Lipid transport in the intestine

描述(申请人提供)：脂肪在肠道中的转运脂肪酸(FA)和单甘油(MG)是饮食中三酰甘油(TG)的主要水解物。在我们对肠道吸收MG和FA的基本机制的理解上仍然存在很大的差距。这些包括对两种高表达的肠细胞脂肪酸结合蛋白(FABP)的分子水平的了解，这两种蛋白是肠道FABP和肝脏FABP(IFABP和LFABP)。在之前的利用体外脂质转运动力学的研究中，我们认为IFABP和LFABP可能至少在肠细胞中具有一些独特的功能。最近使用小鼠对这些FABP中的每一个都为空的研究强烈支持这一假设。在我们对肠道脂肪同化的理解中，另一个重要的差距是关于FA和MG在肠细胞中的代谢命运。吸收上皮细胞在添加在细胞的饮食或顶端(AP)表面的FA的代谢与细胞的基侧(BL)表面相比有显著的不同，我们最近也显示出MG的显著代谢极性。然而，这种划分背后的机制在很大程度上仍不清楚。在这个提案中，我们将使用细胞和动物研究的综合方法来研究IFABP和LFABP的功能，以及构成肠道FA和MG代谢极性的基本机制。其具体目标是：1)利用细胞为基础的方法，确定IFABP和LFABP在FA和MG的肠道细胞转运和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定义了这些蛋白质的不同动力学机制，以及导致它们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转染的方法将野生型和特定突变形式的FABP导入培养的细胞模型中，以概括肠细胞的表型，并确定对脂质摄取和定位、脂代谢和脂蛋白分泌的影响。2)利用空白小鼠模型，确定IFABP和LFABP在肠道脂肪同化中的特殊功能。我们实验室已经建立了IFABP和LFABP缺失的小鼠，并观察到了一些有趣的肠道表型变化，包括IFABP-/-小鼠TG合成增加和LFABP基因敲除的FA_2-氧化减少。此外，我们最近发现LFABP是乳胶粒生物发生中的一个关键成分。利用两个FABP缺失的小鼠模型，将结合动物研究、蛋白质组学、脂体学和结构-功能方法来探索FABP的作用机制。3)探讨FA和MG在肠上皮细胞中的代谢区划机制。补充细胞培养方法的小鼠模型的研究将被用于解决质膜转运蛋白和特定的细胞内酶的作用。这项研究计划的总体目标是提供肠道脂肪运输的分子水平图，以便通过调节特定的运输和代谢过程来控制饮食中脂肪同化的速度和程度以及餐后脂肪水平。与公众健康相关：肠道中的脂肪运输西方人的饮食中通常含有大量的膳食脂肪，所有这些都必须被小肠消化、吸收和运输。餐后血脂水平升高被认为是动脉粥样硬化和糖尿病发展的重要因素。在这项拟议的研究中，我们将研究肠道脂肪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，并将确定脂肪消化的产物--脂肪酸和单酰甘油--如何在肠道细胞内运输，导致脂肪代谢的变化。这些研究将使我们能够了解如何调节膳食脂肪同化的速度和程度。

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.