal standard

area counts. Enrichment for all metabolites was corrected for the natural abundance of the

13 13 13 analyte, C abundance, and the purity of the doubly labeled NR (95/5% [ C1, D 1]-NR/[ C1]-NR).

The corrected concentrations of each analyte were converted to intracellular concentrations by calculating the total intracellular volume of Staph a using an intracellular volume of 0.28 fl (101)

9 and an assumption of 1 x 10 cells/ml per OD 600 nm (102).

Statistical Analysis

Unless otherwise stated, all values are expressed as mean ± standard deviation. Two-

tailed, unpaired t-tests were performed on all comparisons involving fewer than three groups.

Outliers were identified using the ROUT method (103). Two-way, repeated ANOVA followed by

Holm-Sidak’s multiple comparisons test was performed on experiments involving Staph a . Media

samples were compared to non-inoculated media within time points. Intracellular samples were

compared to initial concentrations within condition. Spearmen’s rank correlation coefficient was

calculated for the concentration of Nam and NR versus the milk quality and herd health metrics.

P-values < 0.05 were considered significant. Statistical analyses were performed using GraphPad

Prism version 6.00 for Windows (GraphPad Software, CA).

3.5 Results

NR is a Major Component of the B 3 Vitamin Content in Bovine Milk

The B 3 vitamin content of 19 individual bovine milk samples was determined using LC-

MS/MS and isotope dilution techniques. We define the B 3 vitamin content as the levels of

salvageable NAD + precursor vitamins (Nam, NA, and NR) plus levels of the higher molecular

weight species (NAR, NAMN, NAD +, NAAD and NADP +) from which a vitamin can be released by enzymatic or chemical decomposition. NADH and NADPH are oxidized in extraction, such that these metabolites, if present, would contribute to the peaks of NAD + and NADP +.

As shown in Table 3.1, in all 19 samples, Nam and NR and no other NAD + metabolite were quantifiable. Thus, neither NAD + nor NA is a PP factor in milk. Excluding one unusual milk sample which contained 24 µM Nam and 27 µM NR (Supplemental Table 3.1), the mean sample contained 7.3 ± 1.5 µM Nam and 4.3 ± 2.6 µM NR. To determine whether other parameters correlate with NAD + precursor vitamin contents in the 18 remaining samples, breed was recorded

and, metrics of the health and milk quality of each cow were measured (Supplemental Table 3.2).

As shown in Table 3.2, levels of NR positively correlated with levels of lactose ( P = 0.013) and

milk urea nitrogen ( P = 0.018), whereas Nam negatively correlated with somatic cell count ( P =

0.029) and positively correlated with NR ( P = 0.011). NR concentration negatively correlated with

Staph a infection ( P = 0.014). Nam concentration also negatively correlated with Staph a infection

but the correlation was not significant ( P = 0.09). When we recalculated levels of NR and Nam in

the 12 samples without Staph a infection or extremely high levels of NAD + precursor vitamins,

Nam rose to 7.7 ± 1.2 µM and NR rose to 5.1 ± 2.6 µM. Thus, though it was clear from previous work that there is no NA in bovine milk (94), there has been a substantial under-reporting of NAD +

precursor vitamin on account of lack of an assay for NR.

Staph a Depletes NR and Nam

Because presence of Staph a was associated with lower levels of NR and Nam, we tested

whether Staph a growth might directly alter the levels of these metabolites in rich media. Before

testing stability in the presence of Staph a , we investigated the stability of [ 18 O]-NR in pasteurized

bovine milk or in water adjusted to pH values of 5.0, 7.0, and 11.0. As shown in Figure 3.1A, [ 18 O]-

NR was stable in pasteurized milk and in water at neutral pH, but exhibited lesser stability at pH

5.0 and pH 11.0, with pH 11.0 producing nearly complete hydrolysis within 1 h. We measured the pH of bovine milk in 4 store-bought milk brands and determined the pH to be 6.72 ± 0.01.

Bacteria might alter levels of NR found in milk by incorporating NR intact into NAD + and/or

by converting NR into Nam or NA, either of which could be subsequently incorporated into NAD +.

To distinguish between these possibilities, we synthesized a double-labeled NR containing a 13 C

+ in the Nam moiety and a D 1 in the ribose. Incorporation of NR into the bacterial NAD pool would be accompanied by a 2 Da mass shift (m/z 664 → 666), whereas breakdown of NR to Nam or NA would be accompanied by appearance of 1 Da shifts in the peaks of these metabolites and a 1

Da mass shift in bacterial NAD +.

Three individual colonies of Staph a strain RN3170 were cultured separately in Todd-

13 Hewitt media supplemented with 10 µM [ C1, D 1]-NR and buffered at pH 6.7 with 50 mM Bis-Tris.

The inoculated media and a non-inoculated medium control were incubated at 37 °C with constant

shaking over an 8 h period. Clarified media and cell pellets were collected and analyzed by LC-

MS/MS and LC-MS. The pH of the clarified media was also recorded at each time point. The pH

consistently remained between 6.5 and 6.7 until between the 6 and 8 h time points, at which time

13 the pH rose to 7.8. As shown in Figure 3.1B, [ C1, D 1]-NR was stable in non-inoculated medium

over the time course of the experiment. However, Staph a inoculation significantly decreased the

concentration of extracellular NR within 1 h and eliminated the presence of NR as an extracellular

metabolite within 4 h. As shown in Figure 3.1C, singly labeled Nam appeared in growth media

within 1 h. As shown in Figure 3.1D-F, at 4 h, there was a simultaneous rise in singly labeled cellular NAD +, singly labeled cellular Nam, and extracellular NA.

Todd-Hewitt media contains beef heart extract, Nam and NA (104). As shown in Figure

3.1G and 3.1H, non-labeled Nam was exhausted within 4 h while the non-labeled NA slowly declined. Thus, Staph a principally uses NR as an extracellular source of Nam. Consistent with

Nam deamidation (105, 106), Staph a can also degrade NR and Nam to NA. Because NR was

eliminated by 4 h and the rise in pH occurred after 6 h, pH-mediated mechanisms cannot be

responsible for Staph a -mediated NR instability.

NR Content as a Function of Organic Certification

Milk with organic certification requires avoidance of synthetic chemical inputs, irradiation,

genetically modified seed, and adherence to certain standards of feed, housing and breeding

(107). Because one or more of these variables could affect the B3 vitamin content of milk, we

purchased 4 brands of conventional and 4 brands of organic milk and quantified the NAD +

metabolome. As observed in milk samples from individual cows, only Nam and NR were above

the limit of quantification (Table 3.3). In three of four conventional samples and three of four

organic samples, the concentration of Nam exceeded that of NR. Moreover, the concentration of

Nam was similar in conventional (5.2 ± 3.4 µM) and organic (5.6 ± 2.5 µM) milk. In the samples

we obtained, NR tended to have a higher concentration in conventional (3.1 ± 1.6 µM) versus

organic (1.9 ± 1.0 µM) milk. We note that only one brand of store-bought milk had B 3 vitamin

content (8.9 µM Nam plus 5.4 µM NR) that was higher than the mean of Staph a -negative

individual cow samples (7.7 µM Nam plus 5.1 µM NR). Though the difference between

conventional and organic milk in NR concentration did not rise to statistical significance (P = 0.26),

the data suggest that a difference in the feeding and care of cows or milk preparation might

depress NR levels in organic store-bought milk.

NR is a Bound Metabolite in Bovine Milk

Though a higher level of Staph a infection in organic milk production (32) could potentially

account for lower levels of NR in organic milk, there was no change in Nam and no appearance

of NA that would be consistent with bacterial exposure. As shown in Figure 3.1, NR is more stable

in milk than in water, suggesting that the metabolite might be complexed to a protective factor.

Organic dairies frequently ultrapasteurize milk at 135 oC for 2 sec, whereas most

conventional dairies pasteurize at 72 oC for 15 sec (108). Though ultrapasteurization is employed to kill bacterial spores, it might damage a macromolecule responsible for the stabilization of NR.

WaterLOGSY NMR measurements were employed to detect and map protons in NR that are potentially bound by slower rotating macromolecules in milk (96, 97). As shown in Figures 3.2B and 3.2F, when NR was added to conventional or organic skim milk, four aromatic protons (H2,

H4, H5 and H6) from the Nam moiety of NR produced positive WaterLOGSY signals consistent with protein-binding from both sources of milk. Interestingly, when conventional and organic milk were separated into soluble and resuspended particulate fractions, the conventional soluble fraction retained more NR-binding activity than did the organic soluble fraction (Figures 3.2C and

3.2G). Consistent with denaturation of an NR-binding protein by heat, the solubilized organic particulate fraction produced stronger NR WaterLOGSY signals than did the solubilized conventional particulate fraction (Figures 3.2D and 3.2H).

3.6 Discussion

The data presented in this paper show that about 60% of the B 3 vitamin content of bovine milk is present as Nam, while about 40% is present as NR. Though we occasionally detected bovine milk samples with higher levels of NR than Nam, we did not detect NAD +, NA or any other

NAD + metabolite in any bovine milk sample. In samples from individual cows, presence of Staph

a, the most common cause of cattle mastitis (109, 110), was associated with lower concentrations of NR and Nam. We also showed that Staph a degraded NR into Nam and NA and used the Nam

as a precursor of intracellular NAD +. These data are consistent with the ability of Staph a to utilize either Nam or NA for NAD + synthesis (111) with Nam utilization requiring deamidation to NA (106,

112).

Multiple aspects of bovine nutrition are expected to contribute to levels of Nam and NR in

milk. In particular, feeding of herds with Nam and NA (113, 114) is likely to produce cows that

transmit higher levels of Nam and NR into milk. In addition, it will be interesting to determine

whether NR is actively transported by mammary glands (115).

Though PP factors in food include NAD +, NAD + precursor vitamins and Trp, high doses of

NR appear to have some protective activities for metabolic and neurodegenerative conditions.

The ability of milk to bind and preserve the integrity of NR makes dairy products potentially good sources of supplemented NR. Further research is needed to maximize NR content of conventional and organic milk and to identify the molecular basis of NR binding to milk.

3.7 Tables and Figures

Table 3.1 Mean NAD + metabolomes of 18 raw bovine milk samples. Values are expressed as mean ± SD µM. Concentration Total Staph a- Staph a- (µM) (n = 18) negative positive (n = 12) (n = 6) Nam 7.3 ± 1.5 7.7 ± 1.2 6.4 ± 1.7 NR 4.3 ± 2.6 5.1 ± 2.6 2.7 ± 1.9 1 NA <1.0 <1.0 <1.0 NMN <0.4 <0.4 <0.4 NAD + <0.08 <0.08 <0.08 NaR <0.04 <0.04 <0.04 NADP + <0.02 <0.02 <0.02 NaAD <0.008 <0.008 <0.008

Table 3.2. Correlation coefficients between B 3 vitamin concentrations and milk quality. Spearman correlation coefficients were determined between NAD+ precursor vitamins and milk quality and cow breed using data from Supplemental Table 1 (except cow 3) and Supplemental Table 2. Spear - Fat Protein Lactose Non -fat Total Somatic Cell Milk Urea Staph a- NR man’s (%) (%) (%) Solids Solids Count Nitrogen positive (µM) rho (%) (%) (x10 3 cells/ml) (mg/dl) NR (µM) -0.14 -0.21 0.58 * -0.13 -0.08 -0.38 0.55* -0.57 * Nam (µM) -0.39 -0.13 0.50 -0.07 -0.30 -0.52* 0.27 -0.41 0.58 * * p-value < 0.05

A B mol/L) ( O]-NR Stability O]-NR 18 Intracellular Nam Intracellular [ (Fold Change of 0 h) 0 of Change (Fold

C D mol/L) mol/L) ]-NR( 1 ]-NA ( ]-NA 1 ,D Extracellular Extracellular C 1 13 C [ 13 [

E F mol/L) mol/L) ]-Nam ( NA ( NA 1 Extracellular C 13 [ Extracellular Non-Labeled Extracellular

G H + mol/L) mol/L) ( Nam ( Intracellular NAD Intracellular Extracellular Non-Labeled Extracellular

Figure 3.1. NR is stable in milk and is degraded by Staph a . 18 A. Stability of [ O1]-NR in four store-bought brands of skim milk and in water at pH 5, 7, and 11 13 (n = 3) was assessed using LC-MS/MS. NR metabolism by Staph a was determined using [ C1, 13 D1]-NR and LC-MS (B – H). B – F. Extracellular concentration of [ C1, D 1]-NR, non-labeled 13 13 Nam, [ C1]-Nam, non-labeled NA, and [ C1]-NA in Todd-Hewitt + 50 mM Bis-Tris pH 6.7 media 13 containing 10 µM [ C1, D 1]-NR incubated at 37 °C without or with Staph a RN3170 inoculation (n = 3 for inoculated media). In each panel, the concentration of the metabolite is shown in the 13 non-inoculated media (n = 1). G. Intracellular concentration of endogenous or C1 enriched 13 13 + Nam. H. Intracellular concentration of endogenous C1, or C1, D 1 enriched NAD from extracts of Staph a RN3170 cell pellets (n = 3 per time point). For intracellular measurements, a Holm- Sidak multiple comparisons post-hoc test was performed to test for statistical significance compared to time point zero for each metabolite (B and G). For extracellular measurements, a

Figure 3.1 – continued

Holm-Sidak multiple comparisons test was performed to test for statistical significance compared to non-inoculated medium within each time point (C – F and H). Data are represented as mean ± SEM. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Table 3.3. Vitamin B 3 content in store bought bovine milk. Average values expressed as mean ± SD µM, n = 4. Organic Conventional Metabolite Brand Brand Brand Brand Average Brand Brand Brand Brand Average (µM) A B C D A B C D Nam 2.4 7.1 5.0 7.9 5.6 ± 2.5 5.6 0.67 8.9 5.4 5.2 ± 3.4 NR 3.1 0.84 2.2 1.4 1.9 ± 1.0 2.5 1.7 5.4 2.7 3.1 ± 1.6

Figure 3.2. NR-binding to milk demonstrated by NMR. Organic and conventional skim milk was separated into soluble and resuspended particulate fractions. NR was added to the non-fractionated milk and to the fractions. NR-binding was analyzed using NMR as described. A and E: normal 1D 1H NMR spectra of NR. B-D and F-H: WaterLOGSY spectra of NR in the presence of milk. A: NR alone, B: NR + total conventional milk, C: NR + conventional milk (soluble fraction), D: NR + conventional milk (particulate fraction), E: NR alone, F: NR + total organic milk, G: NR + organic milk (soluble fraction), H: NR + organic milk (particulate fraction). The assigned 1H resonances of the Nam ring aromatic protons are labeled.

3.8 Supplemental Tables

Table 3.4 NAD + precursor concentrations in 19 individual milk samples . These raw data were averaged in Table 3.1. Cow NR Nam (µM) (µM) 1 5.4 8.2 2 4.3 7.8 3 27 24 4 4.7 7.5 5 3.6 9.3 6 6.8 8. 4 7 6.6 9.2 8 2.5 6.9 9 5.3 9.0 10 0.80 5.2 11 11 6.7 12 7.0 7.1 13 2.9 7.3 14 6.0 8.8 15 2.6 5.9 16 1.9 4.6 17 0.96 4.5 18 1.1 7.0 19 3.6 7.6

Table 3.5. Individual milk quality assessments and breed. These data were used for analysis in Table 3.2. Cow Breed Fat Protein Lactose Non - Total Somatic Cell Milk Urea Staph a (%) (%) (%) fat Solids Count Nitrogen positive Solids (%) (x10 3 (mg/dl) (%) cells/ml) 1 Holstein 4.7 3.22 5.03 8.87 13.66 22 24.4 0 5 2 Ayrshire 4.4 3.37 4.63 8.65 13.19 15 22.7 0 9 4 Guern- 4.9 3.34 4.89 8.82 13.8 1310 16.4 1 sey 2 5 Ayrshire 3.8 3.36 4.75 8.75 12.62 18 21.7 0 3 6 Ayrshire/ 4.5 3.09 4.67 8.44 13.02 307 26 0 Red Poll 3 7 Jersey 4.0 3.62 4.78 9 13.11 39 17.1 0 8 8 Ayrshire 4.1 3.79 4.43 8.88 13.11 302 19.9 1 9 9 Ayrshire 3.1 3.22 4.86 8.74 11.89 19 23.8 0 2 10 Jersey 6.3 4.46 4.6 9.64 16.08 176 15.4 1 7 11 Ayrshire 5.0 3.3 4.71 8.64 13.76 43 22.9 0 5 12 Ayrshire 4.3 3.59 4.87 9.08 13.53 36 22.4 0 9 13 Jersey 5.5 3.89 4.81 9.28 14.85 25 17.3 0 1 14 Ayrshire 5.5 2.78 4.77 8.2 13.85 45 22.2 0 7 15 Jersey 5.2 3.07 4.66 8.36 13.65 218 24.2 0 3 16 Jersey 5.8 4.64 4.72 9.92 15.86 93 17.5 1 6 17 Ayrshire 4.2 2.7 4.64 8 12.33 641 19.5 0 8 18 Ayrshire 3.3 3.13 4.59 8.38 11.72 407 16.2 1 3 19 Jersey 5.2 3.82 4.72 9.15 14.48 215 18.7 1 8

CHAPTER 4 EFFICACY OF NMN AND NR AS EXTRACELLULAR NAD + PRECURSORS Samuel A.J. Trammell 1,2 , Marcelo Rodrigues 3, Allyson Mayer 1, Lori J. Manzel 1, and Charles Brenner 1,2

1 Department of Biochemistry, Carver College of Medicine, University of Iowa, Iowa City, IA

2 Interdisciplinary Graduate Program in Genetics, University of Iowa, Iowa City, IA

3 Queen's University Belfast, School of Pharmacy, Belfast, Northern Ireland, UK

4.1 Distribution of Work

Experiments were designed by CB and I. The following chapter was written completely by myself. Cell culture work was performed by LM, AM, or MR with guidance from me. All mass spectrometry and data analysis was performed by myself.

4.2 Abstract

The novel NAD + precursor nicotinamide riboside (NR) opposes age and diet induced morbidities such as obesity and diabetes. Nicotinamide mononucleotide (NMN), phosphorylated

NR, is also effective against diabetic effects. Though both increase intracellular NAD + concentration upon administration to rodents, NMN internalization into cells has not been demonstrated. Mounting genetic and pharmacological evidence suggests NMN is dephosphorylated to NR prior to its intracellular utilization. However, these methods rely upon indirect measurement and does not necessarily exclude whether NMN and NR are kinetically equivalent in supplementing NAD +. In this work, we show through LC-MS/MS and stable isotope enrichment that NMN is dephosphorylated extracellularly to NR and that NR is incorporated into

+ the intracellular NAD faster than NMN and as such NR is a more efficacious B 3 vitamin in cell culture.

4.3 Introduction

Nicotinamide adenine dinucleotide (NAD +) is a cofactor in hydride transfer reactions and consumed substrate for ADP ribose transferases, poly-ADP ribose transferases, cyclic ADP

ribose synthases, and sirtuins (Chapter 1.2). Through these reactions, NAD + is involved in many cellular processes such as gene expression, fuel utilization, DNA repair, protein modification, and cell signaling. NAD + is synthesized either through the de novo pathway from tryptophan or

salvage of nicotinic acid (NA), nicotinamide (Nam), and nicotinamide riboside (NR) (Figure 1.1)

(2, 7). In vertebrates, Nam phosphoribosyltransferase (Nampt/Visfatin/PBEF1) converts Nam to

nicotinamide mononucleotide (NMN) using 5-phosphribosyl-1-pyrrophosphate as a

phosphoribose donor (16). NR is phosphorylated to NMN by either Nrk1 or Nrk2 (7, 33). NMN is

then converted to NAD + through one of three NMN adenylyltransferases (NMNAT1-3).

Both NR and NMN oppose diet and age induced diabetes (34, 37, 116, 117). The

overlapping beneficial effects of these two NAD + precursors suggests utilization of the same pathway. Indeed, both have been shown to increase intracellular NAD +. Both have been suggested to circulate with NMN at ~80 µM (17) and NR at an unknown concentration (Chapter

5). However, way in which NMN contributes to intracellular NAD + remains debatable. NR

depends upon equilibrative nucleoside transporters (2). Though NMN is a nucleotide and not

normally predicted to cross the plasma membrane, NMN has been suggested to enter into cells

and across the highly selective blood brain barrier (118). To date, no NMN transporter has been

identified but could depend upon a similar uptake mechanism to that found in astrocytes (119).

If NMN were directly imported, its utilization would be expected to depend upon NMNAT1-3 but

not on the NRK pathway, the pathway converting NR to NMN. However, pharmacological and

genetic interventions suggests NMN depends upon NRK (35, 120, 121), CD38 (122), and the

predicted NMN extracellular nucleotidase (2), CD73, calling into question whether NMN is

extracellular available or simply an NR or Nam prodrug (Figure 4.1).

Though current data is highly suggestive that NMN utilization requires dephosphorylation

by CD73 to NR or hydrolysis to Nam by CD38, these studies do not preclude that nor tested

whether NMN and NR are equally efficacious as precursors. The rate of NMN

dephosphorylation could be non-rate limiting to its utilization, rendering NMN and NR

equivalently efficacious. We sought to directly test efficacy of NMN versus NR as salvageable

NAD + precursors using stable isotope labeling and LC-MS/MS. In so doing, we show direct

evidence that NR is a more efficacious precursor and confirm metabolite-metabolite

relationships in a human hepatocyte cell line.

4.4 Materials and Methods

Compounds

18 [ O1]-NR was made as described in (65). Labeled NR was phosphorylated to NMN

using Nrk1 and ATP. NMN was purified by HPLC on a strong anion exchange column with a 10

mM to 750 mM gradient of KH2PO4 [pH 2.6]. Isotopic purity was assessed by LC-MS and

concentration was assessed using an extinction coefficient (260 nm) of 4200 M-1*cm-1.

Cell Culture Conditions

Human hepatocyte cell line Hepg2 cells were procured from ATCC. For all labeling

experiments, cells were grown in Eagle’s minimum essential medium (MEM) containing

glutamine (0.292 g/l) and 10% fetal bovine serum to 75% - 80% confluency. Media was

aspirated and replaced with media lacking nicotinamide and fetal bovine serum. Cells were

incubated at 37 °C for 24 hours before aspiration and repletion with MEM containing either 10

18 18 µM [ O1]-NMN or [ O1]-NR. Media and cells were collected at 0, 0.5, 2, 4, 7, and 24 hours. At

collection time, 1 mL of media was collected and the rest aspirated. Cells were trypsinized for

10 minutes at 37 °C, pelleted, washed with ice cold PBS, pelleted, and then flash frozen in liquid

nitrogen. All samples were stored at -80 °C until analysis.

Extraction

Cells were extracted as described in Chapter 2.1 using the buffered boiled ethanol

method. Media were diluted 1:1 with LC-MS grade water containing internal standard (10 μM

cytidine) and injected without further preparation for analysis.

LC-MS/MS

LC-MS/MS was performed as described in Chapter 2.1. Transitions were established for

18 O labeled NAD + metabolites: 18 O Nam (125>98 m/z ), NA (126>53 m/z ), NR (257>125 m/z ),

NAR (258>126 m/z ), NMN (337>125 m/z ), NAMN (338>126 m/z ), NAD + (666>428 m/z ), NAAD

(667>428 m/z ), NADH (668>651 m/z ), and NADP (746>604 m/z ). Enrichment was determined by dividing 18 O labeled metabolite peak areas by 16 O labeled metabolite peak areas

(Tracer/Tracee ratio). The ratios were corrected by subtracting the Tracer/Tracee ratio at each time point by the initial ratio (Tracer/Tracee ratio at time point zero). To ensure that this method was accurate, cells supplemented with the same concentration of non-labeled NR or NMN were grown in parallel with cells supplemented with isotopologues. At each time point, the

Tracer/Tracee ratio was corrected by subtracting the ratio found in heavy labeled fed cells by the ratio found in non-heavy labeled cells. These ratios were compared to correcting the ratio with the initial time point. Ratios were not significantly different between conditions (data not shown).

4.5 Results

NMN is Dephosphorylated Extracellularly and Contributes to the Intracellular NAD + Pool Slower

than NR

18 18 Hepg2 cells were grown in the presence of either [ O1]-NR or [ O1]-NMN at a

concentration of 10 µM after a 24 hour B 3 vitamin starvation. Given that labeled NMN nor NR

was not detectable at time point zero, any detectable heavy labeled NMN, NR, or other labeled

metabolite is derived from the supplemented heavy precursor. Intracellularly, Tracer/Tracee

ratios were determined as a corrected ratio of heavy-to-light metabolite peak areas. Overall, the

rate of enrichment was greater when cells were fed labeled NR compared to NMN with NAD + enrichment reaching 2.6 ± 0.3 compared to 0.91 ± 0.04 after 24 hours (P < 0.01), suggesting

NR is more efficiently utilized intracellularly to produce NAD + (Figure 4.2a and 4.2b).

Extracellular labeled NMN, NR, NA, and Nam were quantified. Labeled Nam nor labeled NA were not detected upon feeding with NMN or NR (Figure 4.2c and 4.2d), revealing NMN and NR are likely not greatly hydrolyzed in the presence of these cells. NR slowly decreased in concentration from 11 ± 0.84 µM at 30 minutes post incubation to 2.3 ± 1.2 µM after 24 hours (P

< 0.01)(Figure 4.2b). Strikingly, NMN dramatically decreased over the 24 hours falling from 10 ±

0.97 to 0.34 ± 0.01 µM (P < 0.001)(Figure 4.2d). Concurrent with this decrease, labeled NR rose in a nearly linear fashion compared to the disappearance of labeled NMN (Figure 4.2d).

Intracellular enrichment of NR slowly rose with the appearance of its extracellular pool after

NMN feeding, but spiked after NR feeding. Together, these findings reveal NMN is dephosphorylated extracellularly and indicate this process is rate-limiting for its intracellular utilization. NR was kinetically superior in enriching the intracellular NAD metabolome compared to NMN.

4.6 Discussion

NR (32, 37, 50, 52, 54-56) and NMN (34, 123-125) both counter metabolic and age

related disorders. NR and NMN are studied as separate pharmacological entities, both

augmenting NAD + through separate pathways but converging due to the action of NAD + in sirtuin activities. NR is either phosphorylated to NMN through the NRK pathway (7) or phosphorylized to Nam (126). However, NR increases five-fold after NMN injection (34) and other studies indicate NMN depends upon CD38, CD73, and NRK (35, 121, 127) for its utilization and suggest that NMN is metabolized to Nam and NR extracellularly. These studies did not eliminate the possibility that extracellular dephosphorylation is non-rate limiting and that

NMN, though biochemically acting as NR, behaves identically as NR. We used LC-MS/MS and stable isotope labeled NR and NMN to measure their kinetic effect on the NAD metabolome. NR contributed to the intracellular NAD metabolome more rapidly than NMN and increased NAD + by

more than 2 fold after 24 hours, indicating NR is kinetically superior to NMN. In line with the

intracellular findings, extracellular NMN rapidly and dramatically decreased over 24 hours as extracellular NR rose. No labeled Nam was detected, suggesting hydrolysis, presumably as catalyzed by CD38, was not the primary route of extracellular NMN metabolism in these cells.

The relationship of the two labeled compounds was seemingly linear and agreed with genetic evidence that NMN is dephosphorylated before it is salvaged. NR and NMN are not identical, interchangeable entities.

