A NOVEL FLUORESCENT HPLC-BASED METABOLIC LABELING TECHNIQUE REVEALS EFFECTS OF NUTRITIONAL CONTEXT ON DIETARY FATTY ACID PARTITIONING IN THE INTESTINAL ENTEROCYTES

When dietary fatty acids are absorbed by intestinal enterocytes, they are re-assembled into complex lipids such as phospholipids, cholesterol esters, and triglycerides. Although the cellular machinery required for these processes is well known, the mechanisms regulating the partitioning of individual fatty acids among the thousands of possible complex lipid products are not as well understood. Metabolic labeling of dietary lipids, historically performed using radioactive reagents or expensive stable isotopes, is a key technique for examining the biochemical behavior of individual fatty acids in the context of a physiologically relevant mixed-lipid diet. Likewise, fluorescent lipids have been established as important tools for live imaging of the trafficking and spatial deposition of dietary lipids, yet their metabolism has not been fully characterized. I have developed a novel HPLC-based quantitative metabolic labeling method in a larval zebrafish model, wherein the same fluorescent lipid reagents and delivery methods may be used for biochemical and live imaging experiments. The characterization of fluorescent fatty acid metabolism described here opens the way for more widespread use of fluorescent lipids in metabolic studies. In this thesis I review the current literature on the zebrafish as a model for lipid uptake and metabolism. This review includes my own analysis and interpretation of a recently published developmental lipidomics data set. I then describe the application of metabolic labeling with fluorescent lipids toward a greater understanding of dietary lipid partitioning in the intestinal enterocytes, specifically addressing the interactions among dietary fatty acids, cholesterol, and ethanol. Preliminary results suggest that phospholipid synthesis from dietary fatty acids is increased in larval zebrafish exposed to ethanol before feeding. Future work will address potential physiological effects of this interaction between ethanol and dietary fatty acid partitioning. Additionally, I report results of a collaborative study in which HPLC lipid profiling methods initially developed in zebrafish were expanded into a mouse model. This enabled measurement of changes in individual plasma lipid species resulting from perturbation of a putative lipid transport mechanism in the intestinal enterocytes. These diverse applications of the metabolic labeling methods I have developed demonstrate significant contributions to the lipid biology field: I have expanded both the amount and type of data that can be obtained using HPLC lipidomics, creating a relatively low-cost and high-throughput alternative to metabolic labeling with mass spectrometry analysis. Furthermore, ongoing work may contribute to greater understanding of both potentially beneficial interactions between ethanol and dietary lipids (e.g. the Mediterannean diet hypothesis) and possible diet-driven variability in the progression of alcoholic liver disease

Repetitive Sequence Variations in the Promoter Region of the Adhesin-Encoding Gene sabA of Helicobacter pylori Affect Transcription

The pathogenesis of diseases elicited by the gastric pathogen Helicobacter pylori is partially determined by the effectiveness of adaptation to the variably acidic environment of the host stomach. Adaptation includes appropriate adherence to the gastric epithelium via outer membrane protein adhesins such as SabA. The expression of sabA is subject to regulation via phase variation in the promoter and coding regions as well as repression by the two-component system ArsRS. In this study, we investigated the role of a homopolymeric thymine [poly(T)] tract -50 to -33 relative to the sabA transcriptional start site in H. pylori strain J99. We quantified sabA expression in H. pylori J99 by quantitative reverse transcription-PCR (RT-PCR), demonstrating significant changes in sabA expression associated with experimental manipulations of poly(T) tract length. Mimicking the length increase of this tract by adding adenines instead of thymines had similar effects, while the addition of other nucleotides failed to affect sabA expression in the same manner. We hypothesize that modification of the poly(T) tract changes DNA topology, affecting regulatory protein interaction(s) or RNA polymerase binding efficiency. Additionally, we characterized the interaction between the sabA promoter region and ArsR, a response regulator affecting sabA expression. Using recombinant ArsR in electrophoretic mobility shift assays (EMSA), we localized binding to a sequence with partial dyad symmetry -20 and +38 relative to the sabA + 1 site. The control of sabA expression by both ArsRS and phase variation at two distinct repeat regions suggests the control of sabA expression is both complex and vital to H. pylori infection

