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«Jay Steven Kirkwood for the degree of Doctor of Philosophy in Pharmacy presented on August 21, 2013. Title: Development and Broad Application of a ...»

-- [ Page 1 ] --

AN

Abstract

OF THE DISSERTATION OF

Jay Steven Kirkwood for the degree of Doctor of Philosophy in Pharmacy presented

on August 21, 2013.

Title: Development and Broad Application of a Mass Spectrometry Based

Metabolomics Platform

Abstract approved:.

Jan F. Stevens

Metabolomics is a comprehensive analysis of small molecules, or metabolites, in a

system. Metabolomics is a hypothesis-generating experiment and offers an unbiased analysis of cell metabolism that can aid in the understanding of fundamental biological processes. Metabolomics is widely and broadly applicable in the biological sciences and has been used to study gene function, elucidate mechanisms of drug action, develop novel therapeutics, and to better understand disease states.

This dissertation presents studies aimed at developing and determining the suitability of a mass spectrometry based untargeted metabolomics platform. Four chapters of original research are presented in this dissertation. The first chapter describes the development of a liquid chromatography-quadrupole-time-of-flight mass spectrometry metabolomics platform and details the various steps involved in a typical metabolomics experiment including metabolite extraction/sample preparation, metabolite separation and data collection, data processing and statistical analysis, and metabolite identification.

The second chapter applies the metabolomics platform to uncover the metabolic consequences of vitamin C deficiency in zebrafish, which, like humans cannot synthesize vitamin C and must acquire it through diet for survival. In addition to uncovering several metabolic changes in vitamin C deficient zebrafish previously reported in genetic animal models of vitamin C deficiency, we found evidence for increased purine nucleotide cycle activity. These results demonstrate the suitability of zebrafish for the study of dietary vitamin C deficiency and highlight the roles of vitamin C in energy metabolism.

The third chapter describes a metabolomics driven effort to characterize the anti- obesity effects and mechanisms of xanthohumol, a prenylated flavonoid found in hops.

Based on a metabolomics analysis of plasma from fatty rats treated with xanthohumol, we measure the bioenergetic effects of xanthohumol on cells in culture and find that it is a general mitochondrial uncoupler. We hypothesize that it is through this mechanism that xanthohumol exerts its anti-obesity effects in vivo.

The fourth chapter investigates the temporal metabolome changes that occur during adipocyte differentiation. Using time course metabolomics, we uncovered increases in several uncharacterized di- and tripeptides, presumably products of protein degradation. We then treated differentiating adipocytes with 18O labeled water and found incorporation of 18O into the peptides, confirming them as products of peptide or protein hydrolysis. In addition, H218O metabolomics revealed enhanced flux through the CDP-choline cycle and activation of glutaminolysis during adipocyte differentiation, highlighting the utility of 18O labeled water metabolomics to uncover alterations in metabolic pathways undetectable with a typical metabolomics experiment.

© Copyright by Jay Steven Kirkwood August 21, 2013 All Rights Reserved Development and Broad Application of a Mass Spectrometry Based Metabolomics Platform

–  –  –

I understand that my dissertation will become part of the permanent collection of Oregon State University libraries. My signature below authorizes release of my dissertation to any reader upon request.

–  –  –

First I thank my advisor, Fred Stevens for his mentorship and guidance over the last four years. I thank you for giving me the freedom to explore any idea no matter how absurd you may have thought it was. I also thank the other members of my committee, Maret Traber, Jane Ishmael, and Tory Hagen for your support and interest in my development as a scientist.

I would also like to express my gratitude toward Jeff Morré for your help with mass spectrometry experiments as well as the laughter over the years. Your knowledge of mass spectrometry instrumentation and troubleshooting skills are humbling.

I thank my Mom, Dad and brother Dax for their support over the years. Thanks Mom, for the abundant supply of home cooked food that fueled the writing of this thesis.

I would like to acknowledge the National Institutes of Health grants RO1HL081721, R21AT005294, S10RR027878, and P30ESOO210.

CONTRIBUTION OF AUTHORS

Cristobal Miranda contributed to the experimental design and writing of Chapters 3, 4, and 5. Claudia Maier contributed to the writing of Chapter 2. LeeCole Legette and Yuan Jiang contributed to the experimental design and writing of Chapter 4. Yuan Jiang contributed to the analysis of Chapter 4. Maret Traber, Katie Lebold, Robert Tanguay, Charlotte Wright, Carrie Barton, and Galen Miller contributed to the experimental design and writing of Chapter 3.