The implication of these data to a biological setting remain to be shown in vivo . The possibility remains that NMN is an endogenous circulating NAD + precursor as has been

suggested (17). An extracellular Nampt (eNampt) is enzymatically active and could produce a

constant amount of NMN from Nam. In the same report, NMN was reported to circulate at as

high as 80 µM, leading some to suggest that NMN is a type of NR carrier that is

dephosphorylated to supply NR around the body (2). However, the ability of eNampt to produce

NMN appears unlikely given undetectable levels of its co-substrate 5-phosphribosyl-1-

pyrrophosphate (18). Further, our group and others have failed to detect NMN in plasma

(Chapter 5)(18). These discordant results may be due to the employment of HPLC versus LC-

MS. HPLC is technically more quantitative as the detector is not at the mercy of gas phase

reactions such as mass spectrometers are, but HPLC UV-vis methods are incapable of

providing selectivity for the metabolite of interest (Chapter 1.1-1.2). This is a very prescient

example of the need for improved and accepted analytical procedures for the study of the NAD

metabolome.

4.7 Figures

Figure 4.1 Proposed model for NMN utilization. It has been proposed that NMN could be imported intact in a similar fashion to NAD + internalization, but others have proposed NMN is extracellularly dephosphorylated to NR by CD73 or that CD38 hydrolyzes NMN to Nam. Dotted lines represent theoretically pathways that have not been directly tested in a cell or in vivo system. Solid lines represent known pathways.

a c NAD NMN NR Nam NADP Intracellular Enrichment ( 18 O NR (10 M))

1 Tracer/Tracee Tracer/Tracee 0 0 5 10 15 20 25 Time (h) b d 18 O NMN 18 O NR 18 O Nam Conditioned Media ( 18 O NMN (10 M)) 15

0 0 5 10 15 20 25 Time (h)

Figure 4.2. Extracellular NMN is dephosphorylated extracellular and incorporated into the intracellular NAD + pool at a slower rate compared to extracellular NR. 18 18 Hepg2 cells were supplemented with either [ O1]-NR (a and b) or [ O1]-NMN (c and d) and time points were taken at 0, 0.5, 2, 4, 7 and 24 hours (n = 3 or 4). At each time point, cells (a and c) and media (b and d) were collected for analysis via LC-MS/MS. Enrichment is represented as the ratio of 18 O labeled metabolite to endogenous metabolite peak areas and displayed as the average ± SEM.

CHAPTER 5 NICOTINAMIDE RIBOSIDE IS UNIQUELY BIOAVAILABLE IN MOUSE AND MAN

Samuel A.J. Trammell 1,2 , Mark S. Schmidt 1, Benjamin J. Weidemann 1, Philip Redpath 3, Frank

Jaksch 4, Ryan W Dellinger 4, Marie E. Migaud 3, and Charles Brenner 1,2

1Department of Biochemistry, 2 Interdisciplinary Graduate Program in Genetics, Carver College of Medicine, University of Iowa, Iowa City, IA 52242, USA

3John King Laboratory, School of Pharmacy, Queens University Belfast, Belfast, UK

4ChromaDex, Inc., Irvine, CA 92618

5.1 Distribution of Work

BJW, FJ, RWD, MEM, CB, and I designed the experiments. MSS, BJW, and I performed the

experiments and analyzed the data with FJ, RWD, MEM, and CB. PR performed the synthesis

under direction of MEM. CB and I wrote the manuscript. All authors edited the manuscript and

figures.

5.2 Abstract

Nicotinamide riboside (NR) is in wide use as an NAD+ precursor vitamin. Here we

conducted experiments to determine the time and dose-dependent effects of NR on blood and

liver NAD + metabolism in people and mice, respectively. We report that human blood cell NAD +

can rise as much as 2.7-fold with a single dose of NR, that NR elevates mouse hepatic NAD + with distinct and superior pharmacokinetics to those of nicotinic acid (NA) and nicotinamide

(Nam), and that single doses of 100, 300, and 1000 mg of NR provide a dose-dependent increase in the blood cell NAD + metabolome in the first clinical trial of NR pharmacokinetics. We

also report that nicotinic acid adenine dinucleotide (NAAD), which was not thought to be en

route for conversion of NR to NAD+, is formed from NR and that the rise in NAAD is a highly

sensitive biomarker of effective NAD + supplementation.

5.3 Introduction

Nicotinamide adenine dinucleotide (NAD +) is the central redox coenzyme in cellular

metabolism (128, 129). NAD + functions as a hydride group acceptor, forming NADH with concomitant oxidation of metabolites derived from carbohydrates, amino acids and fats. The

NAD +/NADH ratio controls the degree to which such reactions proceed in oxidative versus

reductive directions. Whereas fuel oxidation reactions require NAD + as a hydride acceptor, the

processes of gluconeogenesis, oxidative phosphorylation, ketogenesis, detoxification of reactive

oxygen species, and lipogenesis require reduced co-factors, NADH and NADPH, to act as

hydride donors (Figure 5.1). In addition to its role as a coenzyme, NAD + is the consumed substrate of enzymes such as poly-ADPribose polymerases (PARPs), sirtuins and cyclic

ADPribose synthetases (128). In redox reactions, the nucleotide structures of NAD +, NADH,

NADP + and NADPH are preserved. In contrast, PARP (130), sirtuins (131) and cyclic ADPribose

synthetase (132) activities hydrolyze the glycosidic linkage between the nicotinamide (Nam) and

the ADPribosyl moieties of NAD + to signal DNA damage, alter gene expression, control posttranslational modifications, and regulate calcium signaling.

In animals, NAD + consuming activities and cell division necessitate ongoing NAD + synthesis, either through a de novo pathway that originates with tryptophan or via salvage pathways from three NAD + precursor vitamins, Nam, nicotinic acid (NA) and nicotinamide

riboside (NR) (129). Dietary NAD + precursors, which include tryptophan and the three vitamins,

prevent pellagra. Though NR is present in milk (7) (Chapter 3), the cellular concentrations of

NAD +, NADH, NADP + and NADPH are much higher than those of any other NAD + metabolites

(1, 31), such that dietary NAD + precursor vitamins are largely derived from enzymatic breakdown of NAD +. Put another way, though milk is a source of NR, the more abundant

sources of NR, Nam and NA are any whole foodstuff in which cellular NAD + is broken down to these compounds. Human digestion and the microbiome (133) play roles in the provision of these vitamins in ways that are not fully characterized.

Different tissues maintain NAD + levels through reliance of different biosynthetic routes

(87, 134) (Figure 5.1). Because NAD + consuming activities frequently occur as a function of

cellular stresses (130) and produce Nam, the ability of a cell to salvage Nam into productive

NAD + synthesis through Nam phosphoribosyltransferase (NAMPT) activity versus methylation of

Nam to N-methylnicotinamide (MeNam) regulates the efficiency of NAD +-dependent processes

(135). NAD + biosynthetic genes are also under circadian control (57, 136) and both NAMPT

expression and NAD + levels are reported to decline in a number of tissues as a function of aging

and overnutrition (34, 124, 137, 138).

High dose NA but not high dose Nam has been used by people for decades to treat and

prevent dyslipidemias, though its use is limited by painful flushing (139, 140). Though it only

takes ~15 mg per day of either NA or Nam to prevent pellagra, pharmacological doses of NA

can be as high as 2 - 4 g. Despite the >100-fold difference in effective dose between pellagra

prevention and treatment of dyslipidemias, we proposed that the beneficial effects of NA on

plasma lipids might simply depend on function of NA as an NAD + boosting compound (128).

According to this view, sirtuin activation would likely be part of the mechanism because Nam is an NAD + precursor in most cells (87, 134) but is a sirtuin inhibitor at high doses (141).

Based on the ability of NR to elevate NAD + synthesis, increase sirtuin activity and extend lifespan in yeast (7, 142), NR has been employed in mice to elevate NAD + metabolism and

improve health in models of metabolic stress. Notably, NR allowed mice to resist weight gain on

high fat diet (37) and to prevent noise-induced hearing loss (56). Data indicate that NR is a

mitochondrially favored NAD + precursor (35) and, indeed, in vivo activities of NR have been interpreted as depending on mitochondrial sirtuin activities (37, 56), though not to the exclusion of nucleocytosolic targets (143, 144). Similarly, nicotinamide mononucleotide (NMN), the

phosphorylated form of NR, has been used to treat declining NAD + in mouse models of overnutrition and aging (34, 124). Because of the abundance of NAD +-dependent processes, it

is not known to what degree NAD + boosting strategies are mechanistically dependent on

particular molecules such as SIRT1 or SIRT3. In addition, the quantitative effect of NR on the

NAD + metabolome has not been reported in any system.

To translate NR technologies to human beings, it is necessary to determine its oral

availability and the extent and means by which NR is converted to NAD + metabolites in different

cell types. Here we performed targeted quantitative NAD + metabolomic measurements on

human blood samples in an n=1 human experiment in which a healthy 52 year-old man took

1000 mg of NR chloride daily for 7 days. These data indicated that blood cellular NAD + rose 2.7- fold after a single dose of NR and that nicotinic acid adenine dinucleotide (NAAD) unexpectedly increased at least 45-fold. To determine the precise time course of oral NR availability to the liver and to determine if NR is converted to NAD + in a unique manner with respect to Nam and

NA, we performed a detailed analysis of 128 mice, who were provided with saline or vitamin gavage in a manner that eliminated the possibility of circadian artifacts. These data indicated that NR boosts hepatic NAD + and NAD + consuming activities to a greater degree than Nam or

NA and with unique kinetics. As expected, oral NA results in a peak of hepatic NAAD prior to a broad peak of NAD + accumulation. However, NR and Nam resulted in NAAD peaks coincident

with elevated NAD +. Just as NR supplementation produced more hepatic NAD + than did Nam

supplementation, so NR also produced a greater degree of hepatic NAAD.

To address whether the unexpected appearance of NAAD might have been due to

inhibition of de novo biosynthesis, we synthesized NR with heavy atoms incorporated into the

Nam and ribosyl moieties and discovered that oral NR serves as a biosynthetic precursor of elevated NAAD. Finally, we performed a crossover clinical study with 12 healthy human subjects who took single doses of 100 mg, 300 mg or 1000 mg of NR chloride. This study

demonstrated that NR supplementation increases blood cell NAD + metabolism at all doses and validated elevated NAAD as an unexpected, highly sensitive biomarker of boosting NAD +.

5.4 Methods

Materials and Reagents

NR chloride (NR Cl) was synthesized under GMP conditions. Me2PY and Me4PY were

purchased from TLC PharmaChem Inc. (Vaughan, Ontario, Canada). All other unlabeled

analytes were purchased from Sigma-Aldrich (St. Louis, MO, US) at highest purity. Internal

18 18 18 standards [ O1]- Nam and [ O1]-NR were prepared as described (64, 65). [ O1-D3]-MeNam

18 13 was prepared through alkylation of [ O1]-Nam with deuterated iodomethane. C-NA and [D 4]-

NA were purchased from Toronto Chemical Research (Toronto, Ontario, Canada) and C/D/N

13 Isotopes, Inc. (Pointe-Claire, Quebec, Canada), respectively. To prepare [ C, D 1]-NR, we first

13 13 converted C-NA to C-Nam (145) and D-[2-D1]-ribose (Omicron Biochemicals, South Bend,

IN, US) to the labeled D-ribofuranose-tetraacetate (146). The labeled D-ribofuranose- tetraacetate and Nam were then used to synthesize double-labeled NR (147). [ 13 C]-labeled nucleotides and nucleosides were prepared by growing yeast in U-13 C-glucose and extracting

as described (1).

Mice

Twelve week old male C57Bl/6J mice (Jackson Laboratories, Bar Harbor, ME) were

housed 3-5 mice per cage on a chow diet (Teklad 7013) for one week prior to the experiment.

Body weight-matched groups were given either 185 mg NR Cl/kg body weight or equimole

amounts of NA or Nam by saline gavage. On each sacrifice day, a saline injection was

performed and served as time point zero and an additional saline gavage time course was

performed. To avoid circadian effects, time courses were established such that all sacrifices

were performed at ~ 2 pm. With protocols approved by the University of Iowa Office of Animal

Resources, mice were live-decapitated and the medullary lobe of the liver was freeze-clamped at liquid nitrogen temperature. Tissue was stored at -80 °C prior to analysis.

N of 1 Human Experiment

After overnight fasting, a healthy 52 year old male self-administered 1000 mg of NR

chloride orally at 8 am on 7 consecutive days. Blood and urine were taken for quantitative NAD + metabolomic analysis. He took 0.25 gram of NA to assess sensitivity to flushing and self- reported painful flushing that lasted 1 hour. No flushing was experienced on NR.

Clinical Trial

A randomized, double-blind, three-arm crossover pharmacokinetic study of oral NR

chloride was performed at 100, 300, and 1000 mg doses. Twelve healthy, non-pregnant

subjects (6 male, 6 female) between the ages of 30 and 55 with body mass indices of 18.5 -

29.9 kg/m 2 were recruited and randomized to one of three treatment sequences after screening and passing eligibility criteria. Subjects taking multi-vitamins, vitamin B 3 in any form, or subsisting on diets that could contain unusually high amounts of NA, Nam or NR were excluded.

Complete exclusion criteria are provided in Supplementary Materials.

Overnight fasted subjects received a single morning dose of either 100 mg, 300 mg, or

1000 mg of NR on three test days separated by 7-day periods in which no supplement was given. To evaluate pharmacokinetics, blood was collected and separated into plasma and

PBMC fractions for analysis of the NAD + metabolome at pre-dose and again at 1, 2, 4, 8, and 24 hr. Urine was collected pre-dose and in 0-6 hr, 6-12 hr and 12-24 hr fractions. Safety, vitals, biometrics, complete blood counts and a comprehensive metabolic panel were assessed at time zero and 24 hr after each dose.

The study was reviewed and approved by the Natural Health Products Directorate,

Health Canada and Institutional Review Board Services, Aurora, Ontario. Written informed consent was obtained from each subject at the screening visit prior to all study-related activities.

Sample Preparation and LC-MS

Two extractions were performed on all samples for quantitative targeted metabolomic analysis (1). PBMCs were thawed on ice and extracted with three volumes of acetonitrile in the presence of each internal standard mixture. For group B analytes, extracts were passed through

Phenomenex Phree© SPE cartridges (Torrance, CA, USA) and combined with a subsequent acetonitrile wash prior to drying. All extracts were dried via speed vacuum. On the day of analysis, samples were re-suspended and placed in a chilled autosampler. Murine liver was processed as detailed in (Chapter 2.2: Quantitation of the Oxidized NAD Metabolome in Liver).

Standard curves were prepared in water and processed in the same manner as samples.

Separation and quantitation of analytes was performed with a Waters Acquity LC interfaced with a Waters TQD mass spectrometer operated in positive ion multiple reaction monitoring mode. Enrichment analysis was performed with a Waters Q-TOF Premier mass spectrometer operated in positive ion, full scan mode with the same LC conditions as described for non-enrichment experiments. Details are provided in Supplemental Methods.

Statistical Analyses

Statistical analyses were performed in GraphPad Prism version 6.00 for Windows, (La

Jolla, CA, USA). Murine liver data were analyzed using a two-way ANOVA, whereas human blood cells were analyzed using a repeated, two-way ANOVA. Holm-Sidak and Tukey’s multiple comparisons tests were performed when comparing more than two groups. AUCs in blood cells were calculated after subtracting pre-dose metabolite concentrations of each experimental series. For mouse data, AUCs were calculated as described (148) after subtracting the saline group for that day and propagating error. All other tests are stated in the text. Data are expressed as means ± standard error of the mean.

5.5 Results

Oral NR Increases the Blood NAD Metabolome in a Healthy Adult Male

A healthy 52 year old male (65 kg) contributed blood prior to seven days of oral NR

chloride (1000 mg/morning dose). Blood was taken an additional 9 times in the first day and at

24 hours after the first and last oral dose. Blood was separated into a peripheral blood

mononuclear cell (PBMC) fraction and a plasma fraction prior to quantitative NAD + metabolomic

analysis by LC-tandem MS (1), which was expanded to quantify methylated and oxidized

metabolites of Nam. As shown in Table 5.1, the PBMC NAD+ metabolome was unaffected by

NR for the first 2.7 hrs. In six measurements from time zero through 2.7 hrs, NAD + had a mean

concentration of 18.5 µM while Nam had a mean concentration of 4.1 µM and the methylated

and oxidized Nam metabolite, N-methyl-2-pyridone-5-carboxamide (Me2PY) had a mean

concentration of 2.6 µM. However, at 4.1 hours post-ingestion, PBMC NAD + and Me2PY increased by factors of 2.3 and 4.2, respectively.

In yeast, deletion of nicotinamide riboside kinase 1 (NRK1) does not eliminate utilization of NR (142). NR can be phosphorylyzed to Nam by purine nucleoside phosphorylase and still contribute to NAD + synthesis through Nam salvage (126, 142). However, as shown in Table 5.1,

Nam concentration in the human subject’s PBMCs merely fluctuated in a range of 2.6 µM to 7.1

µM throughout all 11 observations. The 4.2-fold increase in Me2PY concentration at the 4.1

hour time point suggests that increased cellular NAD + accumulation is accompanied by increased NAD + consuming activities that are linked to increased methylation and oxidation of the Nam product.

In the human subject’s PBMCs at 7.7 and 8.1 hours post ingestion, NAD + and Me2PY reached peak levels, increasing above baseline concentrations by 2.7-fold and 8.4-fold respectively. At these time points, unexpectedly, NAAD, the substrate of glutamine-dependent

NAD + synthetase (12), which is only expected to be produced in biosynthesis of NAD + from

tryptophan and NA (129), was elevated from less than 20 nM to as high as 0.91 µM. Whereas

NAAD lagged the rise in PBMC NAD + by one time point, the rise in PBMC NAD + was not as

pronounced as the spike in NAAD, which was at least 45-fold above the baseline level. Though

contrary to expectations, these data suggested that NR might be incorporated into NAAD after

formation of NAD + and chased back to the NAD + peak as NAD + declines.

Complete NAD + metabolomic data from the human subject’s PBMC fraction, blood plasma fraction and urine are provided in Table 5.1 and as supplementary Tables 5.2 and 5.3, respectively. These data show that all of the phosphorylated compounds—NAMN, NAAD,

NAD +, NADP +, NMN and ADPR—are found exclusively in PBMCs. Notably, the peak of NADP +, which represents cellular NADP + plus NADPH that was oxidized in extraction, and the peak of

ADPR, which signals an increase in NAD + consuming activities, co-occur with the peak of NAD +.

Using methods that are optimized for recovery of nucleotides, NR was not recovered. The major

time-dependent waste metabolite in plasma and urine was Me2PY, which rose about 10-fold

from pre-dose to time points after NAD + peaked in PBMCs.

Oral NR, Nam and NA Elevate Hepatic NAD + with Distinctive Kinetics

Based on known NAD + biosynthetic pathways (35), it was difficult to understand how

NAAD increased in human PBMCs after an oral dose of NR. Though NR did not elevate Nam in

blood samples at any time during the n=1 experiment, it remained possible that a fraction of NR

was converted to Nam prior to salvage synthesis to NAD +. Such conversion to Nam might allow

bacterial hydrolysis of Nam to NA by pncA gene products—potentially in the gut (133)—and

subsequent conversion to NAD + through an NAAD intermediate.

We designed an experiment in which a mouse’s daily dose of NR (185 mg/kg) 17 and the

mole equivalent doses of Nam and NA were provided to mice by oral gavage. To ascertain the

17 This dosage is equivalent to a 65 kg human being ingesting 1 gram NR Cl based upon surface-to-area ratio between a human and mouse (149).

time course by which these vitamins boost the hepatic NAD + metabolome without the

complication of circadian oscillation of NAD + metabolism (57, 136), we sacrificed all mice at

approximately 2 pm. Thus, vitamin administration by gavage was performed at 0.25 hour, 1

hour, 2 hours, 4 hours, 6 hours, 8 hours and 12 hours prior to sacrifice. To stop metabolism

synchronously, mouse livers were harvested by freeze-clamping. As shown in Figure 5.2, we

additionally performed saline gavages at all time points and sacrificed mice for quantitative

NAD + metabolomic analysis to ensure that animal handling does not alter the levels of NAD +

metabolites. The flat time courses of saline gavages established the suitability of this method.

Baseline levels of hepatic NAD + metabolites at 2 pm were 1000 ± 35 pmol/mg for NAD+, 230 ±

29 pmol/mg for Nam, 210 ± 20 pmol/mg for NADP +, 66 ± 13 pmol/mg for ADPR, and < 15 pmol/mg of all other NAD + metabolites. Hepatic levels of NA, NAR, NAMN, and NAAD have baselines of below 4 pmol/mg. As a point of orientation to quantitative metabolomics in tissue samples, we note that 1000 pmol/mg is ~1 mM, 200 pmol/mg is ~200 µM, and 10 pmol/mg is

~10 µM.

Targeted NAD + metabolomics (1, 31) allows simultaneous assessment of functionally important and highly regulated metabolites such as NAD + and NADP + along with metabolites that could serve as biomarkers of biosynthetic processes, such as NA, NAR, NAMN, NR, NMN and NAAD. In addition, quantification of increases in ADPR, Nam, MeNam, Me2PY and N- methyl-4-pyridone-5-carboxamide (Me4PY) on a common absolute scale with NAD + allows an

assessment of increased NAD + consuming activities associated with NAD + precursor vitamin

supplementation.

Hepatic concentrations of 13 NAD + metabolites were quantified in 3 to 4 mice at 7 time points after gavage of saline and after gavage of each NAD + precursor vitamin. In addition, 4

mice were analyzed after control 2 pm sacrifices without gavage. Each vitamin produced

temporally distinct pattern of hepatic NAD + metabolites. Consistent with the rapid phosphorylation of NR and NAR by NR kinases (33), the only NAD + metabolites that do not

produce hepatic peaks as a function of gavage of NAD + precursor vitamins are NR and NAR

(Supplementary Tables and Figures: Figure 5.5a-b). The accumulation curves of some metabolites as a function of each vitamin were strikingly similar. For example, the accumulation of NMN (Figure 5.2a) is nearly identical to that of NAD + (Figure 5.2b) and NADP + (Figure 5.2c)

though at a scale of ~1:400:40. In addition, the accumulation of Me4PY (Figure 5.2f) is nearly

identical to that of Me2PY (Figure 5.5c).

As shown in Figure 5.2b, NA produced the least increase in hepatic NAD + but also was

4-6 hours faster than NR and Nam in the kinetics of hepatic NAD + accumulation. When NA was provided by oral gavage, liver NA peaked (340 ± 30 pmol/mg) in 15 minutes (Figure 5.2g).

Hepatic NA appearance was followed by an expected peak of 220 ± 29 pmol/mg of NAAD at 1 hr post-gavage (Figure 5.2i) and a rise in hepatic NAD + from 990 ± 25 pmol/mg baseline to 2200

± 150 pmol/mg at 2 hrs (Figure 5.2b). Hepatic NADP + due to NA (Figure 5.2c) rose in parallel to

that of hepatic NAD +. In the hours after gavage of NA, as hepatic NAD + and NADP + fell, there was clear evidence of enhanced NAD + consuming activities with significant rises in ADPR

(Figure 5.2j), Nam (Figure 5.2d), MeNam (Figure 5.2e), Me2PY (Figure 5.5c) and Me4PY

(Figure 5.2f). Thus, a bolus oral administration of NA doubled hepatic NAD + from ~1 mM to ~2 mM through expected intermediates and produced an increase in NAD + consumption and the methylated products, MeNam, Me2PY and Me4PY.

As shown in Figure 5.2g, oral Nam was clearly not used by the liver as NA because it did not produce a peak of NA at any time after gavage. Though there was an increase in hepatic

NAD + 2 hrs after Nam gavage, the Nam gavage drove increased hepatic NAD + accumulation from 2 to 8 hrs with a peak at 8 hrs (Figure 5.2b). Nam gavage produced two peaks of Nam in the liver (Figure 5.3d). The first peak was at 15 min, consistent with simple transport of the vitamin to the liver. The second broad peak was coincident with elevation of NAD + and NADP +

(Figures 5.2c-d) and with elevation of the NAD + consuming metabolomic signature of ADPR

(Figure 5.2j), MeNam, Me4PY and Me2PY (Figure 5.2e-f and 5.8 Supplemental Tables and

Figures: 5.5c).

Of the metabolites associated with NAD + consuming activities, ADPR is the only one that

must be formed from NAD + because Nam, MeNam and the oxidized forms of MeNam could

appear in liver from the gavaged Nam without conversion to NAD +. Interestingly, of three NAD + precursor vitamins provided in bolus at equivalent oral doses, Nam provided the least increase in ADPR (Figure 5.2j). Whereas the area under the curve (AUC) of the Nam-driven rise in hepatic NAD + indicated a ~50% advantage of Nam over NA (Figure 5.2b), there was a >50%

deficit in Nam-driven ADPR accumulation versus NA (Figure 5.2j). This is consistent with the

idea that high dose NA, though not an ideal hepatic NAD + precursor, is capable of improving

reverse cholesterol transport to a much greater degree than Nam because high dose Nam

inhibits sirtuins(128).

As shown in Figure 5.1, Nam is expected to proceed through NMN but not NR, NAR,

NAMN or NAAD en route to forming NAD +. Though there was no elevation of hepatic NR or

NAR with oral Nam, there was also little elevation of hepatic NMN—this metabolite never

reached a mean value of 5 pmol/mg at any time after Nam administration (Figure 5.2a).

Surprisingly, as shown in Figure 5.2i, 2-4 hrs after oral Nam, NAAD was elevated to nearly 200

pmol/mg from a baseline of less than 2 pmol/mg. Elevated NAAD occurred during the broad

peak of elevated hepatic NAD + and NADP + (Figures 5.2b-c). These data suggest that the rise in

NAAD is a biomarker of increased NAD + synthesis and does not depend on the conventionally

described precursors of NAAD, namely NA and tryptophan.

As shown in Figure 5.2b, NR elevated hepatic NAD + by more than 4-fold with a peak at 6

hr post-gavage. NR also produced the greatest elevation of NMN (Figure 5.2a), NADP + (Figure

5.2c), Nam (Figure 5.2d), NAMN (Figure 5.2h), NAAD (Figure 5.2i) and ADPR (Figure 5.2j) both in terms of peak height and AUC. Importantly, though gavage of Nam produces a peak of Nam in the liver at 15 min, the peak of Nam from NR gavage corresponds to the peak of NAD +, NMN,

NADP + and ADPR. These data establish that oral NR has clearly different hepatic pharmacokinetics than oral Nam. More NAD + and NADP + were produced from NR than from

Nam. In addition, there was three times as much accumulation of ADPR, indicating greater

NAD + consuming activities. In an accompanying manuscript, we showed that hepatic cells convert NMN extracellularly to NR and that both NMN and NR depend on expression of NRK1 for conversion to cellular NAD + (150).

As was seen in the n=1 human blood experiment, at time points in which the abundant

NAD + metabolites, NAD + and NADP +, were elevated by NR by ~2-fold or more, NAAD rose from

undetectable levels to approximately 10% of the level of NAD +, thereby becoming a highly sensitive biomarker of increased NAD + metabolism. Though compounds such as MeNam,

Me2PY and Me4PY are also correlated with increased NAD + synthesis, they can be produced without NAD + synthesis. While MeNam, Me2PY and Me4PY are waste products that can no

longer contribute to elevated NAD + or NADP +, NAAD is functional NAD + precursor.