Portal protein diversity and phage ecology

© 2008 The Authors. This article is distributed under the terms of the Creative Commons License, Attribution 2.5. The definitive version was published in Environmental Microbiology 10 (2008): 2810-2823, doi:10.1111/j.1462-2920.2008.01702.x.Oceanic phages are critical components of the global ecosystem, where they play a role in microbial mortality and evolution. Our understanding of phage diversity is greatly limited by the lack of useful genetic diversity measures. Previous studies, focusing on myophages that infect the marine cyanobacterium Synechococcus, have used the coliphage T4 portal-protein-encoding homologue, gene 20 (g20), as a diversity marker. These studies revealed 10 sequence clusters, 9 oceanic and 1 freshwater, where only 3 contained cultured representatives. We sequenced g20 from 38 marine myophages isolated using a diversity of Synechococcus and Prochlorococcus hosts to see if any would fall into the clusters that lacked cultured representatives. On the contrary, all fell into the three clusters that already contained sequences from cultured phages. Further, there was no obvious relationship between host of isolation, or host range, and g20 sequence similarity. We next expanded our analyses to all available g20 sequences (769 sequences), which include PCR amplicons from wild uncultured phages, non-PCR amplified sequences identified in the Global Ocean Survey (GOS) metagenomic database, as well as sequences from cultured phages, to evaluate the relationship between g20 sequence clusters and habitat features from which the phage sequences were isolated. Even in this meta-data set, very few sequences fell into the sequence clusters without cultured representatives, suggesting that the latter are very rare, or sequencing artefacts. In contrast, sequences most similar to the culture-containing clusters, the freshwater cluster and two novel clusters, were more highly represented, with one particular culture-containing cluster representing the dominant g20 genotype in the unamplified GOS sequence data. Finally, while some g20 sequences were non-randomly distributed with respect to habitat, there were always numerous exceptions to general patterns, indicating that phage portal proteins are not good predictors of a phage's host or the habitat in which a particular phage may thrive.This research was supported in part by funding from NSF (CMORE contribution #87), DOE, The Seaver Foundation and the Gordon and Betty Moore Foundation Marine Microbiology Program to S.W.C.; an NIH Bioinformatics Training Grant supported M.B.S.; MIT Undergraduate Research Opportunities Program supported V.Q., J.A.L., G.T., R.F. and J.E.R.; Howard Hughes Medical Institute funded MIT Biology Department Undergraduate Research Opportunities Program supported A.S.D.; NSERC (Canada) Discovery Grant (DG 298394) and a Grant from the Canadian Foundation for Innovation (NOF10394) to J.P.B.; NSF Graduate Fellowship funding supported M.L.C

Corrigendum "Portal protein diversity and phage ecology"

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of John Wiley & Sons for personal use, not for redistribution. The definitive version was published in Environmental Microbiology 13 (2011): 2832, doi:10.1111/j.1462-2920.2011.02616.x

A point mutation decouples the lipid transfer activities of microsomal triglyceride transfer protein.

Funder: G. Harold and Leila Y. Mathers Charitable Foundation; funder-id: http://dx.doi.org/10.13039/100001229Apolipoprotein B-containing lipoproteins (B-lps) are essential for the transport of hydrophobic dietary and endogenous lipids through the circulation in vertebrates. Zebrafish embryos produce large numbers of B-lps in the yolk syncytial layer (YSL) to move lipids from yolk to growing tissues. Disruptions in B-lp production perturb yolk morphology, readily allowing for visual identification of mutants with altered B-lp metabolism. Here we report the discovery of a missense mutation in microsomal triglyceride transfer protein (Mtp), a protein that is essential for B-lp production. This mutation of a conserved glycine residue to valine (zebrafish G863V, human G865V) reduces B-lp production and results in yolk opacity due to aberrant accumulation of cytoplasmic lipid droplets in the YSL. However, this phenotype is milder than that of the previously reported L475P stalactite (stl) mutation. MTP transfers lipids, including triglycerides and phospholipids, to apolipoprotein B in the ER for B-lp assembly. In vitro lipid transfer assays reveal that while both MTP mutations eliminate triglyceride transfer activity, the G863V mutant protein unexpectedly retains ~80% of phospholipid transfer activity. This residual phospholipid transfer activity of the G863V mttp mutant protein is sufficient to support the secretion of small B-lps, which prevents intestinal fat malabsorption and growth defects observed in the mttpstl/stl mutant zebrafish. Modeling based on the recent crystal structure of the heterodimeric human MTP complex suggests the G865V mutation may block triglyceride entry into the lipid-binding cavity. Together, these data argue that selective inhibition of MTP triglyceride transfer activity may be a feasible therapeutic approach to treat dyslipidemia and provide structural insight for drug design. These data also highlight the power of yolk transport studies to identify proteins critical for B-lp biology

