«Jay Steven Kirkwood for the degree of Doctor of Philosophy in Pharmacy presented on August 21, 2013. Title: Development and Broad Application of a ...»
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
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 AUTHORSCristobal 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 CONTENTSPage 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
Chapter 3: Vitamin C deficiency activates the purine nucleotide cycle in zebrafish..40 Capsule
TABLE OF CONTENTS (Continued)Page Chemicals
Measurement of α-tocopherol
Measurement of AA, uric acid, and malondialdehyde (MDA).................44 Metabolite extraction
Data processing and statistical analysis
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
Chapter 4: A metabolomics driven elucidation of the anti-obesity mechanisms of xanthohumol
TABLE OF CONTENTS (Continued)Page Summary
Zucker rat study
LC-MS/MS based metabolomics
Cell culture and treatment
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
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
Cell culture and treatment
LC-MS/MS based metabolomics
Determination of cellular protein content
OCR and ECAR measurements
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
Chapter 6: Conclusions
TABLE OF CONTENTS (Continued)Page Concluding Remarks
Supplemental Experimental Procedures
Cell viability by the MTT assay
Determination of cellular proteins
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