NR Directly Contributes to Murine Liver NAAD

The appearance of hepatic NAAD after murine gavage of Nam or NR, and of hepatic

NAMN after gavage of NR suggested that there is retrograde NAD + metabolic flux when NAD +

and NADP + levels are high. Alternatively, high levels of NAD+ metabolites might inhibit

glutamine-dependent NAD + synthetase, thereby resulting in accumulation of NAMN and NAAD

derived from tryptophan. To test whether NR is incorporated into the peak of NAAD that

appears after gavage of NR, we synthesized NR chloride with incorporation of deuterium at the

ribosyl C2 and 13 C into the carbonyl of the Nam moiety. This double-labeled NR was provided to

15 mice by oral gavage at an effective dose of 185 mg/kg with the same experimental design

used in pharmacokinetic analysis of the three vitamins. The effect of labeled oral NR on the

hepatic NAD + metabolome was first assessed at 2 hours after gavage—a time prior to the rise in

the steady-state level of NAD + (Figure 5.2b).

As shown in Figure 5.3a-b, at 2 hrs, 54% of the NAD+ and 32% of the NADP + contained at least one heavy atom while 5% of the NAD + and 6% of the NADP + incorporated both heavy

atoms. Because > 50% of hepatic NAD + incorporates label prior to a rise in NAD + accumulation, it is clear that the NAD + pool is dynamic. As shown in Figure 5.3c-d, the majority of hepatic Nam

and MeNam following gavage of double-labeled NR incorporated a heavy atom, which is

necessarily the 13 C in Nam. Because NR drives increased NAD + synthesis and ADPR

production (Figure 5.2), the liberated singly labeled Nam would thereby become incorporated

into NMN and NAD + in competition with double labeled NR, thereby limiting subsequent

incorporation of both labels into the NAD + pool.

Appearance of a peak of NAAD after NR administration could either be due to inhibition of de novo synthesis of NAD + or from a novel retrograde pathway that produces NAAD from

NAD +. If NAAD is not derived from ingested NR, then it should not incorporate heavy atoms.

However, if NAAD is derived from ingested NR, then it should incorporate heavy atoms that reflect the rate at which the retrograde reactions occur with respect to NAD + consuming

activities and the degree of heavy atom incorporation into NAD +. As shown in Figure 5.3e, at the

2 hr time point, NAAD contained roughly the same heavy atom composition as NAD + (Figure

5.3a), i.e. , 45% contained at least one heavy atom and 8% incorporated both heavy atoms.

Thus, NR is the biosynthetic precursor of NAD +, NADP + and NAAD. The data suggest that the

process that converts NAD + to NAAD occurs at high NAD + concentrations at a rate comparable to the rate of NAD + turnover to Nam.

Incorporation of the Nam and ribosyl moieties of NR into NAAD establishes this

metabolite as both a biomarker of increased NAD + metabolism and as a direct product of NR

utilization.

NR Increases Blood Cell NAD + Metabolism in Human Subjects

The n=1 human experiment illustrated the potential of 1000 mg NR to boost human

NAD + metabolism. We therefore conducted a controlled experiment with 12 consented healthy men and women to determine the effect of three single doses of NR on PBMC, plasma and urine NAD + metabolites with monitoring of subjects for potential adverse events. Considering

that the recommended daily allowance (RDA) of vitamin B3 as Nam or NA is ~15 mg per adult,

we chose to test three doses of the higher molecular weight compound NR chloride (100 mg,

300 mg and 1000 mg) that correspond to 2.8, 8.4 and 28 times the RDA. Participants were

randomized to receive doses of NR in different sequences with 7-day washout periods between

data collection. Participants and investigators were blinded to doses. Blood and urine collections

were performed over 24 hours following each dose. Participants were asked to self-report any

perceived discomforts.

At 500 mg of niacin, 33 of 33 participants experienced flushing compared to 1 of 35

participants who received a placebo (151). In this study, two individuals self-reported flushing at

the 300 mg dose but not at the 100 mg or 1000 mg dose, and two individuals self-reported

feeling hot at the 1000 mg dose but not at lower doses. Over the total of 36 days of observation

of study participants, there were no serious adverse events and no events that were dose-

dependent. To assess whether NR was associated with authentic and dose-dependent

episodes of flushing, future experiments will incorporate a validated flushing symptom

questionnaire(152).

As shown in Figure 5.4 and Tables A.1 and A.2 (Appendix A), the NAD + metabolome was quantified in the PBMC and plasma fractions at pre-dose and at 1, 2, 4, 8 and 24 hours after receiving oral NR. Urinary NAD + metabolites (Appendix A: Table A.3) were quantified in

pre-dose, 0-6 hour, 6-12 hour, and 12-24 hour collections.

As shown in Figure 5.2, inbred, chow-fed male mice supplemented with NAD + precursor vitamins by gavage and sacrificed at the same time of day produced hepatic NAD + metabolomic

data with little variation. However, blood samples from people in a clinical study exhibited a wider degree of variation, apparently due to differing baseline levels of metabolites and variable pharmacokinetics, both of which might be due to genetic and nutritional changes between human subjects (Figure 5.4). In PBMCs, 8 key metabolites were quantified in at least 10 subjects at all time points at each dose. For each of these metabolites, we plotted the average concentration as a function of dose and time, calculated whether NR elevated that metabolite, plotted the averaged peak concentration of the metabolite as a function of dose, and calculated the dose-dependent AUC of the metabolite attributable to NR supplementation.

Collapsing the data into pre-dose versus 24 hr levels of each metabolite at all doses, NR significantly elevated PBMC NAD + (Figure 5.4b), MeNam (Figure 5.4d) and Me2PY (Figure

5.4e), and significantly elevated PBMC NAAD (Figure 5.4f) at 8 hrs. In contrast, NR did not

produce a statistically significant all-dose elevation of NMN (Figure 5.4a) or Nam (Figure 5.4c)

at any time point.

The averaged peak concentration of MeNam (Figure 5.4d), Me2PY (Figure 5.4e) and

NAAD (Figure 5.4f) increased monotonically with increased doses of NR. Of these metabolites,

only NAAD was below the detection limit in individuals before they took NR. Nam (Figure 5.4c)

exhibited no tendency toward higher cellular concentrations with higher doses of NR. NMN

(Figure 5.4a) and NAD + (Figure 5.4b) rose to higher concentrations of ~2 µM and 20 µM, respectively, in people given 300 mg and 1000 mg doses of NR than in people given 100 mg doses of NR. Thus, 100 mg supplementation produced an average ~4 ± 2 µM increase in

PBMC NAD +, whereas the two higher doses produced average ~6.5 ± 3.5 µM increases in

PBMC NAD +.

As was first seen in the n=1 human experiment and in the mouse liver experiments,

NAAD is the most sensitive biomarker of effective NAD + supplementation because it is

undetectable in the blood of people prior to dosing. At all doses, the peak shape of NAAD

indicated that NAD + metabolism is most greatly boosted at 8 hrs with significant

supplementation at 4 hours and significant supplementation remaining at 24 hours. At the 8 hour peak, the average concentration of NAAD was elevated to 0.56 ± 0.26, 0.74 ± 0.27 and

1.24 ± 0.51 µM in PBMCs from volunteers taking 100, 300 and 1000 mg single doses of NR,

respectively.

Finally, we plotted pre-dose-subtracted AUCs of each metabolite as a function of dose of

NR. With the exception of Nam, the levels of which were unaffected by NR, NR produced or

tended to produce dose-dependent elevation of the entire NAD + metabolome (Figure 5.4).

In the plasma, levels of MeNam, Me2PY and Me4PY also rose in a dose-dependent

manner and were identified at concentrations similar to those in the PBMC fraction. The

methylated and oxidized Nam derivatives were accompanied by low levels of NAR, which

increased with increased doses of NR. Urinary metabolites were similar to plasma metabolites.

5.6 Discussion

Despite more than 75 years of experience with human use of NA and Nam (153) and

more than a decade of preclinical work on NR (7), there has never been a quantitative

metabolomic or pharmacokinetic comparison of the three NAD+ precursor vitamins in any

system. In terms of elevation of mouse liver NAD+, here we show that NR is more orally

bioavailable than Nam, which is more orally bioavailable than NA (Figure 5.2b). The three

precursors also differ in the degree to which they promote accumulation of ADPR, a measure of

NAD+ consuming activities. As shown in Figure 5.2j, the ability of NR to elevate ADPR

exceeded that of Nam by ~3-fold. On a molar basis, this qualifies NR as the favored NAD+

precursor vitamin for increasing NAD+ and NAD+ consuming activities in mouse liver.

NR, Nam and NA each have unique pharmacokinetic profiles in mouse liver, both in

terms of the kinetics of NAD + formation and the population of NAD + metabolites as a function of

time. As shown in Figure 5.2d, Nam is the only vitamin precursor of NAD + that produces

elevated hepatic Nam 15 min after oral administration and, as shown in Figure 5.2g, NA is the only precursor that produces elevated NA 15 min after oral administration.

When PBMCs were analyzed from the first human volunteer taking 1000 mg of NR,

NAAD was observed to increase at least 45-fold from a baseline of less than 20 nM to a peak value of nearly 1 µM. This occurred concomitant with a rise in NAD + from ~18.5 µM to 50 µM.

NAAD was also observed to be elevated in the liver when mice were provided with NAD + precursor vitamins. Surprisingly, NA, the only precursor expected to proceed to NAD + through

an NAAD intermediate, produced the least NAAD. Indeed, though Nam and NR never produced

peaks of hepatic NA or NAR, both produced peaks of hepatic NAAD during the periods in which

these compounds elevated hepatic NAD +. The temporal basis of the NAAD excursions

suggested that elevating NAD + (Figure 5.2b) not only stimulates accumulation of NAD +

consumption products ADPR (Figure 5.2j), Nam (Figure 5.2d), MeNam (Figure 5.2e) and

Me4PY (Figure 5.2f), but also stimulates retrograde production of NAAD (Figure 5.2i) and

NAMN (Figure 5.2h). According to this view, when NAD + is elevated at least 2-fold, a previously unknown activity would deamidate NAD + to NAAD.

In the mouse liver system, the potential flux of this pathway is quite significant: the NR-

driven peak of NAAD amounted to 10% of the NR-attributable peak of NAD +. Production of high

levels of NAAD from NAD + could therefore account for the NR-stimulated peak in NAMN because NAMN adenylytransferase is known to be a reversible enzyme (154).

The hypothesis that NAAD is formed from NAD + in vivo was tested by administering NR that had been labeled in the Nam and ribosyl moieties. As shown in Figure 5.3, NR stimulates appearance of double-labeled NAAD (8% of total) at the same 2 hr time point in which 5% of

NAD + was double-labeled. The biochemical basis for apparent NAD + deamidation is not known.

However, the glutamine-dependent NAD + synthetase reaction would appear to be irreversible

(12, 155). One intriguing possibility is that NAAD is formed by the long-sought enzyme that forms intracellular NAADP (156). According to this view, an NADP deamidase may be

responsible for formation of NAADP—this same activity might deamidate NAD + at high concentrations, stimulating formation of NAAD. Unlike ADPR and methylated Nam waste products, NAAD is not only a biomarker of elevated NAD + metabolism but is also a reserve

metabolite that contributes to elevated NAD + over time.

Finally, in the first controlled clinical study of NR, it was established that PBMC NAD + metabolism is increased by 100 mg, 300 mg and 1000 mg doses of NR without dose-dependent increases in PBMC Nam and without dose-dependent serious adverse events. Though some sporadic thermal responses were self-reported in this study, the experiment was not designed to assess flushing with a validated questionnaire.

In people, as in mice, NAAD is the most sensitive biomarker of boosting NAD +. While NR

elevated PBMC NAD + from ~12 to ~18 µM, NAAD was elevated from below the limit of quantification to ~1 µM. The ability to detect NAAD in human samples is expected to aid conduct of clinical trials of NR and other NAD + boosting strategies.

The wide availability of over-the-counter nutritional supplements can complicate clinical trials because patients may enroll in order to obtain compounds they expect to bring benefits and such patients may be inclined to take supplements in the case that they are assigned to placebo. Detection of NAAD should therefore be incorporated in phase II and III clinical studies of NR efficacy to eliminate the confounding effects of off-study use of NR.

5.7 Figures and Table for Chapter 5.5-5.6

NADK NADK2 2’P-ADPR H O NADPH

NH2

Tryptophan N

CH3 2’P-ADPR 2’P-ADPR de NADK NADH NADP + NADK2 novo

O O

O O O NMNAT1-3 NADSYN1 NMNAT1-3 PO ON ADP HON O O O

OH OH OH OH NAMN NAAD NMN

NRK1,2 NAPRT NAMPT NRK1,2

NAD + consuming enzymes NAD +

PNP pncA PNP

NA Nam O + NNMT ADPR NH2 O Products NAR NR O N NH2

CH3

Me2PY N AOX1 Mammalian Enzymatic Step CH3 MeNam Bacterial Enzymatic Step

Me4PY

Figure 5.1 The NAD metabolome. NAD + is synthesized from salvage of the vitamin precursors, NA, Nam and NR, or from tryptophan in the de novo pathway. NAD + can be reduced to NADH, phosphorylated to NADP + or consumed to Nam. Nam can also be methylated and oxidized to non-NAD + precursors. NAAD was not thought to be a precursor of NAD + from NR.

Table 5.1 PBMC NAD + metabolites (µM) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days. Time (hr) NAD + NADP + NAAD NMN ADPR Nam MeNam Me4PY Me2PY 0 12 9.1 <0.02 0.68 0.70 3.2 0.06 0.20 1.1 0.3 27 23 <0.02 1.8 3.3 4.5 0.14 0.44 2.2 0.6 17 13 <0.02 1.4 2.1 7.1 0.08 0.57 3.1 1 24 13 <0.02 1.3 1.3 3.2 0.20 0.59 3.4 1.4 19 11 <0.02 1.1 1.3 3.7 0.15 0.64 3.1 2.7 12 6.9 <0.02 0.77 0.85 2.9 0.13 0.53 2.8 4.1 42 27 <0.02 2.7 3.4 4.9 0.32 1.9 11 7.7 43 27 0.51 2.6 2.5 5.9 0.49 3.5 17 8.1 50 33 0.91 2.9 3.5 5.7 0.60 4.4 22 23.8 32 17 0.37 1.9 2.5 2.6 0.25 1.9 8.8 167.6 38 12 0.45 1.1 2.1 3.2 0.66 2.5 11

a b

** 6000 ### 15 ### ††† * ††† *** ### ‡‡ 10 ### 4000 † # (pmol/mg) 5 + 2000 NAD AUC (pmol*hr/mg) AUC AUC (pmol*hr/mg) AUC 0 0 0 5 10 15 0 5 10 15 R A m N N NR NA Time (hrs) Na Time (hrs) Nam c d ††† ### ### 600 1500 ††† ††† ‡‡‡ ### *** ### ‡ ‡‡ ††† *** *** ### † ‡‡‡ 400 # 1000 ### † ‡‡‡ ‡‡ ‡‡ 200 500 AUC (pmol*hr/mg) AUC AUC (pmol*hr/mg) AUC 0 0 0 5 10 15 0 5 10 15 NR NA NR NA Time (hrs) Nam Time (hrs) Nam e f †† * ‡‡‡ ††† †† ‡‡‡ * *** †† ‡‡‡ ‡‡ ‡‡‡ ** *** ‡ ‡‡‡ # # AUC (pmol*hr/mg) AUC AUC (pmol*hr/mg) AUC

A NR N NR NA g Nam h Nam

### ### * ‡‡‡ ††† *** *** ***

### ‡‡‡ AUC (pmol*hr/mg) AUC AUC (pmol*hr/mg) AUC

R A m m i N N j NR NA Na Na ### ††† ## ‡ 0.077 ### ### ‡‡‡ ††† ††† 0.053 0.15 ‡‡‡ ### ** ### ‡‡‡ ‡‡‡ * ### †† # # ‡ ‡ AUC (pmol*hr/mg) AUC AUC (pmol*hr/mg) AUC

NR NA NR NA Nam Nam

Figure 5.2. NR elevates hepatic NAD + metabolism distinctly with respect to other vitamins. Either saline (orange) or equivalent moles of NR (black), NA (blue) and Nam (green) were administered to male C57BL6/J mice by gavage. To control for circadian effects, gavage was performed at indicated times prior to a common ~2 pm sacrifice. At time of sacrifice, mice were live decapitated and livers freeze clamped. Livers were extracted as described in 5.4 Methods and 5.8 Supplemental Materials and analyzed using LC-MS/MS. In the left panels, the hepatic concentrations of each metabolite are shown as a function of the four gavages. In the right panels, the baseline subtracted 12-hour areas under the curve are shown. Left panels: ‡ p- value < 0.05; ‡‡ p-value < 0.01; ‡‡‡ p-value <0.001 Nam vs NA; † p-value < 0.05; †† p-value < 0.01; ††† p-value < 0.001 Nam vs NR; # p-value < 0.05; ## p-value < 0.01; ### p-value < 0.05

Figure 5.2 – continued

NA vs NR; Right panel: * p-value < 0.05; ** p-value < 0.01; *** p-value <0.001. The data indicate that NR produces greater increases in NAD+ metabolism than Nam or NA with distinctive kinetics, that Nam is disadvantaged in stimulation of NAD+ consuming activities, and that NAAD is surprisingly produced after oral NR administration.

a b (pmol/mg) + NADP

0 2 4 6 8 0 2 4 6 8 c d Nam (pmol/mg) Nam MeNam (pmol/mg) MeNam

0 2 4 6 8 0 2 4 6 8 e NAAD (pmol/mg) NAAD

0 2 4 6 8

Figure 5.3 NR contributes directly to hepatic NAAD. Double-labeled NR was orally administered to mice. At indicated times, mice were sacrificed and livers freeze-clamped for isotopic enrichment analysis using LC-MS. a-b. Isotopic enrichment of NAD + and NADP + over time at both the M+1 and M+2 mass shifts. c-d. Isotopic enrichment of Nam and MeNam over time at the M+1 mass shift. e. Isotopic enrichment of NAAD over time at both the M+1 and M+2 mass shifts. The data indicate that half of the NAD + is turned over before there is a rise in steady-state NAD + and that NR is an incorporated precursor of NAD +, NADP + and NAAD.

a 3 0.040 0 hr vs p-value

Any NS 2 Time 0.561

1 0.461

0 0 10 20 30 0 0 10 300 Time (hrs) 100 b 30 0 hr vs p-value 0.114

24 hrs 0.0267 0.474 M) 20 ( mol*hr/l)

+ 0.364

10 NAD AUC ( AUC 0 0 10 20 30 0 0 00 10 3 00 Time (hrs) 1 c 40 0 hr vs p-value 40 0.119 30 Any NS 30 0.223 0.560 Time 20 20

10 10

0 0 0 10 20 30 100300 1000 0 0 10 300 Time (hrs) Dose (mg) 100 d ††† *** ††† ‡‡ 0 hr vs p-value *** ‡‡ ††† <0.001 ‡‡ M) †‡ 1 hr <0.001

2 hrs <0.001

4 hrs <0.001

MeNam 0.262 0.004 8 hrs <0.001

24 hrs <0.001 0 0 10 300 100 e *** 20 *** <0.001 ††† 0 hr vs p-value ‡‡‡ ††† 15 ††† ‡‡‡ 1 hr <0.001 ‡ 10 2 hrs <0.001 † 0.001 5 4 hrs <0.001 0.004 8 hrs <0.001 0 0 10 20 30 0 0 0 24 hrs <0.001 0 0 0 1 3 0 Time (hrs) 1 f

0 hr vs p-value M) †† 0.055 < 0.001

M) 8 hrs

0.021 0.055 NAAD Maximum NAAD ( NAAD Maximum 0 0 10 300 00 1 Figure 5.4 Dose-dependent effects of NR on the NAD Metabolome of human subjects. Time-dependent PBMC NAD + metabolomes from 12 healthy human subjects were quantified using LC-MS/MS after three different oral doses of NR. In each left panel, the concentration of a metabolite as a function of dose and time is displayed. # p-value < 0.05; ## p-value < 0.01 100

Figure 5.4 – continued mg vs. 300 mg; †† p-value < 0.01 †††; p-value < 0.001 100 vs. 1000; ‡ p-value < 0.05; ‡‡ p- value < 0.01 300 vs. 1000. A Dunnett’s test was performed comparing the average concentration of each metabolite at each time point to the concentration of that metabolite at time zero. Significant elevations of NAD +, MeNam, Me2PY and NAAD are indicated. In each middle panel, the averaged maximum metabolite concentration per dose is plotted. In each right panel, the background-subtracted metabolite AUCs are displayed with a one sample t-test comparing the AUC to background above each bar. Additionally, asterisks indicate dose- dependent increases in metabolite AUC (* p-value < 0.05; ** p-value < 0.01; *** p-value < 0.001). The data indicate that all doses of NR elevated 8 hr NAAD and 24 hr NAD +, and that additional NAD + metabolites were elevated dose-dependently with statistical significance by multiple comparisons

5.8 Supplemental Materials

Clinical Trial

Exclusion criteria: women who were pregnant, breastfeeding, or planning to become pregnant during the course of the trial; use of natural health products/dietary supplements within

7 days prior to randomization and during the course of the study; use of vitamins or St. John's

Wort 30 days prior to study enrollment; use of supplements containing NR within 7 days prior to randomization and the course of the study; use of nutritional yeast, whey proteins, energy drinks, grapefruit and grapefruit juice, dairy products, alcohol for 7 days prior to the study; consumption of > 2 standard alcoholic drinks per day or drug abuse within the past 6 months; smoking; blood pressure ≥ 140/90; use of blood pressure medications; use of cholesterol lowering medications; metabolic diseases or chronic diseases; use of acute over-the-counter medication within 72 hr of test product dosing; unstable medical conditions as determined by the qualified investigator; immune compromised conditions including organ transplantation or human immunodeficiency virus; clinically significant abnormal lab results at screening ( e.g. , aspartate transaminase and/or alanine transaminase > 2 x upper limit of normal (ULN), and/or bilirubin > 2 x ULN); planned surgery during the course of the trial; history of or current diagnosis of any cancer (except successfully treated basal cell carcinoma or cancer in full remission > 5 years after diagnosis); history of blood/bleeding disorders; blood donation in the previous 2 months; participation in a clinical research trial within 30 days prior to randomization; allergy or sensitivity to study supplement ingredients or to any food or beverage provided during the study; cognitive impairment and/or inability to give informed consent; any other condition which in the qualified investigator's opinion may have adversely affected the subject's ability to complete the study or its measures or which may have posed significant risk to the subject.

Sample Preparation and LC-MS

Dual extractions were carried out for complete analysis of the NAD + metabolome. For

analysis of NR, Nam, NA, MeNam, Me2PY, and Me4PY (group A analytes), samples were

18 18 18 spiked with 60 pmol of [ O1]-Nam, [ O1]-NR, and [D 3, O1]-MeNam and 240 pmol [D 4]-NA

(internal standard (IS) A). For analysis of NAD +, NADP +, NMN, NAR, NAMN, NAAD, and ADPR

(group B analytes), samples were dosed with 13 C-yeast extract (IS B) as described (1) (Chapter

2.1).

Human Samples

100 µl of urine was mixed with 20 µl of IS A in 5% (v/v) formic acid or IS B in water for

the analysis of group A and B analytes, respectively. 50 µl of ice-cold methanol was added and

the mixture vortexed prior to centrifugation at 16.1 k g at 4 °C for 10 min. Supernatants were

injected without further dilution and analyzed as described below. Standard curves and quality

controls for the complete analysis were prepared in the same manner as described for urine

samples but in water.

To quantify group A analytes in plasma, 100 µl of plasma was added to 20 µl of IS A

prepared in 5% (v/v) formic acid and mixed with 400 µl of ice-cold methanol. The mixture was

allowed to sit on ice for 20 min then centrifuged as described for urine. After drying under

vacuum overnight at 35 °C, the sample was reconstituted in 100 µl of water. To quantify group B

analytes, 100 µl of plasma was added to 10 µl of IS B in water and mixed with 300 µl of

acetonitrile with vortexing for 15 sec. After briefly resting on ice, the samples were centrifuged

as above. Supernatants were applied to Phenomenex Phree© SPE cartridges (Torrance,

California, USA) and the flow-through collected. 200 µl of aqueous acetonitrile (4 volumes

acetonitrile:1 volume water) was also applied and the flow through collected. The flow-through

from both steps was combined and dried via speed vacuum. Samples were reconstituted in 60

µl of water. Standard curves and quality controls for both analyses were prepared in donor

plasma (University of Iowa DeGowin Blood Center, Iowa City, IA, USA) and extracted using the same method employed for plasma samples.

Blood cell fractions were thawed on ice and simultaneously extracted for both A and B analyses when possible. 100 µl of sample was added to either 20 µl of IS A in 5% formic acid

(v/v) or 10 µl IS B in water for quantification of group A and B analytes, respectively. Samples were then mixed with 300 µl of acetonitrile and vortexed for 15 sec. Samples were shaken for 5 min at 40 °C then centrifuged as described above. For group A analytes, supernatants were dried via speed vacuum overnight at 35 °C after this step. For group B analytes, supernatant was applied to Phenomenex Phree© SPE cartridges and treated in the same manner as described above for quantification of group B analytes in plasma. Immediately prior to analysis, samples were reconstituted in either 100 µl of 10 mM ammonium acetate with 0.1% formic acid

(for group A analyte quantification) or 100 µl of 5% (v/v) aqueous methanol (for group B analyte quantification). Standard curves were prepared in water and processed in the same manner as samples.

Murine Samples

On the days of gavage and sacrifice, mice were weighed and placed into weight- matched groups. By oral gavage, mice were given either 185 mg NR Cl per kg body weight via

13 a 37 mg NR Cl/ml saline solution or equimolar [ C1, D 1]-NR, NA or Nam. On each day of sacrifice, a saline treated group was sacrificed and served as time zero. Mice were administered vitamin or vehicle at appropriate times prior to sacrifice. All sacrifices took place between 1 and

4 pm with each group being sacrificed on average at 2 pm. Mice were live-decapitated prior to collection of the medullary lobe of the liver, which was harvested by freeze-clamping in liquid nitrogen. All tissues were stored at -80 °C prior to extraction.

Murine liver obtained by freeze-clamp was pulverized using a Bessman pulverizer (100 –

1000 mg size) (Spectrum® Laboratories, Rancho Dominguez, California) cooled to liquid N 2

temperatures. Each pulverized liver sample was aliquoted (5 – 20 mg) into two liquid N 2 cooled

1.5 ml centrifuge tubes, which were stored at -80 °C until analysis. Prior to extraction, IS A and

IS B were added to separate aliquots resting on dry ice for quantification of group A and B analytes, respectively. Samples were extracted as described in (Chapter 2.2: Quantification of the Oxidized NAD Metabolome in Liver). Prior to LC-MS/MS analysis, samples were resuspended in 40 µl of 10 mM ammonium acetate (>99% pure) in LCMS-grade water. Sample

13 preparation following [ C1, D 1]-NR administration differed only in the following respect. 60 pmol

18 of [D 4]-Nam and [D 3, O1]-MeNam and 240 pmol of [D 4]-NA was added to sample in lieu of IS A.

Standard curves were performed without extraction in water.

For both human and mouse samples, samples were transferred to Waters polypropylene

plastic total recovery vials (Part # 186002639) after extraction or preparation and stored in a

Waters Acquity H class autosampler maintained at 8 °C until injection. In all cases, 10 µl of

extract was loaded onto the column.

