Localization and regulation of metabolic gene expression in response to dietary triglycerides

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Abstract: The metabolic response to meals high in fat and cholesterol requires the coordination of a complex system of cellular processes. Disruption of these cellular processes can lead to metabolic diseases, such as diabetes, heart disease, and hepatic steatosis. Previous experiments have shown that high-fat diets increase transcription of genes and gene pathways that mediate lipid metabolism, storage, and secretion. One of the most highly upregulated genes within the digestive organs (liver and small intestine), in response to dietary triglycerides (TG), is apolipoprotein A4 (apoa4). The function of ApoA4 protein is currently unknown, but it has been implicated in the regulation of lipoprotein particle synthesis and secretion, as well as satiety, inflammation, and insulin responsivity. Since TG-induced apoa4 gene expression has not been observed in cultured cells and only observed in whole animal models, I will use the zebrafish system to examine the spatial and temporal expression of lipid metabolic genes in response to dietary TG. The digestive system of zebrafish is functionally and developmentally similar to mammalian systems, with high genetic conservation in essential metabolic components. Because larval zebrafish are optically clear and genetically tractable, I have engineered a cell- tracing, fluorescent reporter in the endogenous apoa4 locus, which will allow me to visualize the dietary TG response in live zebrafish larvae. I plan to characterize the timing and location of apoa4 expression to determine whether expression of lipid metabolic genes is a cell-autonomous response to direct, cellular uptake of TGs; or if it is driven by short- and/or long-distance intercellular signaling. The proposed apoa4 reporter will also allow me to screen for the effect of specific pathway genes and transcription factors (TFs) on the TG-induced transcriptional response of intestinal enterocytes that results in apoa4 expression. I plan to use my apoa4 reporter as a measure of dietary TG absorption to help delineate the cellular mechanisms that underlie the dietary TG metabolic gene response. Although, several TFs implicated in regulating apoa4 have been identified in the liver, TFs responsible for regulating apoa4 in enterocytes have not been characterized. We know that lipoprotein synthesis and secretion in digestive organs is largely dependent on microsomal triglyceride transfer protein (MTP) activity, but the mechanisms underlying the dependence on MTP for apoa4 gene expression in different digestive organs is unknown. To determine the regulatory mechanisms underlying MTP-dependence in the intestine, I plan to use the apoa4 transgenic reporter to assay genes that potentially mediate the relationships between dietary TG absorption, MTP activity, and apoa4 expression. The proposed research will identify regulatory relationships between the metabolic gene response to dietary TGs, and lipoprotein production and secretion. Additionally, the proposed apoa4 optical reporter will be a valuable tool for rapid measurement of triglyceride absorption in response to changes in nutritional, behavioral, microbial, pharmacological, and environmental conditions in future studies.