Lipid Uptake, Metabolism, and Transport in the Larval Zebrafish

The developing zebrafish is a well-established model system for studies of energy metabolism, and is amenable to genetic, physiological, and biochemical approaches. For the first 5 days of life, nutrients are absorbed from its endogenous maternally deposited yolk. At 5 days post-fertilization, the yolk is exhausted and the larva has a functional digestive system including intestine, liver, gallbladder, pancreas, and intestinal microbiota. The transparency of the larval zebrafish, and the genetic and physiological similarity of its digestive system to that of mammals make it a promising system in which to address questions of energy homeostasis relevant to human health. For example, apolipoprotein expression and function is similar in zebrafish and mammals, and transgenic animals may be used to examine both the transport of lipid from yolk to body in the embryo, and the trafficking of dietary lipids in the larva. Additionally, despite the identification of many fatty acid and lipid transport proteins expressed by vertebrates, the cell biological processes that mediate the transport of dietary lipids from the intestinal lumen to the interior of enterocytes remain to be elucidated. Genetic tractability and amenability to live imaging and a range of biochemical methods make the larval zebrafish an ideal model in which to address open questions in the field of lipid transport, energy homeostasis, and nutrient metabolism

A NOVEL FLUORESCENT HPLC-BASED METABOLIC LABELING TECHNIQUE REVEALS EFFECTS OF NUTRITIONAL CONTEXT ON DIETARY FATTY ACID PARTITIONING IN THE INTESTINAL ENTEROCYTES

When dietary fatty acids are absorbed by intestinal enterocytes, they are re-assembled into complex lipids such as phospholipids, cholesterol esters, and triglycerides. Although the cellular machinery required for these processes is well known, the mechanisms regulating the partitioning of individual fatty acids among the thousands of possible complex lipid products are not as well understood. Metabolic labeling of dietary lipids, historically performed using radioactive reagents or expensive stable isotopes, is a key technique for examining the biochemical behavior of individual fatty acids in the context of a physiologically relevant mixed-lipid diet. Likewise, fluorescent lipids have been established as important tools for live imaging of the trafficking and spatial deposition of dietary lipids, yet their metabolism has not been fully characterized. I have developed a novel HPLC-based quantitative metabolic labeling method in a larval zebrafish model, wherein the same fluorescent lipid reagents and delivery methods may be used for biochemical and live imaging experiments. The characterization of fluorescent fatty acid metabolism described here opens the way for more widespread use of fluorescent lipids in metabolic studies. In this thesis I review the current literature on the zebrafish as a model for lipid uptake and metabolism. This review includes my own analysis and interpretation of a recently published developmental lipidomics data set. I then describe the application of metabolic labeling with fluorescent lipids toward a greater understanding of dietary lipid partitioning in the intestinal enterocytes, specifically addressing the interactions among dietary fatty acids, cholesterol, and ethanol. Preliminary results suggest that phospholipid synthesis from dietary fatty acids is increased in larval zebrafish exposed to ethanol before feeding. Future work will address potential physiological effects of this interaction between ethanol and dietary fatty acid partitioning. Additionally, I report results of a collaborative study in which HPLC lipid profiling methods initially developed in zebrafish were expanded into a mouse model. This enabled measurement of changes in individual plasma lipid species resulting from perturbation of a putative lipid transport mechanism in the intestinal enterocytes. These diverse applications of the metabolic labeling methods I have developed demonstrate significant contributions to the lipid biology field: I have expanded both the amount and type of data that can be obtained using HPLC lipidomics, creating a relatively low-cost and high-throughput alternative to metabolic labeling with mass spectrometry analysis. Furthermore, ongoing work may contribute to greater understanding of both potentially beneficial interactions between ethanol and dietary lipids (e.g. the Mediterannean diet hypothesis) and possible diet-driven variability in the progression of alcoholic liver disease

A novel system to quantify intestinal lipid digestion and transport

The zebrafish larva is a powerful tool for the study of dietary triglyceride (TG) digestion and how fatty acids (FA) derived from dietary lipids are absorbed, metabolized and distributed to the body. While fluorescent FA analogues have enabled visualization of FA metabolism, methods for specifically assaying TG digestion are badly needed. Here we present a novel High Performance Liquid Chromatography (HPLC) method that quantitatively differentiates TG and phospholipid (PL) molecules with one or two fluorescent FA analogues. We show how this tool may be used to discriminate between undigested and digested TG or phosphatidylcholine (PC), and also the products of TG or PC that have been digested, absorbed and re-synthesized into new lipid molecules. Using this approach, we explored the dietary requirement of zebrafish larvae for phospholipids. Here we demonstrate that dietary TG is digested and absorbed in the intestinal epithelium, but without dietary PC, TG accumulates and is not transported out of the enterocytes. Consequently, intestinal ER stress increases and the ingested lipid is not available support the energy and metabolic needs of other tissues. In TG diets with PC, TG is readily transported from the intestine and subsequently metabolized.publishedVersio