LC-MS

Separation and quantitation of analytes were performed with a Waters Acquity LC

interfaced with a Waters TQD mass spectrometer operated in positive ion multiple reaction

monitoring mode as described(1). MeNam, Me2PY, and Me4PY were added to the analysis and

detected as described (Chapter 2.2: Addition of MeNam, Me4PY, and Me2PY to the NAD

Metabolomic Assay). For the analysis of urine, plasma, and murine liver, group A analytes were

separated as described for the acid separation(1). In blood cells, group A analytes were

separated on a 2.1 x 150 mm Synergy Fusion-RP (Phenomenex, Torrance, CA, US) using the

same gradient and mobile phase as described for the acid separation(1). For human samples,

group B analytes were separated using the mobile phase and gradient as previously described

for the alkaline separation(1). Murine liver extracts were analyzed using a slightly altered

alkaline separation on a 2.1 x 100 mm Thermo Hypercarb column. Specifically, flow rate was

increased to 0.55 ml/min and run time shortened to 11.6 minutes. Separation was performed

using a modified gradient with initial equilibration at 3% B, a 0.9 minute hold, a gradient to 50%

B over 6.3 minutes, followed by a 1 minute wash at 90% B and a 3 minute re-equilibration at 3%

Analytes in plasma were quantified by dividing their peak areas by IS peak areas and comparing the ratio to a background-subtracted standard curve. Analytes in all other matrices were quantified by dividing their peak areas by IS peak areas and comparing the ratio to a standard curve in water. Urinary metabolites were normalized to creatinine concentrations.

Hepatic metabolites were normalized to the wet liver weights.

For analysis of enrichment in murine liver, metabolites were separated following the same LC procedure described above and detected using a Waters Premier Q-TOF operated in positive ion, full scan mode. Leucine enkephalin was infused to ensure high mass accuracy.

Enrichment data were corrected for natural isotope abundance based on theoretical isotope

13 distribution, 13-carbon abundance skew, and the purity of the labeled standard (3/97% [ C1]-

13 NR/[ C1, D 1]-NR). Quantitation was performed on the Waters TQD as described above and

used to determine the quantity of non-labeled and labeled metabolites.

5.9 Supplemental Tables and Figures for Chapter 5.5-5.6

Table 5.2 Plasma NAD + metabolites (µM) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days. Time (hr) Nam MeNam Me4PY Me2PY 0 0.70 0.10 0.60 2.2 0.6 0.90 0.40 1.1 5.7 1 1.2 0.60 1.4 5.7 1.4 1.1 0.50 1.4 6.6 2.7 0.90 0.60 1.8 7.5 4.1 1.2 0.80 2.6 10 7.7 1.1 0.70 3.2 13 8.1 0.60 0.90 3.8 14 23.8 0.80 0.90 3.9 14 167.6 1.5 2.1 4.6 17

Table 5.3 Urinary NAD + metabolites (µmol/mmol creatinine) in a 52 year-old male who orally ingested 1000 mg NR Cl for 7 consecutive days. Time (hr) Nam MeNam Me4PY Me2PY Pre-1st dose 0.26 5.5 2.8 13 0 - 4.1 0.16 6.5 3.8 20 4.1 - 7.7 2.9 26 21 180 8.1 – 12 1.8 26 24 99 Pre-2nd dose 1.4 29 29 108 Pre-5th dose 1.2 30 24 80

a 400 b 250 ### ††† 300 ‡ 200 200 150 100

100 0 -100 50 -200 0 m 0 5 10 15 NR NA R A m Na N N a c Time (hrs) N 200 ††† ** *** ‡‡‡ 150

‡‡ 100 ‡ ‡‡ 50

0 m NR NA Na

Figure 5.5 Hepatic NR, NAR, and Me2PY concentrations after gavage of NR, Nam and NA. Either saline (orange) or equivalent moles of NR, NA or Nam were administered to male C57BL6/J mice by gavage. To control for circadian effects, gavage was performed at indicated times prior to a common ~2 pm sacrifice. At time of sacrifice, mice were live decapitated and livers collected for analysis via LC-MS/MS. In the left panels, the hepatic concentrations of each metabolite are shown as a function of the four gavages. In the right panels, the baseline subtracted 12-hour areas under the curve are shown. Left panels: ‡ p-value < 0.05; ‡‡ p-value < 0.01; ‡‡‡ p-value <0.001 Nam vs NA; † p-value < 0.05; †† p-value < 0.01; ††† p-value < 0.001 Nam vs NR; # p-value < 0.05; ## p-value < 0.01; ### p-value < 0.05 NA vs NR; Right panel: * p-value < 0.05; ** p-value < 0.01; *** p-value <0.001.

5.10 Perspective on Chapter 5

Introduction

In sections 3-6 of this chapter, we present the first evidence that NR augments the

human NAD metabolome and identify NAAD as a possible non-obvious potential biomarker for

NAD + elevation. During our investigation of NAAD, we compared NR to Nam and NA efficacy in

altering the murine hepatic NAD metabolome and found that 1) NR is most efficacious in

increasing NAD + and is uniquely metabolized, 2) NR and Nam increase murine hepatic NAAD,

and 3), through the use of stable isotope technologies, prove direct contribution of NR to NAAD.

We then performed the first human trial of the effect of NR and found a clear effect of NR on the

NAD metabolome in human blood cells and confirmed that NAAD responds to NR

supplementation in a dose-dependent manner. The following sections are my perspective

regarding what has been written by myself and Dr. Brenner regarding the data that Dr. Mark

Schmidt and I generated. In addition, I present and discuss the effects of intraperitoneal (IP)

injection on the murine hepatic NAD metabolome and the effect of both IP injection and gavage

of double labeled NR on muscle from the same mice.

Results and Discussion

In the above sections, we set out to test the efficacy of NR in altering the NAD

metabolome in a human being and performed the first head-to-head comparison of NR to Nam

and NA. We first supplemented a healthy 52 year old, male with 1 g NR Cl over a week.

Peripheral blood monocytes (PBMCs) were collected from blood at each time point as well as

urine. Time points were taken within 24 hours and after six subsequent dosages. NR effectively

increased NAD + within 24 hours starting 4 hours after ingestion and its level remained increased compared to the pre-dose after six dosages (Table 5.1). NADP + and NMN were also elevated.

Overall, these data represent the first report of the effect of NR on a human subject and reveal

NR can increase NAD + in a human being. Further, this initial experiment revealed blood is an

accessible sample for the effects of NR on the NAD metabolome for future clinical trials.

Methylated Nam derivatives MeNam, Me2PY, and Me4PY were expectedly also highly elevated by NR. Regardless of the amount of NR absorbed intact versus as Nam, any contribution of NR to a cellular fraction could convert to Nam through either enzymatic consumption of NAD + synthesized from NR or direct phosphorolysis via purine nucleoside phosphorylase (PNP) activity (13). These methylated species have been reported as biomarkers for B 3 vitamin deficiency and supplementation in urine and blood (157-161) and

hence are indicative of cellular uptake of NA and Nam and presumably for NAD + synthesis.

However, these metabolites are a diversion of Nam from NAD + biosynthesis and theoretically could appear without contributing to NAD + and, hence, are not truly biomarkers of NAD +

elevation. Others have suggested measurement of NAD+ and NADP + as indicative of whole

body NAD + status (162) but these measurements may not be indicative (163) possibly due to a

buffering capacity of NAD + concentration and/or the inherent analytical problems in detecting a

small change in a very abundant metabolite.

In our study, we identified a rise in NAAD from below the limit of quantification (<0.02

µM) by at least 45-fold in the PBMC fraction (Table 5.1). The appearance of NAAD is

unexpected since neither NR nor Nam are thought to contribute to the deamidated pathway

(Figure 5.1), but could represent a novel, accessible biomarker for efficacious NAD +

supplementation. Unlike the methylated Nam derivatives, NAAD is expected to contribute to

NAD + as it is the proximal deamidated precursor. Even if NAAD were converted to NAMN and

subsequently to NAR intracellularly, the NAR could be recycled back to NAD + through the NRK

pathway or exported and contribute to NAD + in near or distant cells (164). Indeed, we do

observe an increase in NAR in the plasma and urine of subjects ingesting NR (Appendix A:

Table A.2 and A.3).

In furthering our investigation into the NAAD phenomenon, we turned to the murine model. While performing these experiments, we were also interested in the kinetics of NR compared to NA and Nam after a single dose. The NAD+ biosynthetic machinery differs in a cell

and tissue specific manner (87). These biosynthetic processes are also competing with other

modifying activities, such as methylation and/or oxidation (Chapter 1.1), which would divert

pyridine from NAD +. Hence, the efficacy of NR utilization compared to these other precursors

remains an unresolved and crucial question to the future use of NR. We dissected the livers and

muscles 18 from mice after either gavage 19 or intraperitoneal injection (IP) of 185 mg/kg body weight NR Cl or mole equivalent of Nam and NA and analyzed using LC-MS/MS. Saline injections were also performed. The liver NAD metabolome exquisitely responded to all three precursors; however, NR displayed unique and superior effects in increasing NAD + (four-fold compared to two-fold after Nam and NA) after gavage (Figure 5.2b). This profile is in stark contrast to NAD + after IP injection, whereby all three precursors produced indistinguishable elevations (Figure 5.6b), suggesting that absorption of these metabolites may be responsible for the differential observed kinetics. The route and kinetics of NR absorption are current active projects in the laboratory. The IP experiments also revealed that the mode of NR delivery could differentially effect the NAD metabolome. NR depressed ADPR concentration (Figure 5.6j) after injection but not gavage (Figure 5.2j), suggesting injection of NR may act as an NAD +

consuming enzyme inhibitor. Elucidation of the effect of NR IP injection on the liver warrants

further investigation. Intriguingly, Nam and NR increased NAAD after gavage and IP (Figure 5.2i

and 5.6i) and this elevation correlated with the efficacy of each in increasing NAD + (Figure 5.2b

and 5.6b) showing that NAAD correlates with the concentration of NAD + in a non-accessible

18 See Chapter 2.3: Considerations of Quantitative NAD Metabolomics in Mammalian Tissues for information regarding tissue treatment. 19 Gavage is the process by which a drug or food is delivered directly to the stomach.

tissue in clinic. These results coupled with the human n of one experiment indicated that NAAD may serve as a future clinical biomarker for the efficiency of NAD + supplementation.

The appearance of NAAD in such great quantity from Nam and NR begs the question about how we will re-draw the NAD + pathway (Figure 5.1). The amidated precursors Nam and

NR could either directly contribute to NAAD through some as yet known mammalian pathway or act to inhibit NAD synthase, the enzyme converting NAAD to NAD +. We tested these

possibilities by administering a specially labeled NR that contains a deuterium on the ribose and

a 13-carbon on the nicotinamide moiety. If NR contributed directly to NAAD, we expected to

observe enrichment of the NAAD pool. Indeed, we observed NAAD enrichment by both gavage

(Figure 5.3e) and IP (Figure 5.7e) in liver and in muscle (Figure 5.8e and 5.9f), suggesting the

rise in NAAD is an effect of an unknown deamidating pathway that exists in both liver and

muscle.

In order to begin to establish NAAD as indicative of efficient NAD + synthesis, we performed the first clinical trial of NR at several dosages (100, 300, and 1000 mg NR Cl) with a one week wash out period on 12 individuals (6 males and 6 females between 30 and 55 years of age at normal BMI (18.5 – 29 kg/m 2). NR increased NAD + in the blood cell fraction compared

to pre-dose when collapsing all dosages (Figure 5.4a). The methylated derivatives rose and did

so in a dose-dependent manner (Figure 5.4c-e). Additionally and in agreement with the initial

human n of one study and murine study, NAAD increased in a dose-dependent manner,

indicating that NAAD may be an applicable biomarker for NR effectiveness.

Direct contribution of NR to NAAD proves the existence of an NR to NAAD biosynthetic

route and necessitates the existence of an unknown mammalian deamidase. In the 1960s, Nam

deamidating activity was reported in liver (165) but displayed a non-physiological K m at above at

least 40 mM (166). At that time, it was thought that all Nam was converted to NA and utilized

much as it is in yeast (Figure 1.1); however, later work revealed high dose Nam caused the

appearance of non-amidated intermediates and was hypothesized to occur due to bacterial

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deamidases in the gut (167). Our work argues against such a mechanism given that NA was detected at ~320 pmol/mg liver at five minutes after NA gavage/IP but not detected at any time after NR nor Nam. Further, IP administration of the double labeled NR produced a clear enrichment in the M+2 isotopologue of NAAD (Figure 5.7 and 5.9). If NR were hydrolyzed to

Nam and the Nam deamidated to NA, enrichment would only occur in the M+1 NAAD isotopologue. To put it another way, the NAAD produced from NR in the labeled experiment was from intact, non-hydrolyzed NR. And though NR could be deamidated to NAR, NAR did not rise after supplementation of any B 3 vitamin, suggesting but not completely excluding that NR is not a direct NAR precursor. Together, these data strongly suggest NA is not the initial precursor to the observed NAAD and that the NAAD may be synthesized within the liver which necessitates a mammalian deamidation pathway exists.

But if not NA and likely not NAR, then what? Since both NR and Nam increase NAAD, it must be a shared metabolite downstream of both precursors (NMN or NAD +). We argue that

NAD + is the likely source of NAAD and that its increase by at least 2 fold induces a deamidating

pathway. Though this may be the case, the data we have presented does not exclude NMN as

the precursor. Indeed, NAMN, the deamidated analog of NMN, increases at the same time as

NAAD. Though this NAMN could be a result of reversible NMNAT1-3 activity (44) as suggested

above, the “forward” (NMN/NAMN to NAD +/NAAD) direction of the reaction in vivo appears favorable due to limiting inorganic phosphate (87). As it stands, the true route from Nam/NR to

NAAD remains to be elucidated in future investigations.

Regardless of the route of NAAD synthesis from the amidated precursors, further investigation is required to establish NAAD as a biomarker in the clinic for the effectiveness of

NR in NAD + synthesis. At present, we are unaware of the correlation between blood NAAD and hepatic NAAD and NAD + after NR administration. If NAAD is to be established as a robust accessible biomarker for increased NAD + in an inaccessible tissue in clinic, then tissue NAD +

concentration must correlate with blood NAAD concentration. Future work in model organisms

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and in humans could be used to examine the correlative value of the relationship between NR supplementation, blood NAAD concentration, and whole body increases in NAD +. Initial results from Chapter 6 may indicate that NAAD indeed correlates with NAD + abundance in liver.

Perhaps more importantly, the metabolism of NR requires careful and thorough

investigation. At present, NR has not been detected in the blood cell fraction nor in plasma due

to difficulty in its extraction. As it stands, we have measured that NR is absorbed and circulates

in a “shadowy” manner. NR does not simply serve as a Nam precursor given the very distinct

overall effects on the hepatic NAD metabolome after gavage. However, hepatic detection of NR

varied and displayed no response to NR administration nor that of Nam and NA (Figure 5.5b),

but was detected after IP of double labeled NR in liver (Figure 5.7) and muscle (Figure 5.9),

revealing NR does circulate. Additionally, the shadow of NR was detected as M + 2 NAD + in both liver and muscle. In both cases, establishing the accuracy of double label enrichment the nucleotides of the NAD metabolome is difficult to determine without purified standards. These standards are not commercially available and not easily synthesized. To strengthen the claim of intact NR circulation and utilization, loss of function NRK rodent models should be utilized. If NR indeed does not enter a cell intact, then NAD + synthesis after NR administration would be

independent of the NRK pathway and completely dependent upon NAMPT (a route in which NR

is hydrolyzed to Nam then utilized). A rodent model lacking NRK activity would be expected to

experience a complete loss of enrichment in the M+2 NAD + isotopologue and complete

preservation of the M + 1 isotopologue. A whole body NRK1 knockout mouse has been

generated (150) and experiments are underway to definitively test NR intact absorption.

Methods

Unless otherwise stated, all methods are as described in Chapter 5.3: Methods and

Chapter 2.2: Quantification of the Oxidized NAD Metabolome in Skeletal Muscle.

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5.11 Figures for 5.10 a # † b ### 10 ††† ‡ 8

2 NMN (pmol/mg) NMN AUC (pmol*hr/mg) AUC 0 0 20 40 60 80 R A m R A m N N a N N a Time (min) N N c d ### ‡ ††† *** # †† 2000 ‡‡‡ *** *** ††† ‡‡ 1500 ††† ‡‡‡ ††† ††† ‡‡‡ 1000 ‡‡‡

500 AUC (pmol*hr/mg) AUC 0 0 20 40 60 80 R A m R N N a N NA am N Time (min) N e f ### *** ††† *** 10 ### 30 ### *** *** ## ‡‡‡ *** *** ††† ## †† ### †† 8 ‡‡‡ ### †† ‡‡‡ †† ‡‡‡ ### †† 20 ‡‡‡ 6 ‡‡‡ ‡‡‡ ‡

4 10 2 MeNam (pmol/mg) MeNam 0 0 0 20 40 60 80 0 20 40 60 80 m NR NA am NR NA a g Time (min) N h Time (min) N

### ### 30 ## ††† *** 800 ‡‡‡ ‡‡‡ ### †† ‡‡‡ *** *** *** *** ### ‡‡‡ 600 † ### 20 ‡‡‡ ‡‡‡ ### 400 ‡‡‡ 10 200 ### ‡‡‡ Me2PY (pmol/mg) Me2PY AUC (pmol*hr/mg) AUC 0 0 0 20 40 60 80 0 20 40 60 80 R A m R A m N N a N N a i Time (min) N j Time (min) N ### # ††† †† ### 250 ‡ ### ### ††† ### ## ††† ††† ††† * ††† ** 200 ** 150 ### 100 †

50 ADPR (pmol/mg) ADPR 0 0 20 40 60 80 R A m R A m N N a N N a Time (min) N N

Figure 5.6 IP administration of NR, Nam, and NA produce similar effects on murine liver NAD metabolome. Either saline (orange) or equivalent moles of NR (black), NA (blue) and Nam (green) were administered to male C57BL6/J mice by IP. Livers were excised and freeze-clamped and then analyzed by LC-MS/MS. In the left panels, the hepatic concentrations of each metabolite are shown as a function of drug or vehicle. In the right panels, the baseline subtracted areas under the curve are shown. Left panels: ‡ p-value < 0.05; ‡‡ p-value < 0.01; ‡‡‡ p-value <0.001 Nam vs NA; † p-value < 0.05; †† p-value < 0.01; ††† p-value < 0.001 Nam vs NR; # p-value < 0.05; ## p-value < 0.01; ### p-value < 0.05 NA vs NR; Right panel: * p-value < 0.05; ** p-value < 0.01; *** p-value <0.001.

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a 5000 b 4000 M 3000 M+1 2000 M+2 1000

400

200

0 0 5 0 5 0 0 1 30 60 1 3 6 c Time (hours) d

0 0 0 0 0 0 15 3 6 15 3 6 e

0 5 0 0 1 3 6 Figure 5.7 NR directly contributes to hepatic NAAD after IP injection. Double-labeled NR was intraperitoneal injected into mice. At indicated times, mice were sacrificed and livers freeze-clamped for isotopic enrichment analysis using LC-MS. a-b. Isotopic enrichment of NAD + and NADP + over time at both the M+1 and M+2 mass shifts. c-d. Isotopic enrichment of Nam and MeNam over time at the M+1 mass shift. e. Isotopic enrichment of NAAD over time at both the M+1 and M+2 mass shifts. Similar in effect to gavage, NR directly contributes to hepatic NAAD. NR contributed a much lower amount to NAD + over the hour than by gavage but much more of it was intact rather than metabolized to Nam.

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a b 25 20 15 10 5 1.5

1.0 (pmol/mg muscle) (pmol/mg

+ 0.5

0.0 NAD 0 2 4 6 8 0 2 4 6 8 c d Time (hours) 150 6

100 4

50 2

0 0 0 2 4 6 8 0 2 4 6 8 e Time (hours) Time (hours) 600

400

200

0 0 2 4 6 8 Time (hours) Figure 5.8 NR contributes to muscle NAAD following gavage. Quadriceps was dissected and freeze-clamped from the same mice that contributed liver after double labeled NR was gavaged. Quadriceps were extracted and analyzed for enrichment using LC-MS. a-b. Isotopic enrichment of NAD + and NADP + over time at both the M+1 and M+2 mass shifts. c-d. Isotopic enrichment of Nam and MeNam over time at the M+1 mass shift. e. Isotopic enrichment of NAAD over time at both the M+1 and M+2 mass shifts. The data reveal that NR also contributes to NAAD but only as Nam and not in a consistent manner. Additionally, NAD + is not as effectively increased following NR gavage as in liver.

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a b 800 M 700 M+1 600 M+2 500 400 60 40 20 0 0 0 5 0 0 15 30 60 d 1 3 6 c Time (hours)

0 5 0 0 0 0 0 e 1 3 6 f 15 3 6

0 0 0 0 5 0 0 15 3 6 1 3 6

Figure 5.9 NR contributes to muscle NAAD following IP. Quadriceps was dissected and freeze-clamped from the same mice that contributed liver after double labeled NR was gavaged. Quadriceps were extracted and analyzed for enrichment using LC-MS. a. Quantitation of double labeled NR. Endogenous NR was non-quantifiable. b-c. Isotopic enrichment of NAD + and NADP + over time at both the M+1 and M+2 mass shifts. d-e. Isotopic enrichment of Nam and MeNam over time at the M+1 mass shift. f. Isotopic enrichment of NAAD over time at both the M+1 and M+2 mass shifts. Double labeled NR was detected in muscle following IP injection, meaning NR does circulate to muscle intact. As observed with gavage, NR contributed to NAAD only as Nam and poorly increased NAD + compared to liver IP. Together, the data suggest NR may be mainly metabolized through first pass metabolism by liver.

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CHAPTER 6 NICOTINAMIDE RIBOSIDE PREVENTS ALCOHOL INDUCED FATTY LIVER

Samuel A.J. Trammell 1,2 , Sirisha Ghanta 1, Kyle Klingbeil 1, Keisuke Yaku 1, Nicholas M. Riley 3,

Joshua J. Coon 3, and Charles Brenner 1,2

1Department of Biochemistry, 2 Interdisciplinary Graduate Program in Genetics, Carver College

of Medicine, University of Iowa, Iowa City, IA 52242, USA

3Department of Chemistry and Genome Center of Wisconsin, Madison, WI 53706

6.1 Distribution of Work

Experiments were designed by SG, CB, and I. All mouse husbandry in the initial experiment

was performed equally by myself and SG. In the subsequent experiment, KY and I equally

shared all mouse husbandry. Mouse phenotypic data were collected as a joint effort of myself,

SG, and KY. Western blotting was performed by KK with guidance from me. All microscopy was

performed by SG. All metabolomic data were collected and analyzed by myself. All proteomic

data were generated by NMR in JJC’s laboratory. Analysis of proteomic data was performed by

myself.

6.2 Abstract

Chronic alcohol consumption can lead to fatty liver disease (alcoholic fatty liver disease

(AFLD)) through poorly understood mechanisms. Ethanol metabolism causes a reductive skew

in the NAD +/NADH ratio and correlates with mitochondrial protein lysine hyperacetylation in a manner similar to loss of the NAD + consuming enzyme Sirt3, suggesting NAD + metabolic

dysfunction may be related to the hepatic dyslipidemia. We hypothesized mitochondrial protein

lysine hyperacetylation acts to reconfigure carbon metabolism from catabolism to anabolism,

leading to unfettered fat accumulation. NR, precursor to NAD +, opposes age-related and

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metabolic dysfunctions, including non-AFLD. Here, we employed NR as a chemical tool to determine the role of NAD + metabolism and mitochondrial protein acetylation in the etiology of

AFLD. In so doing, we identified NR as a potential anti-AFLD agent and provide evidence that

NR could act to reconfigure mitochondria back to respiration. Unfortunately, we observed that

NR increased consumption of the control and ethanolic diet, complicating further

experimentation.

6.3 Introduction

Alcohol in the form of ethanol is an important part of American culture and often a

substance of abuse. Approximately 20% of alcoholics and heavy drinkers develop alcoholic fatty

liver disease (AFLD), which with continued alcohol abuse, leads to liver damage (168). The liver

is especially vulnerable to chronic ethanol ingestion since it is the site of ethanol metabolism

(169). Ethanol is metabolized to acetaldehyde then acetate with two moles of NAD + reduced to

NADH per mole of ethanol. Unlike acute ingestion, chronic ethanol ingestion induces microsomal ethanol oxidizing system which oxidizes NADPH to NADP + and ethanol to acetaldehyde, which is then converted to acetate as above (170, 171). Acute and chronic ingestion causes a decrease in the NAD +/NADH ratio in the cytoplasm and, due to the malate-

aspartate shuttle and mitochondrial acetaldehyde dehydrogenase activity, convey this ratio to

mitochondria (172). The reductive effect of ethanol inhibits glycolysis, oxidative phosphorylation

(172, 173), and fatty acid oxidation (174) and was thought to explain the accumulation of fat.

However, preservation of the NAD +/NADH ratio does not inhibit AFLD (175, 176) indicating other aspects of ethanol metabolism are responsible for fatty liver.

Chronic ethanol ingestion causes hyperacylation (including acetylation) of mitochondrial protein lysines (177-179). Mitochondrial acylation is reversible through the action of Sirt3 and

Sirt5. Sirt3 and Sirt5 are NAD +-dependent deacylases with differing substrate specificities. Sirt3

primarily removes acetylation (180, 181) and Sirt5 removes malonyl, succinyl, glutaryl, and

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other acyl groups (10, 182). Acylation can be enzymatically inhibitory (10, 183-185) or, at least in one case, activating (186) and appears to affect multiple targets within pathways related to amino acid, carbohydrate, and lipid metabolism (187). Indeed, Sirt3 -/- mice experience mitochondrial hyperacetylation and decreased lipid catabolism (188), indicating mitochondrial acetylation inhibits and Sirt3 activates lipid utilization.

We propose that mitochondrial protein acetylation is part of the etiology of AFLD.

Mitochondrial protein acetylation has been described as a cell intrinsic long lasting satiety program that diverts mitochondria from carbon oxidation to carbon storage (189). In this model, chronic high caloric, low vitamin dense diets induce small molecule metabolic changes ( i.e.

decreases NAD +/NADH) and, as consequence, leads to increased mitochondrial protein

acetylation. Unlike nuclear protein acetylation, mitochondrial protein acetylation appears to be

non-enzymatically driven and instead depends upon the mitochondrial matrix pH and Ac-CoA

concentration (190-192). Protons from NADH are exported across the inner mitochondrial

membrane to create the proton-motive force for ATP synthesis. As consequence, the

mitochondrial matrix pH is alkaline (pH 7.6 – 8) (192), allowing for deprotonization of lysines

with depressed pKas. As observed with high fat diet (193), a decrease in the NAD +/NADH encumbers the TCA cycle and increases mitochondrial Ac-CoA (190). These chemical conditions promote transfer of the acetyl group from Ac-CoA to a deprotonated protein lysine and, in chronic conditions, can account for hyperacetylation.

Ethanol metabolism recapitulates the conditions of the overfed state and could directly contribute to the protein acetylation. The vast majority of ethanol is oxidized to CO 2 in muscle and not stored as fat (194, 195). Fat as a result of ethanol ingestion appears to be from dietary sources and fat mobilization from adipocytes (196-199) indicating ethanol induces a metabolic switch in mitochondria towards fat storage. Though restoring the oxidative balance of NAD + does not prevent fatty liver, the reductive environment coupled with the increased availability of

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acetate (Ac-CoA precursor) (200) may be crucial in causing ethanol induced mitochondrial hyperacetylation, resulting in AFLD.