TABLE OF CONTENTS

Page Chapter 1: Introduction to Metabolomics

Metabolomics in the Biological Sciences

LC-MS Based Metabolomics Workflow

Contents of the Dissertation





Chapter 2: Simultaneous, untargeted metabolic profiling of polar and nonpolar metabolites by LC-Q-TOF mass spectrometry

Summary

Introduction

Metabolite Extraction

LC-MS/MS

Metabolite Identification

Background Information

Critical Parameters

Troubleshooting

Anticipated Results

Time Considerations

Acknowledgements

References

Chapter 3: Vitamin C deficiency activates the purine nucleotide cycle in zebrafish..40 Capsule

Summary

Introduction

Experimental Procedures

TABLE OF CONTENTS (Continued)

Page Chemicals

Fish husbandry

Feeding experiment

Measurement of α-tocopherol

Measurement of AA, uric acid, and malondialdehyde (MDA).................44 Metabolite extraction

LC-MS/MS

Data processing and statistical analysis

Metabolite identification

Sample preparation for the determination of AMPD activity

AMPD activity assay

Results and Discussion

Zebrafish α-T, AA, uric acid, and MDA concentrations

Discovery of differentiating metabolites and identification

Purine metabolism

Carnitine metabolism

Glycerophospholipid metabolism

Glutathione metabolism

Acknowledgements

References

Supplemental Material

Chapter 4: A metabolomics driven elucidation of the anti-obesity mechanisms of xanthohumol

Capsule

TABLE OF CONTENTS (Continued)

Page Summary

Introduction

Experimental Procedures

Chemicals

Zucker rat study

LC-MS/MS based metabolomics

Cell culture and treatment

Respiration measurements

Statistical analysis

Results

XN reduces products of dysfunctional lipid metabolism and ROS...........80 XN increases oxygen consumption rate (OCR) and, at high concentrations, decreases OCR through ROS

XN increases uncoupled respiration

XN electrophilicity is not necessary for effects on respiration.................83 Time course metabolomics reveals a catabolic phenotype and induction of an ASR

Discussion

Conclusions

Acknowledgements

References

Supplemental Material

Chapter 5: H218O metabolomics reveals flux through the CDP-choline cycle and shortchain peptides as products of protein degradation in differentiating 3T3-L1 preadipocytes

TABLE OF CONTENTS (Continued)

Page Summary

Introduction

Experimental Procedures

Chemicals

Cell culture and treatment

LC-MS/MS based metabolomics

Determination of cellular protein content

OCR and ECAR measurements

Statistical analyses

Results and Discussion

Time course metabolomics reveals significant alterations in the 3T3-L1 preadipocyte metabolome shortly after initiating differentiation..................124 Short-chain peptides increase during 3T3-L1 preadipocyte differentiation…

Short-chain peptides are products of hydrolysis

Metabolomics of H218O-treated differentiating 3T3-L1 preadipocytes reveals activation of the CDP-choline cycle and glutaminolysis..................128 Short-chain peptide levels in differentiating 3T3-L1 preadipocytes are associated with proteasome ad matrix metalloproteinase activity, but not autophagy

Short-chain peptide formation is not driven by ROS in differentiating 3T3L1 preadipocytes

Conclusions

References

Chapter 6: Conclusions

TABLE OF CONTENTS (Continued)

Page Concluding Remarks

Appendices

Supplemental Experimental Procedures

Cell viability by the MTT assay

Determination of cellular proteins

LC-MS/MS

Supplemental References

LIST OF FIGURES

–  –  –

1.1 LC-MS based untargeted metabolomics workflow

2.1 Sample total ion chromatogram (TIC) from rat plasma and normalized extracted ion chromatogram for several polar and nonpolar metabolites

2.2 Example of a PCA-DA (principal component analysis-discriminant analysis) scores plot

2.3 Example volcano plot of a metabolomics dataset to investigate differences in metabolites for 15 versus 90-minute treatment times

