Lipid transport in the intestine

脂肪酸（FA）和单酰基甘油（MG）是膳食中甘油三酯（TG）的主要水解产物。我们对肠道吸收MG和FA的基本机制的理解仍然存在实质性的差距。其中包括对两种高表达的肠细胞脂肪酸结合蛋白（FABP）、肠道FABP和肝脏FABP （IFABP和LFABP）的单个功能的分子水平理解。在先前使用体外脂质转运动力学的研究中，我们认为IFABP和LFABP可能在肠细胞中至少具有一些独特的功能。最近对这些fabp的小鼠进行的研究有力地支持了这一假设。我们对肠道脂质同化理解的另一个重要空白涉及FA和MG在肠细胞中的代谢命运。与细胞的基底外侧（BL）表面相比，吸收性上皮细胞在添加在细胞的膳食或顶端（AP）表面的FA代谢方面表现出明显的差异，我们最近也证明了MG的显著代谢极性。然而，这种分隔的机制在很大程度上仍然是未知的。在本提案中，我们将采用细胞和动物研究的综合方法来研究IFABP和LFABP的功能，以及肠道FA和MG代谢极性的基本机制。具体目的是：1)使用基于细胞的方法确定IFABP和LFABP在肠细胞运输和FA和MG代谢中的功能和结构/功能关系。我们之前使用模型膜系统的工作已经定义了这些蛋白质的不同动力学机制，以及负责其不同性质的结构域和氨基酸残基。在这些研究中，将使用直接蛋白质转移和DNA转染方法将野生型和特定突变形式的FABPs引入培养细胞模型，再现肠细胞表型，并确定对脂质摄取和定位，脂质代谢和脂蛋白分泌的影响。2)采用小鼠模型确定IFABP和LFABP在肠道脂质同化中的具体功能。我们的实验室已经建立了IFABP和LFABP无效的小鼠，并观察到一些有趣的肠道表型变化，包括IFABP-/-小鼠中TG合成增加，LFABP敲除小鼠中FA 2氧化减少。此外，我们最近发现LFABP是乳糜微粒生物发生的关键成分。利用这两种FABP无效的小鼠模型，我们将结合动物研究、蛋白质组学、脂质组学和结构-功能方法来探索FABP作用的机制。3)确定FA和MG在肠细胞中代谢区隔的机制。在小鼠模型中辅以细胞培养方法的研究将用于解决质膜转运蛋白和特定细胞内酶的作用。本研究计划的总体目标是提供肠道脂质运输的分子水平图像，以便通过调节特定的运输和代谢过程来控制膳食脂质同化的速度和程度以及餐后脂质水平。西方饮食通常含有大量的膳食脂质，所有这些都必须通过小肠消化、吸收和运输。饭后血脂水平升高被认为对动脉粥样硬化和糖尿病的发展很重要。在拟议的研究中，我们将研究肠道脂质同化的两个不完全了解的领域。我们将阐明两种细胞内脂肪酸结合蛋白在脂肪酸运输中的具体功能，我们将确定脂质消化产物脂肪酸和单酰基甘油如何在肠细胞内运输，导致脂质代谢的改变。这些研究将使我们了解如何调节膳食脂质同化的速率和程度。

DESCRIPTION (provided by applicant): Lipid Transport in the Intestine Fatty acids (FA) and monoacylglycerol (MG) are the primary hydrolytic products of dietary triacylglycerol (TG). Substantial gaps remain in our understanding of the basic mechanisms of MG and FA assimilation by the intestine. These include a molecular level understanding of the individual functions of two highly expressed enterocyte fatty acid-binding proteins (FABP), intestinal FABP and liver FABP (IFABP and LFABP). In previous studies using in vitro lipid transport kinetics, we suggested that IFABP and LFABP are likely to have at least some unique functions in the enterocyte. Recent studies using mice null for each of these FABPs strongly support this hypothesis. Another important gap in our understanding of intestinal lipid assimilation concerns the metabolic fate of FA and MG in the enterocyte. The absorptive epithelial cell exhibits marked differences in the metabolism of FA added at the dietary or apical (AP) surface of the cell, compared to the basolateral (BL) surface of the cell, and we have recently demonstrated striking metabolic polarity for MG as well. However, the mechanisms that underlie this compartmentation remain largely unknown. In this proposal, we will use an integrated approach of cellular and animal studies to address the functions of IFABP and LFABP, and the fundamental mechanisms that underlie the polarity of intestinal FA and MG metabolism. The specific aims are 1) to determine the functions and structure/function relationships for IFABP and LFABP in enterocyte transport and metabolism of FA and MG using cell-based approaches. Our previous work using model membrane systems has defined the different kinetic mechanisms for these proteins, and the structural domains and amino acid residues responsible for their distinct properties. In these studies, direct protein transfer and DNA transfection approaches will be used to introduce wild-type and specific mutant forms of the FABPs into cultured cell models that recapitulate the enterocyte phenotype, and effects on lipid uptake and localization, lipid metabolism, and lipoprotein secretion will be determined. 2) To determine the specific functions of IFABP and LFABP in intestinal lipid assimilation using null mouse models. Mice null for IFABP and LFABP have been established in our laboratory, and several interesting intestinal phenotypic changes have been observed, including increased TG synthesis in the IFABP-/- mouse and decreased FA 2-oxidation in the LFABP knockout. Further, we have recently identified LFABP as a critical component in chylomicron biogenesis. Using the two FABP null mouse models, the mechanisms of FABP action will be explored using a combination of animal studies, proteomic, lipidomic, and structure-function approaches. 3) To determine the mechanisms underlying the metabolic compartmentation of FA and MG in the enterocyte. Studies in mouse models supplemented by cell culture approaches will be used to address the role of plasma membrane transporters and specific intracellular enzymes. The overall goal of this research program is to provide a molecular level picture of intestinal lipid traffic in order to enable the control of both the rate and extent of dietary lipid assimilation and postprandial lipid levels, by modulating specific transport and metabolic processes. PUBLIC HEALTH RELEVANCE: Lipid Transport in the Intestine Westerns diets often contain large quantities of dietary lipid, all of which must be digested, absorbed, and transported by the small intestine. Elevated serum lipid levels following meals are thought to be important in the development of atherosclerosis and diabetes. In the proposed research, we will investigate two incompletely understood areas of intestinal lipid assimilation. We will elucidate the specific functions of two intracellular fatty acid binding proteins in fatty acid transport, and we will determine how the products of lipid digestion, fatty acids and monoacylglycerols, are trafficked within the intestinal cell, leading to alterations in lipid metabolism. These studies will enable us to understand how to regulate the rate and extent of dietary lipid assimilation.