Though loss of Sirt3 activity is certainly involved in ethanol induced hyperacetylation

(201), involvement of the other seven sirtuins (Chapter 1.2) cannot be ruled out (202).

Additionally, as discussed in Chapter 5, increasing sirtuin enzymatic activity without increasing

NAD + does not necessarily result in increased deacylation. Here we employed novel NAD + precursor NR to unravel the role of mitochondrial hyperacetylation in the etiology of AFLD. NR is a far superior whole cell (Figure 5.2) and perhaps mitochondrial (Chapter 1.2) NAD + precursor than Nam and NA and has been shown to oppose non-alcoholic fatty liver disease (53). We predicted that NR would oppose mitochondrial hyperacetylation and, if hyperacetylation is necessary for AFLD, inhibit hepatic dyslipidemia. Here, we utilized a chronic alcoholic mouse model whereby animals derived increasing calories from ethanol over a six week period and remained on a diet of 30% calories from ethanol for two to three weeks. We reveal that NR increases hepatic NAD + in an alcoholic mouse model and moderately opposes fatty liver

development. However, mortality was high in the initial experiment which we had hoped to

overcome through alterations of the animal protocol. In so doing, we found that NR increases

consumption of liquid diet and confounded the experimental outcomes. We discuss possible

improvements to the model in future experiments.

6.4 Materials and Methods

Animal Husbandry and Experimental Design

All animal protocols were approved by the University of Iowa Institutional Animal Care

and Use Committee. Male C57BL/6J mice were purchased from Jackson Laboratories. Control

(catalog #: F1259SP) and ethanol (catalog #: F1697SP, Lieber-DeCarli ’82 formulation) diets

were purchased from Bio-Serv (Frenchtown, NJ, USA).

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Initial Experiment

At ten weeks of age, mice were transitioned to a liquid food following manufacturer’s

instructions. After transition, mice were split equally into a control liquid diet group, ethanol diet

group, and ethanol + NR (0.33 g/l). Those fed ethanol were transitioned from control liquid diet

to ethanol following manufacturer’s instructions. Ethanol content was adjusted as follows: 2%

(w/v) for two weeks, 3.1% (w/v) for two weeks, and finally 4.2% (w/v) for three weeks. Diet was

contained in standard mouse water bottles and changed every 48 hours or as necessary.

Volume of diet consumed was measured at each diet change. Mice were weighed once a week

to monitor health. Mice were euthanized using CO 2 or live decapitation. Livers were dissected

and weighed and portioned for metabolomics, proteomics, western blotting, and microscopy.

Subsequent Experiment

All parameters remained the same as in the initial experiment except that the animals

remained on 4.2% (w/v) diet for four weeks rather than three and standard water bottles were

exchanged with feeding tubes (catalog #: 9019, Bio-Serv) that were changed every day.

Mitochondrial Isolation

Mitochondria were isolated as described in (203).Briefly, 150 mg of liver tissue was

homogenized in 1 mL of isolation buffer (70 mM sucrose, 210 mM mannitol, 5 mM HEPES, 1

mM EGTA, 10 mM Nam, and 0.5% (w/v) fatty acid free BSA) on ice. An aliquot of homogenate

was saved (~200 µL). The rest of the preparation was then centrifuged at 1,000 x g and 4 °C for

10 minutes. The supernatant containing cytoplasm and mitochondria was removed. The pellet

(nuclear fraction) was snap frozen using liquid nitrogen. The supernatant was centrifuged at

10,000 x g and 4 °C for 10 minutes to pellet mitochondria. The supernatant (cytoplasmic

fraction) was snap frozen. The mitochondria pellet was washed once with isolation buffer at 10,

000 x g and 4 °C for 10 minutes. The supernatant was discarded and the mitochondrial pellet

snap frozen.

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Western Blotting

Mitochondrial fractions were thawed in lysis buffer (50 mM Tris-HCl, 150 mM KCl, 1 mM

EDTA, 1% NP-40, 1 mM Na butyrate, 5 mM nicotinamide, and Roche complete protease

cocktail). Protein was loaded and separated via standard SDS-PAGE and transferred to PVDF membrane. Membranes were blocked with 5% skim milk in TBST for 30 minutes, then washed five times with TBST. Membranes were probed with primary antibodies directed against acetyllysine (Cell Signaling, Boston, MA or PTM Biolabs, Inc., Chicago, IL). The membrane was incubated with secondary antibody for 1 hour. Horseradish peroxidase was applied and membranes were imaged following standard procedures.

Microscopy

A small portion of liver was dissected out and frozen in clear freezing media (purchased

from TBS – A Division of General Data Healthcare). Frozen tissue was sectioned (10 µm) and

placed onto positive slide glass and fixed in formaldehyde. Fixed tissue was washed with

distilled water and stained with Harris’ hematoxylin for 30 seconds. Slides were then washed

with running tap water for five minutes, washed with distilled water, and placed into Oil Red O

solution for 10 to fifteen minutes. Slides were washed with distilled water and imaged using an

Olympus BX61 upright microscopy with 20X magnification.

NAD Metabolomics

On the day of extraction, 300 mg of liver tissue was homogenized in 1 mL of isolation

buffer lacking Nam (see Mitochondrial Isolation). The preparation was snap frozen and stored at

-80 °C until analysis. 50 µL of liver homogenate was extracted with 300 µL of buffered ethanol

(75% Ethanol/25% 10 mM HEPES) heated at 80 °C and constant vigorous vortexing for 3

minutes. Extract was separated from particulate through centrifugation (16.1 x g, 10 minutes, 4

°C). Both extract and pellet were dried overnight using speed vacuum. Dry particulate was

weighed and used for normalization of mole amounts of metabolite. Extracts were re-suspended

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in LCMS grade water and then analyzed as described (Chapter 2.1 and 2.3) with the following differences. The acid separation internal standard solution contained 0.75 µM 18 O NR, 0.75 µM

18 O Nam, and 6 µM D 4 NA (purchased from C/D/N Isotopes, Pointe-Claire, Quebec, Canada).

Quantitation was performed using internal standards and calibration curve.

Acetylomics

Chemicals and supplies

The Tandem Mass Tags (TMT) reagents were purchased from Thermo-Pierce

(Rockford, IL). The BCA assay Protein Assay Kit was purchased from Pierce Biotechnology

(Rockford, IL). Trypsin Gold was purchased from Promega (Madison, WI). Sep-Pak tC18

cartridges were purchased from Waters (Milford, MA). A polysulfoethyl A column (9.4 mm x 200

mm, 5 mm, 200Å) was purchased from PolyLC (Columbia, MD). Bridged Ethylene Hybrid (BEH)

C18 resin (1.7m diameter particles, 130 Å pore size) was purchased from Waters (Milford, MA).

Fused-silica capillary tubing was purchased from Polymicro Technologies (Phoenix, AZ). Formic

acid and trifluoroacetic acid ampoules were purchased from Thermo Scientific (Rockford, IL).

Pan-acetyl lysine antibody-agarose conjugate was purchased from Immunechem (Burnaby,

Canada). Protease (complete mini ETDA-free) and phosphatase (PhosSTOP) inhibitors were

purchased from Roche (Mannheim, Germany). All other chemicals were purchased from Sigma-

Aldrich (St. Louis, MO).

Sample Preparation

Liver tissue samples were homogenized with 3 strokes of a motorized stirrer at 1000 rpm

in a Potter-Elvehjem tissue grinder with 1 mL of buffer (8 M urea, 50 mM Tris pH 8.0, 5 mM

CaCl2, 100 mM NaCl, protease inhibitors, and deacetylase inhibitors). Homogenates were then

sonicated at 5 W for 30 seconds and centrifuged at 10000xg to clear the lysate of debris.

Protein concentrations in lysates were quantified by BCA Protein from each sample (1 mg) was

reduced with 5 mM dithiothreitol for 45 minutes at 58 °C and then alkylated with 15 mM

iodoacetamide for 45 minutes at ambient temperature in the dark. The alkylation was quenched

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with 5 mM dithiothreitol. Following dilution to 1.5 M urea with 50 mM Tris pH 8.0, the samples were digested with trypsin (50:1 protein:enzyme) overnight. Additional trypsin (200:1 protein:enzyme) was spiked into the sample the following morning, digestions were quenched by TFA acidification two hours later, and samples were desalted with a tC18 sep-Pak. Desalted material was resuspended in 200 mM TEAB pH 8.5 and labeled with 10-plex TMT (only nine of the ten tags were used, with the lightest channel being omitted). Labeled peptides were combined and desalted. Labeling efficiency was evaluated by analyzing a test mixture by

LC/MS/MS for each experiment. Labeling efficiency was > 95%, calculated by the number of N- terminal labeled peptides divided by the total number of peptide identifications.

Fractionation and Enrichment

Labeled peptides were fractionated by strong cation exchange (SCX) on a polysulfoethyl

A column (0.4 mm x 200 mm) with mobile phases A: 5 mM KH 2PO 4 pH 2.7 and 30% acetonitrile; B: 5 mM KH 2PO 4 pH 2.7, 350 mM KCl, and 30% acetonitrile; C: 5 mM KH 2PO 4 pH

6.5, 500 mM KCL and 20% acetonitrile; D: water. Peptides were eluted over the following gradient on a Surveyor LC quaternary pump (Thermo) at 3 mL/min: 0-2 min, 100% A; 2-5 min,

0-10% B; 5-35 min, 10-60% B; 35-41 min, 60-100% B; followed by washes with C and D prior to re-equilibration with mobile phase A. Sixteen fractions were collected and desalted. A small portion (5%) of each was retained for protein analysis, while the remaining material was pooled into 6 fractions for acetyl lysine enrichment.

These pooled fractions were dissolved in 50 mM HEPES pH 7.6, 100 mM NaCl, and each fraction was combined with approximately 75 uL pan-acetyl lysine antibody-agarose conjugate. The samples were rotated overnight at 4 °C and then rinsed eight times with cold 50 mM HEPES pH 7.6, 100 mM NaCl. Rinses were followed by elution with 0.1% TFA, and eluted peptides were desalted prior to analysis.

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LC/MS/MS

All samples were analyzed by reverse phase liquid chromatography on a nanoAcquity

(Waters) coupled to an Orbitrap Elite (Thermo). Samples were loaded onto a 75 μm inner diameter analytical column made in-house, packed with 1.7m diameter, 130 Å pore size, BEH

C18 particles (Waters) to a final length of 30 cm. The column was heated to 62 °C for all runs.

The elution portion of the gradient was 5% to 30% B (A: water/0.2% formic acid; B: acetonitrile/0.2% formic acid) over 140 minutes for acetyl enriched fractions and 80 minutes for protein fractions.

Mass spectrometry instrument methods all started with one MS1 survey scan (resolution

= 60,000; 300 Th – 1,500 Th; target value = 1e6) followed by data dependent MS 2

fragmentation and analysis (resolution = 30,000) of the fifteen most intense precursors by

beam-type CAD (HCD; normalized collision energy = 35%, target value = 5e4). Only those

precursors with charge state +2 or higher were sampled for MS 2. The dynamic exclusion

duration was set to 40 seconds with a 10 ppm tolerance around the selected precursor and its

isotopes, and monoisotopic precursor selection was turned on. Isolation width was set to 1.80

Da, precursor injection time was capped at 200 ms, and the first mass value for HCD scans was

120 Th.

Database search, FDR filtering, and acetylation analysis

Spectra were converted to searchable text files using DTA generator. Generated text

files were searched for fully tryptic peptides with up to three missed cleavages against a UniProt

target-decoy database populated with mouse canonical plus isoforms (downloaded February

2014) using the Open Mass Spectrometry Search Algorithm (v. 2.1.8) (204). Mass tolerance

was set to ± 125 ppm for precursors and ± 0.02 Da for fragment ions. Carbamidomethylation of

cysteine, isobaric labeling of lysine, and isobaric labeling of the peptide N-terminus were

searched as fixed modifications for all samples. Enriched fractions were additionally searched

for variable acetylation modifications, in which the acetylation mass shift was set to the

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difference between an acetyl group and an isobaric label (-187.1523 Da) to allow the isobaric label on lysine to remain a fixed modification even for acetylated peptides. Search results were filtered to 1% FDR at the unique peptide level using the COMPASS software suite (205). TMT quantitation of identified peptides was performed within COMPASS, as previously reported

(206). Peptides were grouped into proteins according to previously reported rules and protein identifications were further filtered to 1% FDR (207). Protein quantitation was performed by summing all reporter ion intensities within each channel for each protein.

Acetylation events were localized to specific residues using previously described probabilistic methods (208). Briefly, for each peptide spectral match (PSM) that contains an acetyl modification, every possible peptide isoform was generated and fragmented in silico to produce theoretical fragmentation spectra. Each theoretical spectrum was compared to the experimental spectrum at 10 PPM m/z tolerances; the number of matching peaks was recorded and a p-value was calculated using a cumulative binomial distribution. An AScore (i.e. the difference of p-values) was calculated between every pair of isoforms. A peptide was declared

“localized” if all AScores for a particular isoform were larger than the minimum value (AScore =

13, p-value < 0.05) for every comparison. Localized acetylated peptides were grouped together if they share identical modification sites and the reporter ion intensities were summed; peptides with C-terminal acetylation are excluded from quantitation.

Protein Normalization

All reporter ion intensities were log2 transformed and mean normalized for every acetyl isoform and protein. To account for protein abundance differences, the acetyl isoforms were normalized by subtracting the quantitative value of the reporter ion channel for the corresponding protein from the value for each acetyl isoform reporter ion channel. This gives a protein normalized acetylation mean normalized value which is then used to investigate fold changes between conditions. Fold change calculations were made by averaging the protein normalized values for each condition and then calculating the difference of averages.

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Statistical Analysis

Unless otherwise stated, regular two-way ANOVA with Tukey’s posttest was performed using GraphPad Prism version 5.00 for Windows, GraphPad Software, San Diego California

USA, www.graphpad.com ”. All data are displayed as mean ± SEM. P-values less than 0.05 were considered significant. Pathway analysis was performed using DAVID (209, 210) after filtering the data for acetylation sites hyperacetylated by ethanol (fold-change ≥ 2, P < 0.05) but opposed by NR (P < 0.05).

6.5 Results and Discussion

36 male C57Bl/J mice were transitioned to a control liquid diet (F1259SP, Bio-Serv,

Frenchtown, NJ, USA). After transitioning, 12 mice were transitioned to ethanol (F1697SP, Bio-

Serv, Frenchtown, NJ, USA) and another 12 were transitioned to ethanol containing NR Cl (0.33 g/l). Diets were kept isocaloric by replacing the carbohydrate with ethanol. The ethanol ingesting groups started with 2% (w/v) (14% calories) and remained on it for two weeks. After two weeks, the concentration was increased to 3.1% (w/v) (22% calories derived from ethanol) and remained on it for two weeks before replacement with 4.2% (w/v) (30% calories derived from ethanol) on which the mice remained for two – three weeks. The other 12 mice remained on control diet throughout the experiment. We found that animals on ethanol tended not to gain as much weight as control (data not shown) but ate similar amounts of diet (data not shown). The reason for the lack of weight gain is at odds with the consumption data and may represent an error in measurement of the suspension diet in the water bottles. It was noticed that the ethanol diet tended to “spoil” the diet and cause clogs, disallowing the mice from accessing the diet.

We hypothesized that NR supplementation would oppose AFLD through increased

NAD + and mitochondrial sirtuins activity. Lipid content was noticeably but variably increased in the livers of ethanol fed animals control diet animals (Figure 6.1). NR appears to have moderately opposed ethanol induced lipid accumulation in a non-homogenous manner with

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cells closer to blood vessels experiencing less staining. This may indicate higher access to NR lessens lipid content, but requires follow up. These findings suggest NR may have positive outcomes for ethanol associated symptoms and partially prevents fat deposition in liver.

The effect of ethanol on the NAD metabolome has been implicated in its associated diseases (172-174). We sought to quantify the NAD metabolome and the effect of NR in in homogenized liver using LC-MS/MS. As discussed in Chapter 2, Nam concentration is much higher than any other NAD + metabolite, suggesting degradation of the NAD + (Table 6.1). Non-

NAD + related nucleoside and nucleotides (Cytidine, IMP, Inosine, UMP, and Uridine) were

unaffected, establishing that observed changes are specific to NAD + metabolism. Though NAD + was not significantly depressed by ethanol feeding (105 ± 16 vs. 94 ± 15 pmol/mg of dry particulate; Control vs. EtOH), NADH was very significantly increased (11± 1.5 vs. 83 ± 22 pmol/mg dry particulate, Control vs. EtOH (P < 0.01)), reducing the NAD +/NADH ratio from 12 ±

3.5 to 1.1 ± 0.18 pmol/mg dry particulate (P < 0.01). Hence, the NAD +/NADH ratio is altered

through increases in NADH but constant NAD +. This significant shift in redox state is similar to

that observed previously for ethanol feeding (172). If treated as one pool, the amount of NADH

+ NAD + increased with ethanol feeding (177 vs. 116 pmol/mg dry particulate), which could

suggest NAD + biosynthesis increased as a function of ethanol. Biosynthetic intermediates

ADPR, NAAD, and NMN were decreased as a function of ethanol compared to control (ADPr:

4.7 ± 0.9 vs. 1.6 ± 0.8, Control vs. EtOH; NAAD: 5.6 ± 2.3 vs. 16 ± 5.7, EtOH vs. Control; NMN:

5.9 ± 1.1 vs. 12 ± 3.2, EtOH vs. Control). The detriment to NAAD in the presence of ethanol,

suggest NAAD could be a biomarker in some cases for metabolic disease, expanding upon

what was found in Chapter 5. NADP + was significantly depressed by ethanol (NADP: 54 ± 11

vs. 16 ± 3.2, Control vs. EtOH (P < 0.05)). Further, the nicotinamide methylated product,

Me4PY, decreased as a function of ethanol (7.6 ± 0.83 vs. 3.5 ± 0.98, Control vs. EtOH (P <

0.05)).

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NR supplementation significantly increased NAD + compared to both control and EtOH

(179 ± 20 vs. 105 ± 16 vs. 94 ± 15, EtOH + NR Cl vs. Control (P < 0.01), vs. EtOH (P < 0.05)).

However, NR supplementation did not alter NADH. NR doubled the NAD +/NADH ratio compared

to EtOH (P < 0.05) but was five-fold lower than that of control (P < 0.05). NR increased the

concentration of NMN compared to both control and ethanol diets (NMN: 23 ± 3.9 vs. 12 ± 3.2

vs. 5.9 ± 1.1, EtOH + NR Cl vs. Control (P < 0.01), vs. EtOH (p-value < 0.05)). The potential B 3

vitamin biomarker, NAAD (Chapter 5), increased dramatically versus ethanol alone and control

(NAAD: 29 ± 9.7 vs. 16 ± 5.7 vs. 5.6 ± 2.3, EtOH + NR Cl vs. Control, vs. EtOH), which could

suggest that NAAD concentration can be depressed by metabolic dysfunction and further that it

may serve as a marker for efficacious NAD + supplementation.

Ethanol induces a reductive shift in the NAD + + NADH pool and increases the total

NAD(H) pool but did not affect NAD +. Ethanol may increase the rate of NAD + synthesis in order

to continue detoxification. Indeed, metabolic stress can induce expression of the NAD +

biosynthetic machinery (54) and could be a common pathway in opposing certain metabolic

dysfunctions. Expression of the NAD + biosynthetic machinery should be investigated to test for

induction. Additionally, stable isotope technologies and mass spectrometry (similar to that

employed in Chapter 3 and 5) could be used to elucidate whether ethanol increases NAD + turnover. NR nearly doubled hepatic NAD + but did not greatly affect NADH, causing a modest but noticeable increase in the NAD +/NADH ratio. Though this ratio is one of the more striking metrics for the effect of ethanol here and within the literature, improvement in the ratio does not oppose AFLD (175, 176). Our finding that NR opposes fatty liver (Figure 6.1) and raises NAD +

(Table 6.1) supports the hypothesis that ethanol is not really a disease of too much NADH but

rather of too little NAD +.

In increasing NAD +, activity of its consumers such as sirtuins may increase and be better able to oppose the ethanol-induced dyslipidemia. We predicted that NR would oppose mitochondrial hyperacetylation. Global mitochondrial protein acetylation was determined in

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isolated mitochondrial fractions using Western blotting (Figure 6.2a). Little to no acetylation was detected in control samples. Intense hyperacetylation was observed in ethanol fed animals. NR decreased acetylation of higher molecular weight proteins but did not greatly effect global acetylation.

We speculated that the effect of NR on the acetylome could be more site-specific rather than global. To determine site-specificity, we performed an acetylomic analysis of whole liver using LC-MS (performed by Nick Riley in Dr. Coon’s laboratory at the University of Wisconsin—

Madison). In all, 4796 total proteins were identified. 605 of these proteins were mitochondrial, indicating very good quantitation of mitochondrial proteome (605 out of 701 confirmed mitochondrial proteins) (211). Of the 3412 acetyl isoforms, a staggering 1449 were mitochondrial, representing 42% of the total liver acetylome, consistent with reported results

(211). Overall, ethanol and ethanol + NR Cl fed animals acetylomes were indistinguishable from each other (Figure 6.2b). If the profiles differed greatly, acetylation fold changes of ethanol versus those with NR feeding would not correlate (i.e., fall along the y = x line). These data agree with the western blotting data (Figure 3a) showing that NR feeding did not lead to global deacetylation.

We hypothesized that the site-specific NR mediated alterations to acetylation are responsible for its opposing fatty liver disease. We predicted based on this hypothesis that pathways involved in carbon oxidation and lipid catabolism would be enriched with proteins that are hyperacetylated (sites significantly increased in acetylation compared to control: ≥ 2 fold- change and P < 0.05) as consequence of ethanol but not hyperacetylated with NR treatment

(sites non-significantly increased in acetylation compared to control: P < 0.05). We filtered results using these criteria (Appendix B) then performed a pathway analysis using DAVID (209,

210). In agreement with previous results (179, 201), ethanol ingestion caused hyperacetylation of proteins involved in mitochondrial metabolism, specifically amino acid metabolism, lipid metabolism, nitrogen metabolism, the TCA cycle, and the electron transport chain (Table 6.2).

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Strikingly, 17 of the 24 KEGG pathways enriched for ethanol induced hyperacetylation were opposed by NR treatment (i.e. NR prevented acetylation of proteins in key metabolic pathways)

(Table 6.3 and Figure 6.3). NR prevented acetylation of proteins involved in lipid metabolism, amino acid metabolism, and respiration (the TCA cycle and the electron transport chain). Many of these enzymes affected are involved in branch chain amino acid metabolism, which has been implicated in obesity and diabetes (212) and could explain in part how NR opposes high fat diet induced obesity (37). Together, these data suggest that NR could affect mitochondrial lipid metabolism and respiration and that its points of regulation are few but potentially impactful.

Further, these sites are very likely in vivo Sirt3 substrates, suggesting these sites are indeed mediators of mitochondrial metabolism. Future investigation into the impact of NR on enzymes in these pathways should be followed up in vitro with protein biochemistry techniques and in vivo with fluxomic analysis. In particular, amino acid metabolism (213) and beta oxidation (214) should be investigated to interrogate whether NR activates these pathways in the presence of ethanol. Additionally, exact quantitation of the sites on a mol-to-mol scale is necessary to establish the sites that are most acetylated. And finally, acylation (carbon > two) should be investigated in terms of NR supplementation (179).

During the course of the experiment, animals fed ethanol experienced increased mortality compared to control (33% compared to 100% surviving) (Figure 6.4). The animals that survived appeared to be of poor health with irregular gaits, constant tremors, and lethargy. NR opposed these effects with 67% of animals surviving and surviving animals were similar in behavior to control. The mortality was not due to impurities in the ethanol as non-denatured ethanol was used. Additionally, this high mortality is not reported in the literature though publication bias may certainly preclude reporting. Compared to the NIAAA guidelines (215), we noticed that the way in which we were delivering the liquid diet and the housing arrangement were incongruent. The NIAAA protocol called for feeding tubes rather than the standard animal housing water bottles and clearly stated that diet should be changed daily instead of every 48

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hours. Also, the protocol called for no more than two animals per cage rather than the four per cage that we had implemented.

Following these guidelines eliminated mortality differences between groups. Further, animals appeared healthy and did not display the symptoms above. In this experiment we decided to include a control + NR Cl condition so that we may test for effects of NR versus effects of ethanol. Unlike with the water bottles, measurement of the diet consumed daily appeared to be much more accurate with more recovery of the diet out of the feeder tube. Mice on ethanol tended to eat less than mice on the control diet and this effect only increased with increasing ethanol in the diet (Figure 6.4). We found that NR caused increased consumption of diet and confounded the experiment since NR animals did not ingest equivalent calories or gain equivalent dosages of ethanol. The results of this experiment were too confounded for appropriate interpretation of the effects of NR on AFLD.

Overall, we found initially that NR opposed AFLD (Figure 6.1), increased NAD + (Table

6.1), and protected against mortality (6.4). Further, we found that NR does not greatly prevent

global mitochondrial hyperacetylation but does appear to oppose acetylation in a site-specific

manner and may effect key pathways in lipid metabolism. In attempting to improve upon the

initial results, we uncovered NR increases consumption of diet making this model potentially

inappropriate for the study of NR as a preventative measure in AFLD. Preventative studies are

often attempted first when exploring the effect of a drug on a disease model, but this sort of

regimen is rarely applicable to chronic metabolic disease in clinic. In line with this, treatment

experiments whereby mice have become alcoholic and then are transitioned to a non-alcoholic

liquid and then solid diet should be attempted. NR could be added at the moment of transition to

non-ethanol containing liquid diet. In this way, the effect of NR as a treatment for AFLD could be

adequately assessed while uncovering the effect of NR on ethanolic liver metabolism.

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6.6 Tables and Figures

Figure 6.1 NR tends to oppose ethanol induced hepatic lipid deposition. Liver sections were stained with oil red-o and imaged using microscopy. Animals on control liquid diet experienced very little lipid accumulation. Ethanol increased lipid deposition. NR opposed this action, albeit in a heterogeneous and mild manner with greater effect near what appear to be blood vessels.

Table 6.1 Ethanol induced NAD metabolome alterations are opposed by NR. * P < 0.05, ** P < 0.01, *** P < 0.001 versus Control, ŧ P < 0.05, ŧŧ P < 0.01, ŧŧŧ P < 0.001 versus EtOH Whole Liver Control (n = 12) EtOH (n = 4) EtOH + NR (n = 8) Homogenate UMP 4500 ± 390 5900 ± 2000 5400 ± 730 IMP 3700 ± 290 3400 ± 530 4500 ± 340 Nam 3300 ± 550 2500 ± 570 6000 ± 1500 Uridine 520 ± 47 617 ± 191 478 ± 43 Inosine 250 ± 33 120 ± 29 290 ± 95 NAD + 105 ±16 94 ± 15 179 ± 20 **, ŧ NADP + 54 ± 11 16 ± 3.2 * 109 ± 17 *, ŧŧ NAAD 16 ± 5.7 5.6 ± 2.3 29 ± 9.7 NMN 12 ± 3.2 5.9 ± 1.1 23 ± 3.9 *, ŧ NADH 11 ± 1.5 83 ± 22 ** 77 ± 13 *** Me4PY 7.6 ± 0.83 3.5 ± 0.98 * 17 ± 2.5 ***, ŧŧ ADPR 4.7 ± 0.9 1.6 ± 0.8 1.0 ± 0.3 ** Cytidine 2.1 ± 0.20 3.5 ± 0.50 2.8 ± 0.37 NAR 0.16 ± 0.03 0.40 ± 0.11 ** 0.29 ± 0.09 NAD +/NADH 12 ± 3.5 1.1 ± 0.18** 2.3 ± 0.26 *, ŧ NAD + + NADH 116 177 256

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a b

Figure 6.2 NR does not oppose ethanol induced global hyperacetylation. (a) Isolated hepatic mitochondrial proteins were separated on a SDS-PAGE gel, transferred, and blotted with a pan-specific anti-lysine acetylation antibody. Ponceau staining of the blot is shown as a loading control. (b) Total and mitochondrial acetyl isoforms from liver were measured in ethanol and ethanol + NR treated mice and compared to control. The acetylation fold changes of ethanol + NR/control and ethanol/control are plotted on the y- and x-axis, respectively. Overall, the data indicate that ethanol-induced global lysine acetylation is not opposed by NR.

