ABSTRACT Lipids are important endocrine signals in the enterohepatic axis that control gene expression by activating nuclear hormone receptors. Hepatic and intestinal lipid metabolism are key factors in establishing systemic cholesterol and triglyceride homeostasis in mammals. Excess lipid accumulation in these tissues is linked to diabetes, NASH, cancer and other diseases. Our long-term objective is to reveal fundamental mechanisms by which lipid-activated nuclear receptors orchestrate cellular and systemic lipid homeostasis. In the current application, we focus on regulation of a novel sterol-transport pathway by LXR and FXR. We have discovered a family of mammalian proteins (Aster-A, -B and -C) that play an important role in cholesterol movement. The 3 Asters are expressed in a tissue-specific manner and are differentially regulated by nuclear receptors. Aster- B is a target for regulation by sterol-activated LXRs, while Aster-C is regulated by the bile acid-activated FXR. We hypothesize the Asters play key roles in lipid homeostasis in the enterohepatic axis, including dietary lipid absorption, chylomicron production and reverse cholesterol transport. We further hypothesize that Asters are important contributors to the pharmacological effects of LXR and FXR agonists. We will address these hypotheses with the following specific aims. Specific Aim 1 will define the role of FXR-regulated Aster-C in hepatic cholesterol transport. We will use a combination of cellular, biochemical, imaging, and in vivo studies to define the pathway for Aster-C-dependent cholesterol transport in hepatocytes. We have generated liver- specific Aster-C knockout mice, and preliminary analysis reveals them to have altered hepatic and plasma lipid levels. We will perform metabolic analyses and in vivo cholesterol tracer studies to interrogate systemic cholesterol flux. We will analyze how loss or overexpression of Aster proteins affects the movement of HDL- cholesterol into bile and for systemic reverse cholesterol transport at baseline and in response to FXR agonists. Specific Aim 2 will elucidate the role of Asters in intestinal cholesterol transport. We will analyze mice lacking Aster-B, Aster-C, or both to test the importance of Asters in dietary cholesterol absorption and chylomicron production. Preliminary analysis has revealed reduced uptake of dietary cholesterol and reduced enterocyte cholesterol ester content in the absence of both Asters. We will further test whether Asters contribute to trans-intestinal cholesterol excretion. Finally, we have discovered that the approved drug ezetimibe, which inhibits intestinal cholesterol uptake, is a selective ligand for Aster-C, and we have solved the crystal structure of the Aster-C–ezetimibe complex. Based on these findings we will determine whether the Aster pathway contributes to the pharmacological effects of ezetimibe. Completion of our aims is expected to provide fundamental insight into pathways governing lipid t...