2.4 Metabolite matching by MS and MS/MS

2.5 Comparison of experimental isotope ratio to theoretical isotope ratio.................36

2.6 Comparison of experimentally identified metabolite against chemical standard by retention time and MS/MS

3.1 α-T, AA, uric acid, and MDA concentrations in zebrafish fed experimental diets containing low AA and sufficient or insufficient α-T, compared with fish supplemented with high AA and with sufficient or insufficient α-T..................61

3.2 PCA-DA scores plot of zebrafish fed the AA-deficient and AA-sufficient diet..63

3.3 Volcano plot of polar metabolites detected in positive ion mode in zebrafish fed an AA-deficient and AA-sufficient diet with α-T supplementation

3.4 Identification of metabolites using the untargeted metabolomics approach........67

3.5 Relative differences in metabolite levels between zebrafish fed an AA-deficient diet and fish supplemented with AA, both groups with adequate dietary α-T...69

3.6 Bound and free AMPD activity in zebrafish fed an AA-deficient diet and in fish supplemented with AA, both groups with adequate dietary α-T

4.1 Metabolomics reveals a reduction in products of dysfunctional lipid metabolism and ROS in Zucker fatty rats treated with XN

LIST OF FIGURES (Continued)

–  –  –

4.2 XN has a hormetic effect on respiration and glycolytic energy production.......102

4.3 XN acutely increases uncoupled respiration in muscle and liver cells...............104

4.4 XN acutely increases uncoupled respiration in preadipocytes

4.5 Effects on respiration are not dependent on electrophilicty

4.6 Untargeted, time course metabolomics of muscle cell response to XN.............110

4.7 XN induces oxidative and electrophilic stress and an adaptive response...........112

4.8 XN affects protein degradation, cofactor, and energy metabolism

4.9 XN induces a transient catabolic phenotype

4.10 Proposed downstream cellular responses to acute effects of XN

5.1 Time course metabolomics of differentiating 3T3-L1 preadipocytes................138

5.2 Temporal changes in polyamine, glutathione, and amino acid metabolism during 3T3-L1 preadipocyte differemtiation

5.3 Temporal changes in products of protein degradation during 3T3-L1 preadipocyte differentiation

5.4 Short-chain peptides are products of peptide hydrolysis

5.5 The CDP-choline cycle is activated during 3T3-L1 preadipocyte differentiation…..

5.6 Glutaminolysis is activated during 3T3-L1 preadipocyte differentiation..........148

5.7 Short-chain peptide levels are reflective of proteasome activity

5.8 Short-chain peptide levels are reflective of matrix metalloproteinase activity..152

5.9 Metalloporphyrin complex antioxidants alter the levels of short-chain peptides and bilirubin

LIST OF APPENDIX FIGURES

–  –  –

S3.1 Study design, feeding protocol, and body weights

S3.2 PCA-DA scores plot of zebrafish fed experimental diets

S3.3 Activity of AMP deaminase (AMPD) after various incubation periods..........165 S3.4 Activity of AMPD at various total protein concentrations

S3.5 Activity of AMPD at various substrate concentrations

S4.1 Sex dependent effects of XN in Zucker rats

S4.2 XN alters the plasma metabolome of fasting Zucker rats

S4.3 XN moderately inhibits myocyte OCR and ECAR at concentrations between 5 and 8 µM

S4.4 The effects of antioxidants on myocyte respiration

S4.5 Effect of XN on cell viability and protein content

S4.6 Identification of XN-glutathione (XN-SG) adduct in myocytes

S4.7 Full time course profiles for targeted energy metabolites

S5.1 The relative changes in amino acid levels during 3T3-L1 preadipocyte differentiation are consistent

S5.2 Effect of 3T3-L1 preadipocyte differentiation on total cellular protein content…..

S5.3 Effect of 3T3-L1 preadipocyte differentiation on OCR and ECAR................200 S5.4 Reactive oxygen species do not play a major role in short-chain peptide formation during 3T3-L1 preadipocyte differentiation

–  –  –

S3.1 Metabolites identified from the untargeted metabolomics analysis.................171 S4.1 Metabolites identified in Zucker rat plasma

S4.2 Metabolites identified in C2C12 mouse skeletal muscle cells

S5.1 Metabolites identified in differentiating 3T3-L1 preadipocytes

DEVELOPMENT AND BROAD APPLICATION OF A MASS



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