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Table 6.2 Pathways affected by ethanol-induced acetylation. Pathway analysis of acetylated peptides in liver was performed using DAVID after filtering for sites that were significantly (P < 0.05) acetylated greater than two fold in ethanol treated animals versus control animals. Ethanol ingestion led to hyperacetylation of proteins involved in central carbon metabolism and lipid synthesis and utilization. FDR: false discovery rate. Term a Count b %c P Value FDR Valine, leucine and 19 10.16042781 1.47E-20 1.45E-17 isoleucine degradation Citrate cycle (TCA 13 6.951871658 6.20E-14 6.12E-11 cycle) Oxidative 21 11.22994652 6.21E-14 6.13E-11 phosphorylation Fatty acid metabolism 13 6.951871658 1.03E-11 1.01E-08 Butanoate metabolism 11 5.882352941 5.20E-10 5.13E-07 Alanine, aspartate and 8 4.278074866 7.10E-07 7.01E-04 glutamate metabolism Propanoate 8 4.278074866 7.10E-07 7.01E-04 metabolism Glycine, serine and 8 4.278074866 1.14E-06 0.001124901 threonine metabolism Fatty acid elongation in 5 2.673796791 6.49E-06 0.006406492 mitochondria Arginine and proline 8 4.278074866 3.83E-05 0.037814431 metabolism Lysine degradation 7 3.743315508 7.81E-05 0.077081708 Synthesis and 4 2.139037433 6.15E-04 0.605733673 degradation of ketone bodies Tryptophan 6 3.20855615 6.75E-04 0.664889193 metabolism Pyruvate metabolism 5 2.673796791 0.005957113 5.729197729 beta-Alanine 4 2.139037433 0.006750756 6.469772851 metabolism Glyoxylate and 3 1.604278075 0.032510392 27.84502708 dicarboxylate metabolism a The pathway name, b the number of genes affected in the pathways, c the percentage of genes in the pathway affected

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Table 6.3 Pathways affected by ethanol-induced protein acetylation and responsive to NR treatment. Hepatic peptides hyperacetylated by ethanol ingestion (fold-change ≥ 2 and p-value ≤ 0.05) that were not hyperacetylated with NR treatment (p-value ˃ 0.05) were analyzed using DAVID. Hyperacetylation of pathways involved in central carbon metabolism and fat synthesis and utilization observed after ethanol ingestion was largely opposed by NR. FDR: false discovery rate. Term Count % P Value FDR Valine, leucine and isoleucine degradation 10 19.23077 2.70E-12 2.36E-09 Fatty acid metabolism 6 11.53846 5.55E-06 0.004843 Propanoate metabolism 5 9.615385 2.53E-05 0.022045 Citrate cycle (TCA cycle) 5 9.615385 2.89E-05 0.025204

Oxidative phosphorylation 7 13.46154 9.62E-05 0.083959

Glycine, serine and threonine metabolism 4 7.692308 8.39E-04 0.729522

Fatty acid elongation in mitochondria 3 5.769231 9.33E-04 0.811669 Butanoate metabolism 4 7.692308 0.001287 1.117999 beta-Alanine metabolism 3 5.769231 0.00731 6.202982 Alanine, aspartate and glutamate metabolism 3 5.769231 0.013364 11.08008 a The pathway name, b the number of genes affected in the pathways, c the percentage of genes in the pathway affected

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Figure 6.3 Proteins acetylated by ethanol and sensitive to NR. The proteins involved in the pathways in Table 6.3 are displayed at left. The pathway(s) that the proteins map to are included above the columns at right. As stated in Table 6.3, central carbon and lipid metabolism were affected by ethanol ingestion. NR prevented many of these alterations.

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100 Control EtOH EtOH + NR

2.0% 3.1% 4.2% 14 28 52 0 0 10 20 30 40 50 60 70 Days of Experiment

Figure 6.4 NR opposed increased mortality experienced by ethanol fed mice. A survival curve shows the number of mice surviving to the end of the initial study (Figures 6.1 – 6.3 and Tables 6.1 – 6.3). The small marks above the lines represent planned sacrifices. The second x-axis shows the concentration (w/v) of ethanol over the days of the experiment. Mice in the ethanol group experienced increased mortality compared to control and ethanol + NR treated groups, suggesting NR may oppose part of the toxicity or disease experienced as a result of ethanol.

Figure 6.5 NR increased diet consumption in both control and ethanol animals. In the subsequent experiment, animals were housed two per cage, diet was replaced daily and contained in specialized feeding tubes. Diet consumption was recorded each day for each cage and averaged between mice (n = 2 per cage). Displayed are the weekly averages after all ethanol animals had been transitioned to ethanol. The percentages displayed above the graphs are the percent weight per volume of ethanol in the diet. Control animals tended to consume more diet than ethanol animals after switching ethanol animals to 4.2% (w/v). A repeated measure, two-way ANOVA was performed to test for condition effects over time. In both cases, NR caused a significant increase in consumption that depended upon time in both control (effect

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Figure 6.5 – continued

of NR: P < 0.05; effect of NR and time: P < 0.05) and ethanol groups (effect of NR: P < 0.05; effect of NR and time: P < 0.05).

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CHAPTER 7 NICOTINAMIDE RIBOSIDE OPPOSES TYPE 2 DIABETES AND NEUROPATHY IN MICE Samuel A.J. Trammell 1, Benjamin J. Weidemann 1, Matthew S. Yorek 2, Amey Holmes 2,

Lawrence J. Coppey 2, Alexander Obrosov 2, Randy H. Kardon 2,3 , Mark A. Yorek 2,4 and Charles

Brenner 1,4

Departments of Biochemistry 1, Opthalmology 3 and Internal Medicine 4, Carver College of

Medicine, University of Iowa, Iowa City, IA 52242, USA; Iowa City Veterans Administration 2,

Iowa City, IA 52246

7.1 Distribution of Work

CB, MAY, and I designed experiments. BJW and I performed statistical analyses. Mouse

husbandry and dissections were performed by MSY, AH, LJC, AO, RHK, and MAY. Microscopy

was performed by MSY and AH. Lipid parameters were measured by LJC. All mass

spectrometry was performed by myself. CB wrote the manuscript. BJW and I edited the

document.

7.2 Abstract

Male C57BL/6J mice raised on a high fat diet become prediabetic and develop insulin

resistance and sensory neuropathy. The same mice given low doses of streptozotocin are a

model of type 2 diabetes, developing hyperglycemia, severe insulin resistance and diabetic

peripheral neuropathy involving sensory and motor neurons. Because of suggestions that

increased NAD + metabolism might address glycemic control and be neuroprotective, we treated prediabetic and type 2 diabetic mice with nicotinamide riboside in their high fat diets.

Nicotinamide riboside improves glucose tolerance, reduces weight gain, liver damage and the

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development of hepatic steatosis in prediabetic mice while protecting against sensory neuropathy. In type 2 diabetic mice, nicotinamide riboside greatly reduces non-fasting and fasting blood glucose, weight gain and hepatic steatosis, while protecting against diabetic neuropathy. The neuroprotective effect of NR cannot be explained by glycemic control alone.

Corneal confocal microscopy was the most sensitive measure of neurodegeneration and this assay allowed detection of the protective effect of nicotinamide riboside on small nerve structure in living mice. The hepatic pool of NADP + plus NADPH was significantly degraded in

prediabetes and type 2 diabetes but was largely protected when mice were supplemented with

nicotinamide riboside.

7.3 Introduction

The global epidemic of obesity and diabetes has created severe economic stresses for

health systems and intense neuropathic complications for affected individuals. Obesity is

frequently associated with prediabetic polyneuropathy (PDPN) (216), while about half of

individuals with diabetes will suffer from diabetic peripheral neuropathy (DPN) (217), rendering

them insensitive to heat and touch. Severe DPN can progress to foot ulcers and amputations.

Few treatments are effective for obesity while nothing has been found to arrest or reverse DPN.

Best available care is tight glycemic control, dietary improvement and exercise, and pain

medication when DPN is painful (218).

Deficiency in the NAD + co-enzyme causes pellagra, which was endemic a century ago in

the American south in populations subsiding on corn rations and lard (129). Though pellagra

has been nearly eliminated, there are indications that supplementation with nicotinamide

riboside (NR), a recently discovered NAD + precursor vitamin (7, 142), can improve metabolic

health in overfed mice (37) and function as a neuroprotective agent in conditions involving

Wallerian degeneration (219-222). Though the mechanisms accounting for resistance to weight

gain and improved glycemic control for mice on high fat diet (HFD) are not fully understood, NR

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elevates NAD + levels in skeletal muscle, liver and brown adipose tissue and appears to increase

activity of nuclear and mitochondrial NAD +-dependent protein lysine deacetylases, the sirtuins

SIRT1 and SIRT3 (37). Two mechanisms have been proposed for neuroprotection: boosting

mitochondrial NAD + to support SIRT3 (221) and preserving axonal NAD + in the face of damage- induced SARM1 activation, which results in NAD + degradation (222). In addition, a

neuroprotective mechanism has been proposed that depends on both mitochondrial and axonal

NAD + (143). Though NR is not only a precursor of NAD + but also of NADH, NADP + and NADPH

(129), the NAD + metabolome has not been investigated in any disease model for which NR prevention or therapy has been tested. In addition, NR has not been tested on DPN.

Because of the potential for NR to improve prediabetic (PD) and diabetic glucose and lipid metabolism while also treating neuropathic complications, we made mice obese and PD with HFD and rendered them type 2 diabetic (T2D) with HFD plus two low doses of streptozotocin (STZ) (223). Here we show that NR improves fasting glucose levels and glucose tolerance of PD mice, while providing resistance to a substantial degree of hepatic steatosis, hypercholesterolemia, liver damage and weight gain. NR greatly lowered fasting and nonfasting glucose of T2D mice, while reducing hepatic steatosis and weight gain. Though hepatic steatosis and hyperglycemia were not fully corrected by NR, supplemented mice have greatly reduced neuropathic symptoms in both models. Remarkably, PD and T2D mice have lower levels of hepatic NADP + plus NADPH, and T2D mice trended toward lower levels of hepatic

NAD +. Upon supplementation, NAD + was more correctable than was NADP + plus NADPH, suggesting that maintenance of the latter metabolites is challenged by obesity. Our data also indicate corneal confocal microscopy (CCM) can be used as a minimally invasive and translational assay to monitor NR-dependent improvements in PDPD and DPN in future clinical investigations.

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7.4 Methods

Mouse Models

Mouse methods were as described with investigators blinded to treatments (223-225).

NR chloride was a gift of ChromaDex, Inc.

NAD Metabolomics

Methods were performed as a revision of established procedures (1) as detailed in

Supplemental Methods of this chapter and as described in Chapter 2.2.

Statistics

Data are presented as mean ± SEM unless indicated otherwise. The effect of treatment,

NR supplementation, and interactions of the two factors were determined by two-way ANOVA

with multiple comparisons performed using the Holm-Sidak test. Time dependent

measurements ( i.e. in GTT and body weight) were analyzed across and within the six groups via two-way repeated measures ANOVA followed by Holm-Sidak tests. P-values of less than

0.05 were considered significant.

Study Approval

All animal procedures were approved by the Iowa City Veterans Administration Animal

Care and Use Committee, which has an Animal Welfare Assurance (A3748-01) on file with the

Office of Laboratory Animal Welfare and is fully accredited by AAALAC International.

7.5 Results and Discussion

Sixty male C57Bl/6J mice, housed 3 or 4 per cage, were raised on Teklad 7001 normal

chow (NC). At 12 weeks of age, when mice weighed ~23 g, 40 mice were transferred to HFD

(Research Diets 12492, 60% calories from fat) to render them PD, while 20 mice remained on

NC. After 8 weeks on HFD, 20 of 40 mice were given two low doses (75 mg/kg body weight

followed 2 days later with 50 mg/kg body weight) of STZ to induce T2D. PD and T2D mice

remained on HFD for the duration of the experiment. Five weeks after STZ administration to

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create the T2D population, 10 of 20 mice in each of the three groups (NC, HFD and HFD+STZ) were supplemented with 3 g of NR chloride per kg of their diet, thereby creating six groups of 10 mice (NC, NC+NR, HFD, HFD+NR, HFD+STZ, HFD+STZ+NR; 7.9 Supplemental: Figure 7.4).

Five weeks before sacrifice, intraperitoneal glucose tolerance tests (GTT) were performed on fasted mice. Seven weeks after the beginning of NR supplementation, one mouse from each group was sacrificed per day for 5 days per week over a 2-week period. PD mice were effectively on HFD for 21 weeks without supplementation or with NR supplementation from week 13 to 21 on HFD. All T2D mice were non-supplemented for five weeks post STZ administration and 10 out of 20 were supplemented with NR from week 13 to 21 on HFD. On the day of sacrifice, mice were subjected to CCM, motor neuron conduction velocity (MNCV) and sensory neuron conduction velocity (SNCV) tests, and assayed for thermal sensitivity. The remaining assays were performed post-mortem (224).

As shown in Figure 7.1a and Supplemental Figure 7.4, during the 21 week experiment, mice on HFD gained ~27 g of body weight while mice in the HFD+STZ treatment group gained

~16 g. Though supplementation was for only 8 weeks, NR blunted weight gain in PD by ~7 g (P

< 0.01) and by ~6 g in the T2D group (P < 0.05). As shown in Figures 1b-d, mice on HFD developed severe hepatic steatosis. Whether or not HFD mice were treated with STZ, supplementation with NR strikingly reduced the hepatic oil red O-positive staining area (P <

0.01). NR supplementation reduced oil red O droplet size by two-thirds in PD mice (P < 0.001).

As shown in Figures 7.1e and 7.1f, NR significantly depressed circulating cholesterol (P < 0.05) and alanine aminotransferase (ALT) (P < 0.05), a sign of liver damage, in PD mice.

As shown in Figures 7.1g and 7.1h, NR tended to normalize hemoglobin A1c (HbA1c) and significantly improved nonfasting glucose (P < 0.01) in T2D. As shown in Figure 7.1i, NR had a powerful effect on fasting glucose, depressing levels from 210 mg/dl to 161 mg/dl in PD mice (P < 0.01) and from 345 mg/dl to 194 mg/dl in T2D mice (P < 0.001). Finally, as shown in

Figure 7.1j and Figure 7.5, NR significantly improved glucose tolerance in PD (P < 0.01) and

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tended to improve glucose tolerance in T2D. These data indicate that NR has profound effects on whole body metabolism in PD and T2D mouse models. However, mice supplemented with

NR are neither hyperactive nor hypophagic (data not shown).

As shown in Figures 7.2a and 7.2b, PD mice retained good MNCV but had significantly depressed SNCV (P < 0.001). This deficit was not evident in mice supplemented with NR. T2D mice had significantly depressed MNCV (P < 0.001) and SNCV (P < 0.001) that were prevented by NR supplementation. Thermal insensitivity, a particularly dangerous aspect of human DPN

(225), was strikingly evident in the PD (P < 0.001) and T2D (P < 0.001) models and was significantly reduced by NR in PD (P < 0.01) and T2D (P < 0.001). Consistent with the sensory neuron deficits in both models, as shown in Figures 7.2d and 7.2e, intraepidermal nerve fiber density (INFD) in hindpaws was significantly degraded in PD (P < 0.001) and T2D (P < 0.001).

NR significantly protected against these deficits in PD (P < 0.01) and T2D (P < 0.001).

Early small fiber neuropathic changes are difficult to quantify in human populations and this may contribute to a failure to translate potentially effective treatments from DPN animal models to the clinic (226). The cornea is the most densely innervated structure of the human body, containing Aδ and unmyelinated C fibers derived from the ophthalmic division of the trigeminal nerve (227). CCM is gaining establishment as a valid measure of diabetic nerve damage in the clinic (228, 229) that can also be used to monitor diabetic neurodegeneration in rodent models (223, 224, 230). As shown in Figures 7.3a and 7.3b, quantification of sub- epithelial corneal nerves by CCM indicated that corneal nerves are severely degraded by PD (P

< 0.001) and T2D (P < 0.001). CCM indicates that NR protects corneal innervation in T2D (P <

0.05) and trends positively in PD. Upon sacrifice, sub-basal corneal innervation was analyzed by staining for class III b -tubulin. This assay, shown in Figures 7.3c and 7.3d, produces the same qualitative results as those obtained from live CCM images.

In cultured dorsal ganglion root neurons, the concentration of NAD +, as determined by

LC, was reported to decline in a SARM1-dependent manner in a four hour period after axotomy

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(222). Because NR affects whole body metabolism, the targets of NR supplementation are not assumed to reside in a single tissue, nor is it assumed that obesity exclusively dysregulates targets of the NAD + metabolome that depend exclusively on NAD +. Moreover, because sensory

nerves die back in DPN, all neuronal metabolites are expected to fall as neuronal tissue

declines with disease. We therefore employed LC-MS/MS to measure the NAD + metabolome on a common pmol scale (1, 31) in freeze-clamped liver samples from freshly euthanized mice.

NADPH is oxidized in extraction, such that the obtained NADP + signal represents the sum of

NADP + plus NADPH.

As shown in Table 7.1, the liver NADP + plus NADPH pool was significantly depressed in

PD and T2D (P < 0.0001) with respect to NC controls. NR supplementation significantly boosted

hepatic NADP + plus NADPH but did not fully correct it. In PD, NAD + trended down (P = 0.84) and trended down further in T2D (P = 0.11) mice with respect to NC controls. Hepatic NAD + was

fully normalized by NR in both models—the boost in hepatic NAD + achieved significance in NR-

supplemented T2D mice (P < 0.05). Consistent with a challenge to hepatic NADP + plus NADPH

metabolism, nicotinamide (Nam) waste products, 1-methyl nicotinamide (MeNam) and 1-methyl-

4-pyridone-3-carboxamide (Me4PY), were increased in PD (P = 0.0310 and P < 0.001).

It had previously been shown that HFD produces severe hepatic lipid accumulation in mice,

which primes them for loss of glycemic control with low doses of STZ (223). Here we show that

liver NADP + plus NADPH is significantly compromised in these PD and T2D models and that

NAD + tends to decline in the mouse model of T2D. NR supplementation is accompanied by

substantial resistance to weight gain and improvements in dyslipidemia, liver function and

glycemic control in one or both models. Moreover, the PD and T2D mouse models exhibited

structural and functional sensory nerve deficits that were not manifested when mice were

supplemented with NR for their last 8 weeks on HFD. Though NR lowered hepatic steatosis and

weight gain and greatly assisted glycemic control, NR did not normalize any of these metabolic

parameters. In addition, neuroprotection cannot be explained by glycemic control alone. For

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example, T2D mice supplemented with NR have higher nonfasting glucose than PD mice without NR (P < 0.01). Nonetheless, PD mice without NR have SNCV deficits, whereas T2D mice supplemented with NR do not. Thus, NR is presumed to have neuronal and hepatic targets. Finally, the decline in CCM-monitored neuronal density was more severe than any other measure of neuropathy and the protection of corneal innervation by NR was evident in the T2D model.

A large body of work has investigated NAD +-consuming enzymes including poly(ADPribose) polymerases (PARPs) and sirtuins (128). However, the SARM1-dependent factor that degrades axonal NAD + in Wallerian degeneration is resistant to PARP inhibition and the pool of NADP + plus NADPH was not investigated (222). Whereas NAD+ is the central

hydride-accepting coenzyme for fuel oxidation, NADPH is the key hydride-donating cofactor for

detoxification of reactive oxygen species (ROS) (231), a major contributor to insulin resistance

(232). Because significant depression of NADP + plus NADPH occurs in PD and T2D whereas

NAD + only trended down and was easier to correct, we suggest that the overnutritional stresses of HFD specifically challenge maintenance of hepatic NADPH and that this may be central to

PD and its progression.

Cellular NADPH is known to be limited by expression of NAD + kinase (233) and could be

depressed by loss of a repair system that restores damaged NADPH (234). In addition, there

are reports of an NADP + phosphatase (235) and NADP + glycohydrolase activities (236)— induction of such enzymes could be responsible for loss of these metabolites. By diminishing levels of NADPH, any of these mechanisms could lower the capacity of hepatocytes and potentially other cells to detoxify ROS (231) and diminish circadian functions (237), thereby contributing to two major systems depressed in obesity. Ongoing work is designed to test the effect of NR on ROS damage in PD, T2D, PDPD and DPN, to discover the basis for depressed hepatic NADP + and/or NADPH in PD, and to translate these results to human populations.

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7.6 Acknowledgments

This work was supported by a pilot and feasibility grant from the Fraternal Order of

Eagles Diabetes Research Center, the Roy J. Carver Trust, National Institutes of Health grant

DK081147, and grants from the Department of Veterans Affairs, BX001680-01, RX000889-01 and C9251-C.

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7.7 Figures and Tables for Sections 3-5

Figure 7.1 NR improves metabolic parameters in PD and T2D. Male C57Bl/6J mice were made prediabetic (PD) with HFD or diabetics (T2D) with HFD and STZ treatment (Figure 7.4). All groups including normal chow (NC) animals were supplemented as described in text. Gross metabolic parameters of PD and T2D were measured as described in methods. (a) NR reduces weight gain on HFD independent of STZ. (b – d). NR reduces hepatic steatosis in the PD and T2D models. NR reduces circulating cholesterol (e) and alanine aminotransferase (f) in PD. In T2D, NR tends to lower HbA1C (g) and depresses nonfasting glucose (h). NR depresses fasting glucose in both models (i). NR improves GTT in PD (j). Overall, NR opposed the deleterious metabolic effects of HFD and HFD + STZ. Statistics were by two-way ANOVA followed by a Holm-Sidak multiple comparisons test. n = 10. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Figure 7.2 NR opposes PDPN and T2DPN. Prior to sacrifice, neuropathy was assessed in both PD and T2DPN models. (a) NR protects against a decline in motor nerve conduction velocity (MNCV) in T2D. (b) NR protects against declines in sensory nerve conduction velocity (SNCV) in PD and T2D. (c) NR protects against loss of thermal sensitivity in both models. (d) and (e) NR improves INFD on NC and in both disease models. NR acted to oppose all measured forms of neuropathy in both models. Statistics were by two-way ANOVA followed by a Holm-Sidak multiple comparisons test. n = 10. **, P < 0.01; ***, P < 0.001.

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Figure 7.3 Activity of NR in DPN can be monitored by corneal confocal microscopy (CCM). (a) and (b) CCM is a sensitized assay for PD and T2D nerve loss and the protective effects of NR. (c) and (d) By post-mortem class III β-tubulin staining, NR protects against corneal sub- epithelial nerve loss in T2D. CCM is a non-invasive assay that can be performed on live animals. CCM may be an effective tool in assaying neuropathy and the action of anti- neuropathic agents such as NR in future clinical trials. Statistics were by two-way ANOVA followed by a Holm-Sidak multiple comparisons test. n = 10. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

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Table 7.1 The hepatic pool of NADP + and NADPH is depressed by PD and T2D and is partially restored by NR. Livers were excised and analyzed using LC-MS/MS. NAD + was depressed in PD and T2D animals. NR restored NAD + to control levels in all cases. NADP + was significantly depressed in PD and T2D animals and partially restored by NR in both cases. The data suggest that the ROS detoxification system is undermined by HFD and that NR could be protective against its diminishment. Values are expressed as mean ± SEM pmol/mg liver. Underlined concentrations within a treatment are significantly different from NC after collapsing for the effect of supplementation. The effect of treatment and supplementation of NR were analyzed by two-way ANOVA followed by multiple comparisons using the Holm-Sidak test. † P < 0.05, †† P <0.01, ††† P <0.001 for effect of NR within a treatment group (i.e. NC vs. NC+NR, HFD vs. HFD+NR, HFD+STZ vs. HFD+STZ+NR). # P < 0.05, #### P <0.0001 for effect of treatment versus NC within supplementation group (i.e. NC vs HFD, NC vs HFD+STZ, NC+NR vs HFD+NR, NC+NR vs HFD+STZ+NR). *** P <0.001 for effect of STZ vs. HFD+STZ. NC NC+ HFD HFD + HFD+ HFD+STZ + NR NR STZ NR NAD + 1200 ± 84 1500 ± 99 1000 ± 85 1200 ± 95 760 ± 72 1300 ± 180 † NADP+ 230 ± 11 250 ± 16 150 ± 9.2 #### 200 ± 17 †,# 150 ± 13 #### 200 ± 11 ††† Nam 170 ± 9.3 230 ± 20 † 190 ± 12 180 ± 7.8 # 130 ± 9.0 210 ± 17 †† ADPR 84 ± 17 98 ± 19 60 ± 8.6 70 ± 14 44 ± 10 84 ± 19 MeNam 3.1 ± 0.28 4.4 ± 0.25 2.7 ± 0.14 2.9 ± 0.22 2.4 ± 0.32 3.8 ± 0.63 NMN 2.8 ± 0.13 3.7 ± 0.24 3.7 ± 0.36 3.5 ± 0.14 2.2 ± 0.30 *** 3.1 ± 0.21 Me4PY 2.6 ± 0.25 7.4 ± 0.64 ††† 6 ± 0.45 # 8.3 ± 1.5 4.2 ± 0.37 ### 7.9 ± 0.95 † NR 1.2 ± 0.12 2.0 ± 0.45 1.6 ± 0.38 1.1 ± 0.15 1.0 ± 0.15 2.3 ± 1.3

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7.8 Supplemental Materials

Sample Extraction for NAD + Metabolomics

Murine liver obtained by freeze-clamp was pulverized using a Bessman pulverizer (100 –

1000 mg size) (Spectrum® Laboratories, Rancho Dominguez, California) cooled to liquid N 2

temperatures. Each pulverized liver sample was aliquoted (5 – 20 mg) into two liquid N 2 cooled

1.5 ml centrifuge tubes, which were stored at -80 °C until analysis. Prior to analysis, 13 C-labeled

yeast extract (1) was added to one tube and an internal standard solution containing 60 pmol of

18 18 18 O NR, 60 pmol of O nicotinamide (Nam), 60 pmol of O D 3 1-methylnicotinamide (MeNam), and 240 pmol of D 4 nicotinic acid (NA) (C/D/N Isotopes, Pointe-Claire, Quebec, Canada) was added to the other tube while on dry ice. Samples were extracted by addition of 0.1 ml of buffered ethanol (3 parts 100% ethanol: 1 part 10 mM HEPES, pH 7.1) at 80 °C. Samples were vortexed vigorously until thawed, sonicated in a bath sonicator (10 seconds followed by 15 seconds on ice, repeated twice), vortexed, then placed into a Thermomixer® (Eppendorf,

Hamburg, Germany) set to 80 °C and shaken at 1050 rpm for five minutes. Samples were centrifuged (16.1k g, 4 °C, 10 minutes). Clarified supernatants were transferred to fresh 1.5 ml tubes and dried via speed vacuum for two hours. Prior to LC-MS/MS analysis, samples were resuspended in 40 µl of 10 mM ammonium acetate (>99% pure) in LCMS-grade water. All samples were transferred to Waters polypropylene plastic total recovery vials (Part #

186002639) and stored in a Waters Acquity H class autosampler maintained at 8 °C until 10 µl injections.

