Lipid transport in the intestine

近端肠脂肪酸结合蛋白（FABPs）、FABP1（又名肝脏FABP）和FABP2（又名肠道FABP）长期以来被认为在肠细胞的膳食脂转运和加工中起作用。我们之前在生物物理、细胞和动物水平上的工作已经清楚地表明，这两种结合长链FA的共表达蛋白具有不同的功能。虽然两者都参与肠道脂质摄取和同化，尽管方式不同，但它们似乎在调节全身能量稳态方面也发挥着独特的作用。特别是，高脂肪喂养的FABP1-/-小鼠会贪食并变得明显肥胖，而FABP2-/-小鼠则保持苗条。由于FABP1在肠道和肝脏中高度表达，我们设计了肠道特异性FABP1敲除。我们发现肥胖表型在FABP1int-/-小鼠中重现，强调肠道FABPs在全身能量代谢中的重要作用。出乎意料的是，单细胞转录组学揭示了FABP1和FABP2都在肠内分泌细胞（EEC）中表达，并且我们确实发现了FABP1-/-和FABP2-/-小鼠血浆GIP和GLP-1水平的变化。此外，我们和其他人已经表明，除了FA， FABP1和FABP2结合内源性大麻素（EC）。初步结果还确定了其他生物活性化合物作为这些蛋白质的新配体。因此，我们将探索肠道FABPs可能通过EECs结合来自饮食或其代谢物的特定配体的假设，从而发挥营养传感器的作用，影响系统能量平衡的下游调节。我们将使用多种方法——生化、生理和基于结构的硅方法——开始在这些特定目标中解决这一假设：1)定义FABP1和FABP2的完整脂质组学结合谱，并确定已知和新发现的配体对肠上皮细胞转录组学和激素分泌的影响。研究将使用体外脂质组学生化分析以及肠道类器官培养，这些培养来自与已知和新鉴定的配体孵育的FABP2 null和FABP1int-/-小鼠，以鉴定由特定脂质- fabp相互作用调节的途径；2)确定FABP1和FABP2在肠内分泌信号传导中的作用，以及其如何调节全身代谢作用。这些研究提出的假设是，FABP1和FABP2通过结合EC和/或其他脂质，调节EEC信号和GIP和GLP-1释放到血液中，影响体重增加。研究将使用对FABP1和FABP2进行eec特异性消融的小鼠，并从WT和FABP无效的小鼠中分离eec。3)确定特异性的FABP1和FABP2配体是否能够定制蛋白质表面以进行传感和信号传导。我们将使用已知底物和新鉴定的配体分析核磁共振光谱得出的蛋白质三级结构。我们假设，通过与特定配体的相互作用，fabp将显示出独特的表面特性，从而促进与不同下游效应物的相互作用，即fabp -蛋白相互作用中的配体依赖性差异。

The proximal intestinal Fatty Acid Binding Proteins (FABPs), FABP1 (aka liver FABP) and FABP2 (aka intestinal FABP) have long been thought to function in dietary lipid transport and processing in the enterocyte. Our previous work at the biophysical, cellular, and animal levels has shown clearly that these two coexpressed proteins, both of which bind long chain FA, have distinct functions. While both are involved in intestinal lipid uptake and assimilation, albeit in different ways, they each appear to also function uniquely in regulating systemic energy homeostasis. In particular, whereas high fat-fed FABP1-/- mice are hyperphagic and become markedly obese, FABP2-/- mice remain lean. Since FABP1 is highly expressed in both intestine and liver, we engineered an intestine-specific FABP1 knockout. We find that the obese phenotype is recapitulated in the FABP1int-/- mouse, underscoring an important role for intestinal FABPs in whole body energy metabolism. Unexpectedly, single cell transcriptomics has revealed that both FABP1 and FABP2 are expressed in enteroendocrine cells (EEC), and indeed we find alterations in plasma GIP and GLP-1 levels in FABP1-/- and FABP2-/- mice. Moreover, we and others have shown that in addition to FA, FABP1 and FABP2 bind endocannabinoids (EC). Preliminary results also identify other bioactive compounds as novel ligands for these proteins. Thus, we will explore the hypothesis that intestinal FABPs, potentially via EECs, bind specific ligands derived from the diet or their metabolites, and thereby function as nutrient sensors to effect downstream modulation of systemic energy balance. We will use multiple approaches--biochemical, physiological, and structure-based in silico methods--to begin to address this hypothesis in these Specific Aims: 1) To define the complete lipidomic binding profiles of FABP1 and FABP2, and determine the effects of known and newly identified ligands on intestinal epithelial cell transcriptomics and hormonal secretion. Studies will use in vitro lipidomic biochemical profiling as well as intestinal organoid cultures from FABP2 null and FABP1int-/- mice incubated with known and newly identified ligands to identify pathways modulated by specific lipid-FABP interactions; 2) To identify the roles of FABP1 and FABP2 in enteroendocrine signaling, and how this may regulate systemic metabolic effects. These studies address the hypothesis that FABP1 and FABP2, acting through binding of EC and/or other lipids, modulate EEC signaling and GIP and GLP-1 release into the blood, affecting body weight gain. Studies will use mice with EEC-specific ablation of FABP1 and FABP2, and isolated EECs from WT and FABP null mice. 3) To determine whether specific FABP1 and FABP2 ligands tailor the protein surface for sensing and signaling. We will analyze protein tertiary structures derived from NMR spectroscopy using known substrates and newly identified ligands. We hypothesize that by interacting with specific ligands, FABPs will display uniquely altered surface properties which promote interactions with different downstream effectors, i.e. ligand-dependent differences in FABP-protein interactions.