Location: Obesity and Metabolism Research
Project Number: 2032-51530-022-47-A
Project Type: Cooperative Agreement
Start Date: Jun 1, 2017
End Date: Dec 31, 2017
Recent studies have identified a novel pathway for increased risk for developing cardiovascular disease (CVD). People with high plasma concentrations of trimethylamine N-oxide (TMAO) are more likely to have increased CVD events in clinic-based samples. In spite of this striking association there are limitations in current understanding, as we do not understand why some people are more likely to generate TMAO from dietary precursors (choline and carnitine) than are other people. Specifically, it is not known if (and how) common genetic variants and dietary choline/L-carnitine interact to affect TMAO levels or the mechanism by which TMAO increases CVD. Furthermore, many questions remain regarding the contribution of the microbiome to the TMAO-CVD pathway; for example we do not know which bacteria in the gut are regulating TMAO levels and if host genetics affects the amount of these bacterial species. We suggest that discovery of which genes are responsible for modulation of plasma TMAO concentrations and how these genes interact with gut microbiota is essential if we are to understand the association between TMAO and CVD risk. In the proposed work, we capitalize upon our expertise in mouse genetics to further understand the regulation of TMAO plasma concentrations. In particular we focus on genetic studies in hyperlipidemic mice that integrate microbiome analysis to better understand the genetic and microbes that may mediate both TMAO levels and cardiovascular risk. These studies allow us to leverage the well-controlled studies in mice using the most advanced mouse genetic resources available called the Diversity Outbred (DO) mice. Using a simple breeding scheme we will develop a panel of 600 hyperlipidemic DO mice for genetic mapping and use these for detailed analysis of the microbiome. In this proposal, we seek to further understand the genetic regulators of this metabolite as well as the potential underlying mechanisms for TMAO’s regulation. Successful completion of the proposed aims will provide detailed mechanistic, genetic, and clinical insights into the regulation of TMAO’s metabolism and potential for novel therapeutic targets for CVD and potentially a basis for personalized nutritional recommendations.
TMAO biology is complex and involves interactions among several scales of data including diet, genetics and the gut microbiome. Thus, we approach our goal through specific aims that first isolate the contribution of these scales of data and then identify interactions among them. The genomic loci and microbial taxa identified by the innovative studies in this approach will provide a critical link to understanding the underlying mechanisms regulating TMAO levels and identify new pathways affecting atherosclerosis susceptibility. In order to accomplish our goals we propose the following integrated specific aims: Aim 1: Identify and Validate Pathways that Regulate Plasma TMAO Levels and Atherosclerosis Susceptibility in Mice. We hypothesize that complex genetic interactions regulate plasma TMAO concentration and atherosclerotic lesion development. This aim focuses on identifying and validating genetic factors intrinsic to the host that affect TMAO levels and atherosclerosis. A key concept is that dominant hyperlipidemic models can be used to map genes and pathways associated with TMAO levels and atherosclerosis susceptibility. To accomplish this aim we propose to breed female DO mice with male ApoE*3Leiden/Cholesterol Ester Transfer Protein (CL) double transgenic mice. The incipient F1 mice (CL-F1’s) will be used for high-resolution association mapping. We will accomplish this aim by executing the following: a) Determine the genetic loci regulating the formation of advanced atherosclerotic lesions and plasma TMAO concentration in the CL-F1 transgenic mice. Based on our preliminary data we anticipate being able to identify clinically relevant loci for atherosclerosis with unprecedented resolution; a 100-fold improvement over traditional QTL approaches. b) Validate selected candidate genes that are associated with plasma TMAO levels and atherosclerosis using adenoviral overexpression and genetically engineered mouse models. Aim 2: Identify Resident Gut Microbial Taxa that Regulate Plasma TMAO Levels and Atherosclerosis Susceptibility in Mice. We hypothesize that specific bacterial taxa affect TMAO levels and atherosclerosis susceptibility. To accomplish this aim we propose performing 16S microbial diversity analysis before and after dietary perturbation on the CL-F1 generated for Aim 1. The bacterial taxa identified by these studies will be used as a quantitative trait for bioinformatic analyses that relate specific candidate taxa to TMAO levels and atherosclerosis. We also identify genetic regulation of microbial diversity and abundance. This aim consists of the following: a) Characterize the microbiome and its relationship to basal TMAO concentration and determine the dynamic response of microbial diversity in the CL-F1 mice to a high-fat diet. b) Identify genomic loci regulating composition of the gut microbiome and determine whether these loci are also associated with plasma TMAO concentrations and atherosclerosis. c) Using adoptive microbiome transfer studies, validate selected taxa that are associated with plasma TMAO.