LC-MS/MS Analysis for NAD Metabolomics

Chromatographic separation was performed using a 2.1 mm X 100 mm Thermo

Scientific Hypercarb™ column as described with slight modification to the alkaline separation

(1). Specifically, flow rate was increased to 0.55 ml/min and run time shortened to 11.6 minutes.

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Separation was performed using a modified gradient with initial equilibration at 3% B, a 0.9 minute hold, a gradient to 50% B over 6.3 minutes, followed by a 1 minute wash at 90% B, and a 3 minute re-equilibration at 3% B. Analytes were detected and quantified using a Waters TQD operated in positive ion multiple reaction monitoring (MRM) mode. MRM transitions for all mononucleotides and dinucleotides in the acidic separation were as described (1), though

NADH was not quantified due to high variability in the internal standard mixture. Newly quantified metabolites in the acidic separation, MeNam and Me4PY were assayed with the following transitions: MeNam (137 > 94 m/z) and Me4PY (153 > 136 m/z). Samples were electrospray ionized at a capillary voltage of +3.1 kV, a desolvation gas flow rate of 500 l/hr, a cone gas flow rate of 100 l/hr, a desolvation temperature of 350 °C, and a source temperature of

150 °C. Analytes were quantified using a calibration curve containing the same concentration of internal standard as samples with external standard concentrations ranging from 0.1 – 100 µM.

When NAD + concentration exceeded the linear range, the sample was diluted by 10 and samples re-analyzed. All pmol amounts were normalized to g of wet liver weight extracted

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7.9 Supplemental Figures

Figure 7.4 Experimental design and weight gain. 12 week old, male C57Bl/6J mice were placed on either NC or HFD for 8 weeks. At 8 weeks, half of the HFD fed mice were treated with two low doses of STZ to create the type 2 diabetes (T2D) model. Mice remaining on HFD without STZ treatment are the prediabetic (PD) model. Both groups continued on HFD for the remainder of the study. Five weeks after creation of the T2D group, all conditions were supplemented with NR, creating six conditions (NC, NC + NR, HFD, HFD + NR, HFD + STZ, HFD + STZ + NR). Weight was followed throughout the experiment as shown above. Glucose tolerance tests (GTTs) were performed at 16 weeks of the experiment. Measurements of neuropathy were performed on or after sacrifice. At time of sacrifice, blood and liver were collected for metabolic and LC-MS/MS metabolomic analyses. Mouse age and weeks of experiment are displayed on the x-axis. NR caused resistance to HFD-induced weight gain in both PD and T2D mice.

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Figure 7.5 GTT primary data used for Figure 7.1 i and j. Glucose tolerance tests (GTTs) were performed at 16 weeks (Figure 7.4) of the experiment as described in methods. Data from all groups are displayed at left. At right, GTTs of each diet condition +/- NR are displayed with NC at top, HFD in the middle, and HFD + STZ at bottom. A repeated two-way ANOVA was performed to test for an interaction between condition or treatment and time. NR did not significantly GTT over time. Overall, NR tended to improve GTT in PD and T2D mice with significant differences observed as indicated. A post-hoc Holm-Sidak multiple comparison was performed to test for significant differences between no NR and NR within each time point. n = 10. *, P < 0.05; **, P < 0.01; ***, P < 0.001

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7.10 Perspective on Chapter 7

In the above sections of this chapter, we enumerated the beneficial effects of NR on pre-

diabetic and diabetic mouse models. However, before investigating the effect of NR on type 2

diabetes, we evaluated NR as an anti-PDN agent in a type 1 diabetic rat model. Here, I describe

our finding in the type 1 diabetic model while discussing how these effects inform our findings in

the type 2 diabetic mice. Additionally, I measured NADH and NADPH in the same samples

analyzed by LC-MS/MS in Table 7.1 and discuss the findings in light of the PD and T2D effects

on the oxidized NAD metabolome.

7.11 Results and Discussion

Type 1 Diabetes in Rat Compared to Type 2 Diabetes in Mouse

At first, we chose to investigate a type 1 diabetic (T1D) model to test the hypothesis that

NR opposes DPN. Male Sprague-Dawley rats were treated with streptozotocin (STZ) as

described (238) to induce T1D and began treatment on NR Cl at a dosage of 0.3% (w/w of chow

(Harlan Teklad, #7001, Madison, Wi)) after 96 hours post STZ administration. NR Cl treatment

lasted on average for six weeks. The animals in this initial experiment were not treated with

insulin. Unlike with PD and T2D (Figure 7.2 and 7.9 Supplemental Figure 7.5), NR was unable

to oppose the heightened, non-fasting blood glucose experienced by T1D (Table 7.2).

Additionally, A1c, indicative of the long term concentration of blood glucose, was elevated in

T1D compared to control and was not improved by NR. However, the hyperglycemia

experienced in the T1D model appears to have been more dramatic than that of PD and T2D

(Holm-Sidak test: T1D vs PD: P < 0.001; T1D vs T2D: P < 0.001) and may have overpowered

the modest though significant effects of NR on hyperglycemia. This may also explain the lack of

improvement in the blood lipid profile. Essentially, these results established that these animals

were severely diabetic and of poor health.

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Despite the severe diabetes and similar to its effect in T2D, NR improved neuropathy

(Table 7.3). Though variability was high in the mechanical test, T1D rats were less responsive to mechanical prodding and NR treatment increased sensitivity. NR significantly prevented thermal insensitivity. Motor nerve conduction velocity and sensory nerve conduction velocity tended to be and was significantly aided by NR, respectively. C fiber density was also maintained by NR administration. Together, these results are perfectly congruent with what was observed with

T2D, i.e. NR opposes DPN in a manner independent of hyperglycemia.

Unlike the mouse sciatic nerve, the rat sciatic nerve is large enough for NAD metabolomic analysis. Granted, as stated above (Chapter 7.5), a problem of normalization exists. The diabetic nerves are dying back, meaning DNA, RNA, protein, and other macromolecules are decreasing in mass, indicating that similar masses of sciatic nerve in the diabetic animals may cause an overestimate of the NAD metabolome. Additionally, at this time homogenates containing 10 mM Nam 1 were utilized for NAD metabolomic analysis. This addition complicates interpretation of the results as the alterations of the NAD metabolome between conditions would represent in vivo effects on metabolite concentration combined with the ability of any one sample to metabolize Nam. With these in mind, T1D depressed NMN,

NADP +, and NAD + (Table 7.4). NR tended to elevate NAD + and NADP + and significantly increased NMN. These results are very similar in profile to the hepatic NAD metabolome in T2D with and without NR, suggesting the murine hepatic NAD metabolome may be representative of the murine sciatic nerve.

The results from the T1D rat model strongly revealed that NR affected DPN independent of glycemic control and that this effect correlated with improvement of NAD + and related

metabolites. Though these results were fairly clear, we decided to extend treatment with NR

form six weeks to 8 weeks and also to compare the effects of NR to the effects of Nam and NA.

1 Nam was added to the homogenate in large quantities to maintain the mitochondrial acetylation profile. It was hoped that the same homogenate could be used for multiple analyses.

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12 week old male Sprague-Dawley rats were allocated into five groups with 10 animals per group and either administered STZ as above or treated with vehicle. Treatment with 0.3% (w/w) of NR Cl or mole equivalent of Nam and NA began 96 hour after STZ or vehicle administration.

These animals were treated with insulin every other day. All three B 3 vitamins increased mortality starting at ~3 weeks post beginning of treatment. Unfortunately, the times of death were not recorded, disallowing appropriate analysis of survival. What is known, is that at the end of only 6 to 7 weeks, 4, 3, and 2 rats remained in the NR, Nam, and NA treated groups, respectively. Only three rats were sacrificed in the control and STZ groups as the remaining rats were diverted to other experiments. The dramatic increase in mortality is likely due to the poor health of the T1D model further confounded by the insulin resistance effects of Nam and NA

(239, 240) which worsened hyperglycemia in these animals (Table 7.5). In the T2D model, glucose intolerance increased in NC animals fed NR (Figure 7.1 j and 7.9 Supplemental Figure:

Figure 7.5), which could be a result of insulin resistance. Further work is necessary to measure insulin resistance in NR supplemented animals but is a crucial question to answer before clinical testing.

Though the sample sizes were low and the animals were visibly ill 2 at time of sacrifice,

B3 vitamins appear to cause differential effects on DPN. T1D and those treated with Nam

tended to be less sensitive to mechanical stimuli (Table 7.6). T1D caused significant thermal

insensitivity, which as was not experienced by any B 3 vitamin treated groups. Interestingly,

MNCV dysfunction was prevented only by NR. In this experiment, T1D tended to experience deficit in SNCV (28 ± 1.2 vs 34 ± 2.3 m/s), but Nam caused significant deficit (25 ± 0.2 vs 34 ±

2.3 m/s, P < 0.05) whereas NA tended to do so (27 ± 3.7 vs 34 ± 2.3 m/s). NR was indistinguishable from control (32 ± 1.3 vs 34 vs 2.3 m/s). Together, these data suggest specific

effects of NR and not a general effect of B 3 vitamins.

2 Animals appeared lethargic and many died after administration of anesthesia. Some animals contained blood in kidney and intestines.

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In the second T1D experiment, sciatic nerves were snap frozen in liquid nitrogen then prepared in a manner similar to liver (Chapter 2.2) for analysis using LC-MS/MS. Unlike the previous samples where sciatic nerve was prepared as a homogenate, NMN, NADP +, and NAD + were not depressed by T1D nor augmented by supplementation of any B 3 vitamin. Unlike in

murine liver (Table 7.1), MeNam and Me4PY were unaffected by T1D but both as well as

Me2PY were greatly increased in sciatic nerve after B 3 vitamin supplementation, indicating that

+ though NAD was not increased, the NAD metabolome was activated by B 3 vitamins. These

methylated metabolites could be indicative of either increased flux through NAD + whereby precursor would contribute to NAD + then degrade to Nam through enzymatic consumption

(Chapter 1.2) and then become methylated and subsequently oxidized. As stated in Chapter

5.2, these methylated compounds could also occur without utilization for NAD +, complicating interpretation of these metabolites as a measure of flux. Unfortunately, NAAD was not detected in this experiment (Chapter 5). Using the same technologies described in Chapter 3 – 5, stable labeled NR supplementation could clarify whether NAD + is effectively elevated by NR in the sciatic nerve with expectation that NAD + enrichment would increase as a function of T1D after

NR supplementation. However, further experiments whereby the concentration of NR was

decreased by a third produced similar toxicities to 0.3% NR dosage. Due to the incredible

increase in mortality, we abandoned the T1D model for the T2D model.

In total, NR prevented DPN in the T1D model in a similar matter to that of T2D. First, NR

effects appear to be independent of glycemic control. Tight glycemic control has been shown to

prevent DPN in type 1 diabetic individuals (241). This glycemic control is achieved through an

intensive regimen with 3 or more insulin doses per day compared with the conventional

treatment of 1-2 per day, which is impractical in the treatment of type 2 diabetics. NR increases

glycemic control in T2D, but this effect appears dispensable for its action on DPN given NR

acted as an anti-neuropathic in both models of diabetes. These results suggest NR may be

efficaciously coupled to other glycemic normalizing drugs such as metformin and insulin to

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prevent and treat further neuropathy. Future work in rodent models is necessary to establish whether NR is synergistic with other anti-diabetic drugs.

NADH and NADPH Measurement in T2D Murine Liver

T2D mice experience depression in liver NADP +. In our routine analysis of the NAD

metabolome as described in Chapter 2, an amount of NADPH oxidizes to NADP + (61), and

hence, measurement of NADP + is a proxy measurement for NADPH. Without direct measurement, the effect of HFD on the NADP + plus NADPH pool could either represent increased reduction to NADPH with concomitant detriment to NADP +, diminishment of NADPH alone, or decrease in both. NAD + tended to decrease as a result of HFD -/+ STZ, but the fate of

its reduced form is unknown. HFD could certainly decrease the NAD +/NADH (189, 193). In order

to measure these metabolites and gain further insight into the effect of high fat diet and STZ on

the NAD metabolome, a methodology for the extraction and analysis was developed (Chapter

2.2: Quantification of NAD(P)H and Extraction from Liver) and performed on the same liver

samples used in the original analysis (Figure 7.4). In development of the NAD(P)H assay, we

also determined the amount of NADH and NADPH that became NAD + and NADP + through the processing and analysis of the oxidized NAD metabolome. 12% and 60% of NADPH and NADH contributed to the NADP + and NAD + quantitation in our routine analysis, respectively. Based

upon this finding, more accurate NADP + and NAD + was determined by subtracting the oxidized form concentration of each by the product of the reduced forms and the appropriate factor. The trends in NADP + and NAD + concentration in Figure 7.4 are exactly as described in table 7.1 with

NADP + depressed by HFD and even more so by HFD+STZ (Figure 7.4d) and NAD + trending down (Figure 7.4a). NR easily restored hepatic NAD + and tended to restore NADP + though not back to control levels. The reductive metabolites presented an interesting pattern. NADH was unaffected in PD and T2D (Figure 7.4b). The NAD +/NADH ratio which is often used a

measurement of the metabolic status of a cell decreased not because both metabolites

decreased but solely due to NAD + deficits (Figure 7.4a and c). PD depressed NADPH (P <

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0.001) compared to NC (Figure 7.4e). T2D depressed NADPH compared to both NC (P <

0.001) and PD (P < 0.05). In both disease models, NR tended to increase NADPH but failed to reach statistical significance and in fact was still significantly depressed compared to control in

T2D (P < 0.01). Since both NADP + and NADPH were depressed in approximately equal

amounts (Figure 7.4f), the oxidized-to-reduced ratio remained unchanged regardless of disease

or supplementation. These results indicate that NAD+ is specifically affected by PD and T2D and

not NADH. Increased acetylation in both the nucleus and mitochondria is observed after HFD

(37, 242, 243), which if NAD + is limiting, could decrease NAD + through the deacylation action of

sirtuins. Further, it implies that modest depletion of NAD + is non-limiting to oxidoreductase

reactions given NADH concentration remained constant. In contrast, NADP + and NADPH

appear to be intimately connected with deficit in one leading to deficit in the other in a nearly

one-to-one manner. This presents an interesting problem in measuring the ratio of the two

metabolites rather than their individual concentrations. If this ratio were the readout for

investigating an effect on ROS detoxification, the consistency in the ratio would lead to a false

negative conclusion. In our data, we observe that the capacity of these phosphorylated NAD + analogs to oppose ROS may be crippled. Regardless of the ratio, if the mole amount of ROS outpaces the mole amount of NADPH, GSH will not regenerate, and the ROS glutathione detoxification pathway will collapse. As stated above, future work is aimed at unraveling the damaging effects of NADP + and NADPH deficit in both PD and T2D and whether NR opposes

said damage.

7.12 Methods

Unless otherwise stated, all methods were performed as described in Chapter 7.2

Methods. NAD(P)H were measured as described in Chapter 2.2: Quantification of NAD(P)H and

Extraction from Liver. Sciatic nerve homogenate (0.05 ml) was thawed and combined with

appropriate internal standard solutions (Chapter 7.8 Supplemental Materials) and extracted with

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0.3 ml of buffered boiled ethanol as described Chapter 2.1. Metabolites separated in the alkaline condition were analyzed as described (1). MeNam, Me2PY, and Me4PY were analyzed as detailed in Chapter 7.5 Supplemental Methods of this chapter and as described in Chapter 2.3

Continued Method Development Post-Initial Publication: Addition of MeNam, Me2PY, and

Me4PY to the NAD Metabolomic Assay.

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7.13 Tables and Figures

Table 7.2 Glycemic control, dyslipidemia, and overall health were not improved by NR in T1D. Male Sprague-Dawley rats made Type 1 diabetic with STZ treatment and immediately began supplementation with NR. Starting and ending weight were recorded for each rat after six weeks of experiencing T1D and NR supplementation. Blood parameters were measured post-mortem. Statistical significance was tested using a one-way followed by a multiple comparisons test with the Holm-Sidak method. A two-way ANOVA was performed to test for statistical significance between starting and ending weight followed by a multiple comparisons tests using the Holm- Sidak method. Results are displayed in parentheses. * P < 0.05, ** P < 0.01, *** P < 0.001 versus Control Control (n=9) STZ Treated STZ Treated + NR (n=7) (0.3%) (n=6) Starting weight (g) 328 ± 12 318 ± 10 319 ± 7 Ending weight (g) 467 ± 24 257 ± 39 *** 228 ± 34 *** (0.083) (0.0368) Blood glucose (mg/dl) a 152 ± 31 580 ± 37 *** 562 ± 36 *** Blood triglycerides 63.2 ± 10.5 634 ± 175 ** 571 ± 127 * (mg/dl) Blood cholesterol (mg/ml) 1.13 ± 0.06 4.23 ± 0.88 4.18 ± 0.66 Hemoglobin A1c (%) 6.7 ± 0.4 17.9 ± 1.1 16.6 ± 0.5 a Blood glucose meters measure at maximum 600 mg/dl, meaning some observations are at or outside of the maximum range.

Table 7.3 NR opposes T1D neuropathy. Mechanical response threshold, thermal response latency, motor nerve conduction velocity, and sensory nerve conduction velocity were measured prior to sacrifice. C fiber density was measured post-mortem in rat hind paws. Statistical significance was determined using a one- way ANOVA followed by a Holm-Sidak multiple comparisons test. STZ tended to worsen measurements of sensation and nerve conduction velocity as well as paw pad C fiber density. NR tended to ameliorate all measurements. * P < 0.05 versus Control, † P < 0.05 versus STZ Control STZ Treated STZ Treated + NR (n=9) (n=7) (0.3%) (n=6) Mechanical response 13.8 ± 4.7 26.8 ± 31.8 15.0 ± 3.6 threshold (g) Thermal response latency 9.8 ± 0.9 16.3 ± 2.3 * 8.8 ± 1.4 † (sec) Motor nerve conduction 62.6 ± 7.4 40.6 ± 4.2 54.9 ± 5.2 velocity (m/sec) Sensory nerve conduction 20.2 ± 0.9 16.6 ± 0.8 * 18.4 ± 0.7 velocity (m/sec) C fiber density (/mm 2) 14.9 ± 1.2 10.6 ± 0.6 * 14.1 ± 0.4 †

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Table 7.4 NR tends to improve STZ induced NAD metabolome defects in sciatic nerve homogenate. Sciatic nerves were excised on the day of sacrifice from Sprague-Dawley rats and prepared as described in 7.12 methods for LC-MS/MS analysis. Overall, NMN, NADP +, and NAD + tended to decrease as a function of STZ and increase a function of NR. Metabolites are expressed as mean ± SEM pmoles/mg of dry particulate weight. Significance was determined using a one- way ANOVA followed by a Holm-Sidak multiple-comparisons test. * P < 0.05 compared to STZ Control (n= 6) STZ (n = 6) STZ + NR Cl (0.3%) (n = 6) NAD + 270 ± 150 136 ± 36 390 ± 9 NMN 86 ± 26 36 ± 7.9 110 ± 18 * NADP + 30 ± 12 22 ± 5.7 44 ± 7.3

Table 7.5 B3 vitamins were ineffective in improving glycemic control and overall health. Type 1 diabetic rats were treated with either 0.3% (w/w) NR or molar equivalent of Nam and NA for up to 12 weeks after STZ treatment. Starting and ending weight as well as non-fasted blood glucose were measured in these animals. NR did not improve these parameters. Weight data were analyzed using a two-way ANOVA followed by a Holm-Sidak multiple comparisons test. P values between start and end are shown in parentheses. Blood glucose measurements were tested for significance using a one-way ANOVA followed by the same type of multiple comparisons test as above. *** P < 0.001 versus Control Control STZ STZ + NR STZ + Nam STZ + NA (n = 3) (n = 3) (n = 4) (n = 3) (n = 2) Starting 300 ± 4.9 290 ± 7.1 300 ± 6.6 300 ± 0.9 321 ± 0.5 Weight(g) Ending 420 ± 13 260 ± 17 270 ± 20 *** 250 ± 4.3 *** 270 ± 1.5 *** Weight (<0.001) (0.0315) (g) Blood 150 ± 20 590 ± 10 *** 580 ± 11 *** 590 ± 9.3 *** 540 ± 65 *** Glucose (mg/dl)a a Blood glucose meters measure at maximum 600 mg/dl, meaning some observations are at or outside of the maximum range.

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Table 7.6 Among the B3 vitamins, NR consistently opposed aspects of T1D neuropathy. Type 1 diabetic rats were treated with either 0.3% (w/w) NR or molar equivalent of Nam and NA for up to 12 weeks after STZ treatment. NR tended to prevent T1D induced neuropathy in a similar fashion as observed after six weeks of T1D (Table 7.3). Nam and NA were indistinguishable from non-treated STZ. Nam appears to have caused a deficit in sensory nerve conduction velocity. Measurements were tested for significance using a one-way ANOVA followed by the same type of multiple comparisons test as above. The low sample sizes preclude strong conclusions. * P < 0.05, ** P < 0.01 versus Control Control STZ STZ + NR STZ + Nam STZ + NA (n = 3) (n = 3) (n = 4) (n = 3) (n = 2) Tactile Response 16 ± 2.4 8.0 ± 1.6 14 ± 1.7 16 ± 3.5 9.6 ± 1.3 (g) Thermal 9.9 ± 0.4 17 ± 1.3 * 11 ± 1.4 14 ± 1.6 10 ± 0.5 Response Latency (sec) Motor Nerve 60 ± 3.9 38 ± 1.4 ** 50 ± 4.2 43 ± 1.4 * 42 ± 0.05 * Conduction Velocity (m/sec) Sensory Nerve 34 ± 2.3 28 ± 1.2 32 ± 1.3 25 ± 0.2 * 27 ± 3.7 Conduction Velocity (m/sec)

Table 7.7 All three B 3 vitamins tended to alter the NAD metabolome in sciatic nerve. Type 1 diabetic rats were treated with either 0.3% (w/w) NR or molar equivalent of Nam and NA for up to 12 weeks after STZ treatment. Sciatic nerve was excised at time of sacrifice and flash frozen. Tissue was extracted and analyzed using LC-MS/MS. Measurements were tested for significance using a one-way ANOVA followed by the same type of multiple comparisons test as above. Metabolites are expressed as mean ± SEM pmoles/mg of dry particulate weight. Control STZ STZ + NR STZ + Nam STZ + NA (n = 3) (n = 3) (n = 4) (n = 3) (n = 2) NAD + 49 ± 1.1 64 ± 11 61 ± 12 67 ± 11 80 ± 21 Nam 5.8 ± 0.68 4.3 ± 0.68 6.1 ± 1.6 13 ± 8.0 8.0 ± 0.34 MeNam 0.19 ± 0.06 0.15 ± 0.05 1.7 ± 0.39 3.2 ± 1.8 2.2 ± 0.62 NADP 3.8 ± 0.67 4.1 ± 0.45 4.9 ± 1.1 4.0 ± 0.30 3.9 ± 1.2 NMN 1.6 ± 0.55 1.1 ± 0.13 3.2 ± 1.4 1.2 ± 0.23 2.6 ± 0.28 Me-4PY 0.37 ± 0.19 0.36 ± 0.06 0.94 ± 0.32 0.45 ± 0.28 1.1 ± 0.93 Me-2PY <0.18 <0.18 1.2 ± 0.40 0.65 ± 0.57 0.35 ± 0.14

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Figure 7.6 NADP + and NADPH were equally depressed by PD and T2D and improved by NR. Livers were excised at time of sacrifice and prepared for LC-MS/MS or LC-MS as described in methods. (a) NAD + was depressed by HFD and HFD+STZ regardless of NR. PD and T2D tended to be depressed compared to control and were restored by NR. (b) NADH remained unchanged and consequentially (c) the NAD +/NADH ratio was depressed in HFD and HFD+STZ as a function of NAD +. In contrast, (d) NADP + was significantly depressed in PD and T2D and was partially restored by NR. (e) NADPH was similarly affected and as consequence (f) the NADP +/NADPH ratio remained unchanged. Overall, the results reveal an effect of HFD that is compounded by STZ and partially restored by NR. Metabolites are expressed as mean ± SEM pmoles/mg wet liver weight. Statistical significance was measured using two-way ANOVA and a Holm-Sidak multiple comparisons test * P < 0.05, ** P < 0.01, *** P < 0.001. Comparisons between groups regardless of NR supplementation are shown below graphs.

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CHAPTER 8 GENERAL SUMMARY AND FUTURE DIRECTIONS 8.1 General Summary

NAD + is crucial to the health of a cell and organism. Pellagra, a disease of NAD +

deficiency, was common in the American rural south a century ago and represented one of the

first recognized health crises in the United States. Investigators identified Pellagra as a

nutritional disease caused by a diet mainly consisting of maize and lard. Pellagra was cured by

including milk and animal meat in the diet and today has been eliminated in high income

nations. Later investigations, identified the first B 3 vitamins NA and Nam as anti-Pellagra agents.

We began this century with new health crises related to an aging population consuming low

vitamin, high calorie diets and, as consequence, experiencing obesity, diabetes and heart

disease at a frequency never before observed. We began this millennium with the identification

of sirtuins as anti-aging enzymes in yeast that depend upon NAD + for activity. Intense investigation into the role of sirtuins and other NAD + glycohydrolases as targets against age and

obesity related morbidities ensued. These investigations continue to reveal these disorders may

also be diseases of dysfunctional NAD + metabolism. Concurrent with these investigation, the

Brenner laboratory identified NR as a novel B 3 vitamin that acts to oppose aging in yeast. NR was later shown to oppose metabolic and neurodegenerative disorders, implicating NR as a health-promoting agent in rodent models. In this thesis, I developed and implemented LC-MS based NAD metabolomic technologies to assess the extra- and intracellular NAD + concentration

and related metabolites in health and disease. The information gathered here represents a first

step in translating the health promoting actions of NR to humans and demonstrates the power of

utilizing metabolomic tools in answering biological questions.

In this thesis, I describe current methods for measuring NAD + and related metabolites and their disadvantages compared to LC-MS. The most common methods, enzyme coupled

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assays and HPLC, lack specificity and, as consequence, are prone to erroneous quantitation.

Further, most investigators report the oxidized versus reductive ratios of NAD + to NADH and

NADP + to NADPH. These ratios reflect gross abnormalities in the NAD metabolome but fail to

elucidate the exact impact of disease/drug/treatment. In my investigations, the ratio of

NAD +/NADH was altered upon ethanol ingestion (Chapter 6) in a manner depending upon

NADH increase. In Chapter 7, my NAD metabolomic analysis revealed the NADP +/NADPH was unchanged but the absolute quantity of both metabolites decreased in models of prediabetic and diabetic animals. In both cases, NR positively affected the individual metabolites but did not greatly affect the ratio. These examples reveal these ratios are not necessarily indicative of disease nor treatment.

