Date of Award
Doctor of Philosophy (PhD)
Adult zebrafish have been used extensively as a vertebrate model organism, however, metabolomic studies, especially isotope labeling analyses, have historically been underrepresented. A major contributor to this deficit is the lack of methods for metabolomic studies in adult zebrafish. To address this, a workflow was developed for both untargeted profiling and isotope-tracing targeted metabolomics in adult zebrafish using liquid chromatography/mass spectrometry (LC/MS) of extracted tissue and serum samples. To demonstrate the utility of zebrafish and this workflow, LC/MS-based metabolomics was applied to three unique biochemical problems: (i) to understand the metabolic connections between tumor and liver, (ii) to understand off-target effects of hydroxychloroquine sulfate (HCQ), and (iii) to understand the role of microbiota in whole-body metabolism. For the first, melanoma-bearing zebrafish were found to utilize an alanine-glucose tumor-liver cycle to sustain nitrogen levels in the tumor. Tumors excreted excess nitrogen in the form of alanine and the liver maintained systemic glucose levels by increasing gluconeogenic flux. Second, a platform was developed to analyze whole-body effects of HCQ using absolute quantitation, and untargeted and isotope-tracing metabolomics. For these studies, a matrix-matched absolute quantitation workflow was developed. These analyses found that although HCQ accumulation was low in the brain, the drug increased pyruvate carboxylase activity and N-acetylaspartic acid concentration in this organ. For the third application, LC/MS-based metabolomics was employed to study microbial contributions to organismal metabolism. While many studies have investigated bacterial populations, current understanding of microbial metabolite contributions to whole body metabolism remains limited. To address this, a microbiota-depleted model in adult zebrafish was developed and validated. To probe bacteria-specific metabolism, stable isotope-labeled cellobiose was synthesized and administered to zebrafish. These studies showed a broad impact on organismal metabolism due to microbiota manipulations. Further, the data support evidence for intestinal sorbitol biosynthesis for use by gut microbiota. In the absence of gut bacteria, this sorbitol can contribute to fatty liver via activation of glucose metabolism in the liver. In conclusion, this work established a platform for LC/MS-based metabolomics studies in adult zebrafish and applied this to three biochemical studies.
Chair and Committee
Richard A. Loomis
Alexei V. Demchenko, Meredith E. Jackrel, Gary J. Patti, Lilianna Solnica-Krezel
Jackstadt, Madelyn Maria, "Leveraging Isotope Tracers in Adult Zebrafish to Understand Alterations to Organismal Metabolism" (2023). Arts & Sciences Electronic Theses and Dissertations. 2863.
Available for download on Monday, April 10, 2073