I developed quantitative NAD metabolomics using LC-MS and LC-MS/MS due to the superior sensitivity and specificity. Though these technologies add much needed specificity in measurement, quantitation is influenced by sample induced ion suppression. I improved upon earlier LC-MS based NAD metabolomics by employing isotopologue internal standards to control for ion suppression and sample extraction efficiency. Extraction of these metabolites presented challenges due to the chemical behavior of the metabolites, especially NADH and

NADPH, versus the rest of the metabolome. I developed a novel extraction method and LC system to quantitate these reduced metabolites along with the rest of the NAD metabolome. In so doing, I uncovered that a commonly used ion pairing agent (TBA) is incompatible with

NADPH quantitation and may impact quantitation of other organic acids. I then reveal that TEA may be an appropriate alternative to TBA.

In quantifying what we refer to as the NAD metabolome, I was able to assess within a dataset the quality of the data. Normally, the Nam/NAD + ratio is approximately 1:5 (Chapter 5)

but inappropriate sample handling can increase this ratio to ~30 (Table 6.1). Additionally,

inclusion of metabolites not related to NAD + (UMP, Uridine, CMP, Cytosine, etc.) allows

distinguishing of NAD + metabolism specific effects versus base, nucleoside, and nucleotide

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metabolic defects. These improvements in NAD metabolomic analysis may be crucial to investigations of NR as a naturally occurring substance and a therapeutic agent.

NR is a natural precursor found in milk albeit at trace amounts but makes up a majority of its B 3 content. In quantifying the abundance of NR, I found that NR concentration negatively

correlated with Staphylococcus aureus infection. I employed stable isotope technologies to

determine the way in which NR is metabolized by these microbes. In so doing, I found that

these microbes hydrolyze NR via an unknown enzyme resulting in NR serving as a Nam

precursor to this bacterium. To my knowledge, this is the first indication of NR hydrolysis by this

bacterium and requires further investigation to identify responsible enzyme(s). But to a greater

extent, the technology exemplifies a procedure to uncover unknown pathways, which proved

instrumental in subsequent work.

The health promoting effects of NR are known to overlap with its phosphorylated form,

NMN. These findings are obvious if both depend upon NAD + elevation for their therapeutic

action, but to date, no one has directly measured the efficacy of NR versus NMN as a precursor

to NAD +. The metabolism of NMN has become a source of great debate over direct import versus extracellular metabolism to NR. I compared the kinetics of NR versus NMN using stable isotope technologies and revealed NR is a superior precursor to NMN and that NMN appears to be converted to NR extracellularly. Hence, the therapeutic effects of NMN are almost certainly a result of NR, meaning NR is likely a superior therapeutic agent.

The therapeutic effects of NR are achieved at high dosage in mice, receiving 400 mg/kg body weight per day from ad libitum feeding. This design indicates that these mice are receiving

a near constant lower dose over the entire day, which is nearly impossible to translate to a

treatment regimen for a chronic disease. As a proof-of-principle, a healthy 52 year old male

weighing about 63 kg ingested 1 g of NR to assess whether NR could alter the NAD

metabolome. I then analyzed the blood and urine of this individual collected over a day and a

week after NR supplementation and found robust increases in NAD + and associated

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metabolites. These data are the first indication that the health promoting effects in rodents could translate to a human being in a controlled dose strategy.

Since the health promoting effects of NR depend upon its ability to increase NAD + and

possibly other metabolites (73), measuring NR mediated effects on the NAD metabolome in the

target tissue could serve as an indicator of efficacy for NR in opposing disease. Many targets

are inaccessible in clinic, warranting identification of accessible biomarkers for NAD + status. In

performing the first human trial of NR supplementation, I noticed a large increase in NAAD that

occurred around the same time as NAD + elevation. NAAD is part of the deamidated pathway

and is thought to be synthesized from tryptophan and NA, not from NR. The increase in NAAD

could result from a negative feedback mechanism that inhibits NAD synthase, the glutamine

requiring enzyme that converts NAAD to NAD + or from direct conversion of NR or subsequent

metabolite (Nam, NMN, NAD+) through an unknown deamidase pathway.

To test this hypothesis, I compared the kinetics of non-labeled NR, Nam, and NA and

employed the same isotope labeling technology as with the Staphylococcus aureus experiment.

Based on these experiments, NR is a superior liver precursor to NAD + compared to Nam and

NA and the increase in NAD + correlated with NAAD. I proved that NR directly contributes to

murine hepatic NAAD and that the isotopic distribution of NAAD correlated with NAD +, indicating

the presence of a deamidating pathway and that NAAD may truly serve as both a precursor of

+ and biomarker for NAD . We tested whether NAAD could act as a biomarker for efficacious B3

vitamin supplementation by performing the first multi-subject human trial of NR at several one-

time dosages. Blood NAD + was variable in the subjects and mostly did not respond to NR;

however, NAAD responded in a dose dependent manner, supporting NAAD as a biomarker. In

studying alcoholic fatty liver disease (AFLD), I uncovered that NAD + is mildly abrogated

whereas NAAD decreased by half but qualitatively correlated with the decrease in NAD +,

suggesting NAAD may serve as a biomarker for not only NAD + elevation but of NAD + deficit

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(Table 6.1). In this same experiment, NR increased NAAD, agreeing with the human results and further establishing NAAD as an NAD + biomarker.

The work presented in Chapters 3 – 5 establishes that NR is a naturally occurring

metabolite that is by far the superior precursor to NAD + in vitro and in vivo . With this in mind, we turned our attention to NR in treatment of metabolic disease, specifically, AFLD (Chapter 6) and diabetic peripheral neuropathy (Chapter 7). Chronic ethanol ingestion leads to fatty liver disease, but the mechanism of fatty deposition remains unclear. Ethanol metabolism causes a dramatic reductive skew in the NAD + pool, supplies an acetylating precursor to the

mitochondria, and fosters a pro-protein acetylating milieu in said mitochondria leading to

mitochondrial protein hyperacetylation. Mitochondrial protein hyperacetylation causes reduction

in respiration and lipid metabolism. We hypothesized mitochondrial protein acetylation causes

fatty liver disease and that NR would oppose AFLD by restoring the NAD +/NADH ratio and decreasing mitochondrial acetylation through the action of sirtuins. We found that NR could serve as an anti-AFLD agent and positively affected NAD + but did not restore the NAD +/NADH

ratio. Preliminary work seemed to indicate that NR diminished acetylation in a site-specific

manner but not globally, which could mean that most acetylation is non-regulatory or non-

responsive to sirtuins. The NR responsive sites may be regulatory of mitochondrial metabolism

and did appear to enrich in pathways that effect lipid metabolism.

Like AFLD, diabetic peripheral neuropathy is a disease of NAD metabolomic alteration.

Increased NAD + glycohydrolase activity by activation of SARM1 leads to axonal death.

Protection of NAD + through supplementation of NR opposes axonal death in vitro . Further, the

NMNAT1 overexpressing Wld s mouse resists NAD + depletion and in several models of

neuropathy, suggesting NAD + decrement is part of the etiology of neuropathy. We hypothesized

NR would oppose diabetic neuropathy by increasing available NAD +. We found that NR

protected against neuropathy in both a type 1 and type 2 diabetic model. NR did not oppose

hyperglycemia in the type 1 diabetic model but did so in the type 2 diabetic model, suggesting

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that neuropathy and the positive effect of NR does not depend upon increased glycemic control.

Unlike in the AFLD model, NR dramatically reduced fatty liver disease in prediabetic and diabetic animals and protected against liver damage. Further, prediabetic and diabetic mice experienced a deficit in the hepatic NAD metabolome, which was opposed by NR. NR tended to increase the sciatic NAD metabolome in type 1 diabetic rats, agreeing with the hypothesis that in tissue NAD + elevation protects against neuropathy.

These projects are an expansion of tools and knowledge generated by the Brenner

laboratory upon my arrival. This laboratory was the first to report NR as a mammalian B 3 vitamin

(7) and a potential therapeutic agent (32) in yeast and the first to develop an NAD metabolomic assay (31). The works in this thesis provide improvements on the original LC-MS methodology, greatly establish NR as a superior B 3 vitamin in vivo , and add to our knowledge of NR as a

potential therapeutic in metabolic disease.

8.2 Regulation of the NAD + Metabolome: a Future Avenue of Inquiry

Age related and obesity induced metabolic disorders have been described as diseases

of NAD + insufficiency (82, 144). However, NAD + concentration rarely decreases by more than

30% (50) and indeed appears to be maintained by as yet unknown mechanisms (Table 6.1).

Others have reported up-regulation of NAD + biosynthetic enzymes, such as NRK2, and down-

regulation of ADPR transferases following cellular damage (54, 220). These findings highly

suggest the existence of mechanisms maintaining NAD+ concentration. Indeed, SIRT1 appears

to control expression of the NAD + biosynthetic enzyme NAMPT (57, 136), indicating NAD +

regulates its own synthesis. The induction of NRK2 and suppression of NAD + consuming enzymes may follow a similar pathway, whereby activity of a nuclear localized sirtuin decreases in activity, resulting in increased acetylation of a key protein(s). Future work should be designed to elucidate the molecular entities monitoring NAD + abundance and the subsequent

mechanisms that are induced to maintain its normal concentration. In this future work, gene

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expression of all known NAD + biosynthetic and degradative enzymes could be assayed by

qPCR. Additionally, I predict that there exists a nuclear acetylomic program which controls

expression of both types of enzymes and could be elucidated using quantitative LC-MS based

proteomics.

8.3 Future Investigations of NR as a Health Promoting Agent

How NR mediates its near miraculous effects remains to be elucidated. We

hypothesized NR would oppose ethanol induced mitochondrial hyperacetylation; however, NR

appeared to oppose particular sites of acetylation. These sites could be regulatory for

mitochondrial function. As of now, the proteomic data presented here is relative quantitation in

the form of fold changes. Acetylation at any one site could be greatly affected by ethanol and

responsive to NR but represent only 1% of the lysine at that site. In order to identify the

importance of each site, determination of mol-to-mol occupancy of acetylation in a site-specific

manner before and after NR is necessary. Current efforts in the laboratory are aimed at

developing LC-MS/MS technologies for such quantitation.

Orally delivered NR increases the human blood NAD metabolome, indicating that the

effects of NR in ad libitum and controlled dose rodent studies may translate to a human

population. Age and obesity related diseases normally develop gradually after chronic metabolic

insult. Treatments of these diseases require constant administration and in many cases become

less effective as the disease progresses. Though NR does possesses remarkable properties

against diabetic peripheral neuropathy, NR increases mortality in type 1 diabetic rats and

decreases glucose tolerance in control mice (Figure 7.1j). Decreased tolerance could be a result

of increased insulin resistance. Indeed, NA has been shown to increase insulin resistance (240).

Further, despite higher innervation than control, NR causes lower thermal sensitivity in non-

diabetic mice (Figure 7.2c). This could indicate that healthy individuals could be negatively

impacted by NR supplementation. Further work is necessary to uncover whether NR increases

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insulin resistance in healthy mice and to establish the long term effects of NR over time on both healthy and diseased animal models. If NR proves efficacious over time in opposing metabolic insult, at risk groups for developing metabolic disorders could be supplemented in a preventive regimen.

With the exception of acute noise induced hearing loss (56), all studies thus far have implemented a prevention arm alone and delivered NR in food or water. Though injection of NR appears to treat noise-induced hearing loss, we present the first evidence that injection of NR could cause NAD + glycohydrolase inhibition in liver (Figure 5.6j) and may negatively impact liver health as consequence. Investigation into the appropriate delivery method of NR is currently underway. In most cases, the metabolic disorders aided by NR in rodent studies occur over time with most symptoms appearing after the disease has developed. NR as a treatment rather than prevention warrants study.

Finally, though we and others are NAD +-centric, we cannot rule out that NR acts through

non-NAD + related metabolism. NR increases MeNam and its oxidized derivatives Me2PY and

Me4PY (Figure 5.2 e and f and Figure 5.5 c). As mentioned in Chapter 1.2: NAD + Transactions and Chapter 2.2: Addition of MeNam, Me2PY, and Me4PY to the NAD Metabolomic Assay, these metabolites carry their own biological activities and should not be merely interpreted as waste products. These biological activities could indicate that NR mediated effects are at least in part independent of NAD +. If the effects of NR are phenocopied by MeNam or Me2PY or

Me4PY or some combination thereof, then the biologically relevant activity of NR may be less

about contributing to NAD + and more about producing these methylated and oxidized

metabolites. However, the effect of NR could be a result of simultaneous elevation of both NAD +

and these methylated metabolites, indicating that NR is a superior and distinct actor from these

other pharmacological agents. Supplementation of MeNam and its oxidized derivatives should

be undertaken in a head to head comparison to NR in a metabolically challenged or

neurologically stressed rodent model.

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APPENDIX A Table A.1 Human blood NAD metabolome after NR at 100 (low), 300 (mid), and 1000 (high) mg. PBMCs were prepared as described in Chapter 5 and analyzed using LC-MS/MS. All values are in µM. Any 0 values were below the limit of quantification. NM: not measureable Nam MeNam Me4PY Me2PY Dose hrs Mean SEM N Mean SEM N Mean SEM N Mean SEM N low pre 23.4 2.8 12 0.09 0.02 12 0.28 0.03 10 1.5 0.2 10 1 28.6 3.1 12 0.16 0.03 12 0.43 0.04 10 2.4 0.3 10 2 25.8 3.1 12 0.20 0.02 12 0.43 0.04 10 2.7 0.3 10 4 27.0 3.4 11 0.15 0.03 11 0.43 0.06 9 2.9 0.5 9 8 26.9 3.3 12 0.09 0.02 12 0.41 0.05 10 2.4 0.3 10 24 24.5 3.0 12 0.15 0.03 12 0.31 0.04 10 1.6 0.3 10 mid pre 24.1 2.9 12 0.14 0.03 12 0.38 0.06 10 2.2 0.4 9 1 21.6 3.2 12 0.28 0.05 12 0.70 0.09 10 4.2 0.8 10 2 25.2 2.5 12 0.24 0.07 12 0.69 0.12 10 3.9 0.9 11 4 25.0 2.9 11 0.21 0.04 11 0.84 0.09 9 4.5 0.6 10 8 23.9 2.6 12 0.22 0.04 12 1.01 0.16 10 6.5 1.8 11 24 27.5 4.2 12 0.21 0.04 12 0.77 0.13 10 5.1 1.3 10 high pre 22.8 2.6 12 0.10 0.02 12 0.31 0.06 10 2.2 0.5 9 1 23.7 2.3 12 0.34 0.06 12 0.82 0.14 10 5.2 1.0 11 2 24.9 3.2 12 0.34 0.05 12 0.95 0.16 10 5.4 0.9 12 4 24.3 3.3 12 0.42 0.07 12 1.38 0.25 10 8.0 1.4 12 8 23.5 2.6 12 0.38 0.06 12 1.93 0.37 10 11.0 2.1 12 24 29.4 5.8 12 0.48 0.06 12 1.95 0.22 10 10.5 1.6 12

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Table A.1 – continued

NAAD NAD + NMN NADP + Dose Hrs Mean SEM N Mean SEM N Mean SEM N Mean SEM N

low Pre 0.00 NM 10 12.0 3.3 12 1.2 0.3 9 0.99 0.29 10 1 0.00 NM 10 16.0 4.3 12 1.2 0.4 9 1.07 1.14 10 2 0.00 NM 10 14.1 4.1 12 1.6 0.5 9 1.02 0.87 10 4 0.12 0.05 9 16.4 3.7 11 1.6 0.3 8 1.31 1.10 9 8 0.28 0.15 10 14.0 3.8 12 1.1 0.3 9 1.19 0.91 10 24 0.06 0.03 10 13.4 2.9 12 1.3 0.4 9 2.12 2.48 10 mid Pre 0.00 NM 10 13.9 2.8 12 1.5 0.2 10 1.49 1.34 10 1 0.00 NM 10 13.6 2.4 12 1.7 0.3 10 1.24 0.98 10 2 0.00 NM 10 15.1 2.8 12 1.3 0.3 10 1.41 0.96 10 4 0.12 0.04 10 12.3 3.1 12 1.5 0.4 10 1.00 0.82 10 8 0.53 0.23 10 14.7 3.5 12 1.6 0.3 10 1.12 0.92 10 24 0.10 0.05 10 20.4 5.3 12 1.8 0.4 10 1.60 1.28 10 high Pre 0.00 NM 10 13.8 2.6 12 1.4 0.3 9 1.11 1.01 10 1 0.00 NM 10 14.6 3.2 12 1.7 0.3 9 1.25 1.21 10 2 0.00 NM 10 12.4 3.1 12 1.5 0.3 9 1.11 0.97 10 4 0.30 0.18 10 17.2 4.5 12 1.5 0.4 9 1.10 1.15 10 8 0.99 0.44 10 18.9 3.5 12 2.0 0.4 9 1.42 1.41 10 24 0.41 0.16 10 20.3 4.1 12 1.8 0.3 9 1.22 1.10 10

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Table A.2 Human plasma NAD metabolome after NR at 100 (low), 300 (mid), and 1000 (high) mg. Plasma were prepared as described in Chapter 5 and analyzed using LC-MS/MS. All values are in µM. Any 0 values were below the limit of quantification. NM: not measureable Nam MeNam Me4PY Me2PY Dose Hrs Mean SEM N Mean SEM N Mean SEM N Mean SEM N Low Pre 0.00 0.00 12 0.00 0.00 8 0.00 0.00 6 0.00 0.00 11 1 0.13 0.09 12 0.06 0.02 8 0.10 0.03 6 1.10 0.15 11 2 0.04 0.15 12 0.17 0.04 8 0.23 0.04 7 1.51 0.21 11 4 0.24 0.25 12 0.06 0.01 8 0.29 0.08 7 1.45 0.26 11 8 0.04 0.22 11 -0.04 0.03 7 0.19 0.05 7 1.07 0.32 11 24 -0.07 0.20 12 -0.01 0.02 8 0.09 0.04 6 0.11 0.21 12 Mid pre 0.00 0.00 12 0.000 0.00 8 0.00 0.00 6 0.00 0.00 12 1 0.20 0.11 12 0.292 0.04 8 0.25 0.06 6 2.07 0.23 12 2 0.17 0.09 12 0.28 0.05 8 0.37 0.04 8 2.44 0.24 12 4 0.05 0.13 11 0.33 0.12 7 0.55 0.07 9 3.94 0.52 11 8 -0.14 0.09 12 0.08 0.06 8 0.52 0.10 9 3.72 0.35 12 24 -0.18 0.09 11 0.012 0.05 8 0.31 0.15 8 2.10 0.66 11 High pre 0.00 0.00 12 0.000 0.00 8 0.00 0.00 7 0.00 0.00 12 1 0.49 0.11 12 0.551 0.07 8 0.56 0.10 9 3.44 0.55 12 2 0.45 0.16 12 0.625 0.10 8 0.75 0.13 10 4.05 0.84 12 4 0.44 0.16 11 0.946 0.21 7 1.27 0.18 10 8.63 1.04 11 8 0.28 0.10 12 0.735 0.11 8 2.02 0.22 10 13.40 1.64 12 24 0.25 0.12 12 0.362 0.07 8 1.83 0.22 10 10.00 0.99 12

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Table A.2 – continued

NAR (µM) Dose hrs Mean SEM N Low pre 0.00 NM 12 1 0.00 NM 12 2 0.00 NM 12 4 0.00 NM 12 8 0.00 NM 12 24 0.00 NM 12 Mid pre 0.000 NM 12 1 0.001 NM 12 2 -0.003 NM 12 4 0.002 NM 11 8 0.017 0.01 12 24 0.000 NM 12 High pre 0.000 NM 12 1 -0.005 NM 12 2 0.000 NM 12 4 0.013 0.010 12 8 0.069 0.024 12 24 0.008 0.007 12

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Table A.3 Human urine NAD metabolome after NR at 100 (low), 300 (mid), and 1000 (high) mg. Urine was prepared as described in Chapter 5 and analyzed using LC-MS/MS. All values are in µmol/mmol creatinine. Any 0 values were below the limit of quantification. NM: not measureable Nam MeNam Me4PY Me2PY Dose hrs Mean SEM N Mean SEM N Mean SEM N Mean SEM N

Low pre 0.23 0.04 12 4.4 1.0 12 3.4 0.4 12 28.1 5.9 12 0 – 6 0.08 0.02 12 3.9 0.6 12 1.0 0.2 12 6.3 1.6 12 6 – 12 0.03 0.00 11 2.7 0.5 11 1.4 0.3 11 7.8 2.3 11 12 – 24 0.12 0.03 12 4.4 0.8 12 2.3 0.4 12 13.2 2.7 12 Mid pre 0.19 0.02 12 4.9 0.8 12 3.2 0.5 12 23.9 4.8 12 0 – 6 0.16 0.04 12 6.3 1.5 12 1.4 0.3 12 12.8 4.6 12 6 – 12 0.09 0.02 12 5.3 0.5 12 2.9 0.3 12 21.1 2.3 12 12 – 24 0.11 0.02 12 7.2 1.0 12 3.8 0.5 12 33.0 4.8 12 High pre 0.18 0.04 12 4.7 0.8 12 2.9 0.6 12 20.8 5.3 12 0 – 6 0.39 0.08 12 9.2 2.0 12 2.0 0.4 12 17.7 4.0 12 6 – 12 0.50 0.12 12 20.9 5.2 12 8.3 2.2 12 54.5 10.2 12 12 – 24 0.45 0.10 12 29.0 5.3 12 13.3 2.8 12 95.6 14.2 12

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Table A.3 – continued

NAR Dose hrs Mean SEM N Low pre 0.002 0.001 10 0 – 6 0.002 0.002 10 6 – 12 0.029 0.007 9 12 – 24 0.036 0.013 10 Mid pre 0.002 0.000 10 0 – 6 0.010 0.003 10 6 – 12 0.102 0.037 11 12 – 24 0.103 0.031 10 High pre 0.003 0.004 10 0 – 6 0.014 0.005 10 6 – 12 0.274 0.087 11 12 – 24 0.346 0.081 12

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APPENDIX B Table B.1 NR responsive acetyl lysine sites after ethanol ingestion. Hepatic acetylated sites were determined using LC-MS as described in Chapter 6. Results were filtered based upon a significant fold change (≥ 2, P < 0.05) with ethanol alone that was not significant (P > 0.05) upon NR supplementation. UniProt Gene log 2(EtOH log 2(EtOH ID Names AcK Sites /Cntl) P value + NR/Cntl)l P value 5033411D Q7TNE1 12Rik K392 1 1.107 0.040 0.468 0.171 Q8BWT1 Acaa2 K13 1 1.234 0.002 0.286 0.542 Q8BWT1 Acaa2 K25 1 1.224 0.023 0.313 0.413 P45952 Acadm K178 1 2.499 0.009 1.194 0.128 Q07417 Acads K343 1 1.041 0.012 0.379 0.342 P50544 Acadvl K269 1 1.068 0.000 0.818 0.118 Q99KI0 Aco2 K521 1 1.603 0.042 0.338 0.674 Q9CQR4 Acot13 K17 1 1.857 0.049 0.938 0.060 Q8VCW8 Acsf2 K398 1 1.417 0.000 0.597 0.108 Q9WTP7 Ak3 K29 1 1.519 0.048 0.747 0.070 Q8VC19 Alas1 K208 1 3.397 0.034 1.468 0.057 Q9DCX2 Atp5h K99 1 2.612 0.000 0.796 0.157 Q9DCX2 Atp5h K117,K121 2 1.717 0.040 1.836 0.051 Q9DB20 Atp5o K53 1 2.373 0.039 0.725 0.353 Q9DB20 Atp5o K53,K60 2 1.911 0.017 0.760 0.170 Q9JLZ3 Auh K84 1 1.175 0.008 0.625 0.099 Q80XN0 Bdh1 K89 1 1.410 0.012 0.786 0.327 Q71RI9 Ccbl2 K204 1 1.710 0.000 1.053 0.138 Q8C3X2 Ccdc90b K197 1 1.095 0.046 0.220 0.561 P12787 Cox5a K58 1 1.796 0.001 0.783 0.299 Q8C196 Cps1 K458 1 2.296 0.004 1.429 0.083 Q9DBG1 Cyp27a1 K365 1 1.382 0.002 0.752 0.183 Q9D172 D10Jhu81e K152 1 2.268 0.003 1.004 0.348 Q99LB2 Dhrs4 K106 1 1.171 0.044 0.248 0.643 Q9DBT9 Dmgdh K201 1 1.130 0.033 0.622 0.173 Q9D7J9 Echdc3 K76 1 1.588 0.000 0.945 0.090 Q99LC5 Etfa K162,K164 2 1.463 0.040 1.248 0.065 Q921G7 Etfdh K124 1 1.757 0.023 0.803 0.158 Q9DCM0 Ethe1 K32 1 1.334 0.009 0.413 0.281 Q91W43 Gldc K381 1 1.108 0.045 0.616 0.161 Q91W43 Gldc K78 1 1.068 0.017 0.473 0.100 P26443 Glud1 K346 1 1.647 0.024 1.049 0.057 Q5FW57 Gm4952 K41 1 1.039 0.014 0.446 0.127 Q99LP6 Grpel1 K100 1 1.002 0.036 1.010 0.188 Table B.1 – continued

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UniProt Gene Sit log 2(EtOH log 2(EtOH + ID Names AcK es /Cntl) P value NR/Cntl)l P value Q8BGT5 Gpt2 K509 1 1.504 0.028 1.108 0.142 Q8BMS1 Hadha K353 1 1.476 0.013 1.164 0.051 Q99JY0 Hadhb K273 1 1.253 0.020 0.709 0.108 Q8QZS1 Hibch K359 1 1.454 0.041 0.551 0.291 P54869 Hmgcs2 K306 1 2.461 0.001 0.970 0.309 P54869 Hmgcs2 K310 1 1.976 0.019 0.572 0.243 Q2TPA8 Hsdl2 K457 1 1.356 0.043 0.364 0.521 P38647 Hspa9 K600 1 1.789 0.001 1.099 0.095 P38647 Hspa9 K360 1 1.244 0.017 0.765 0.050 P63038 Hspd1 K361 1 2.265 0.050 0.736 0.318 P63038 Hspd1 K180 1 2.744 0.006 1.412 0.120 P63038 Hspd1 K87 1 1.861 0.000 0.879 0.211 P63038 Hspd1 K462 1 1.485 0.005 0.532 0.258 P63038 Hspd1 K389 1 1.585 0.000 0.713 0.202 P54071 Idh2 K133 1 1.603 0.013 0.729 0.053 P54071 Idh2 K282 1 1.652 0.015 0.798 0.095 Q9D6R2 Idh3a K92 1 2.295 0.018 0.915 0.243 P08249 Mdh2 K328 1 3.179 0.040 1.445 0.114 Q8R2L5 Mrps18c K132 1 1.483 0.014 0.435 0.451 Q9D0D3 Mtpap K68 1 1.225 0.015 0.505 0.067 P16332 Mut K604 1 1.489 0.000 0.803 0.170 Q9CPP6 Ndufa5 K60 1 1.124 0.007 0.637 0.136 Q91ZA3 Pcca K150 1 1.148 0.032 0.575 0.272 E9QPD7 Pcx K887 1 2.080 0.011 0.840 0.254 Q8K2B3 Sdha K547 1 1.255 0.009 0.917 0.071 Q3UUI3 Them4 K98 1 1.573 0.000 1.284 0.113 Q3UUI3 Them4 K65 1 1.733 0.010 1.423 0.058 P97493 Txn2 K147 1 2.334 0.006 1.130 0.183 Q9CQB4 Uqcrb K12 1 1.115 0.003 0.559 0.159 Q9DB77 Uqcrc2 K92 1 1.369 0.000 0.694 0.243

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