Université Pierre et Marie Curie-Paris 6, UMR S 872, Les Cordeliers, Paris 75006, France.
Biol Cell. 2011 Nov;103(11):499-517. doi: 10.1042/BC20110024.
Intestinal absorption of alimentary lipids is a complex process ensured by enterocytes and leading to TRL [TAG (triacylglycerol)-rich lipoprotein] assembly and secretion. The accumulation of circulating intestine-derived TRL is associated with atherosclerosis, stressing the importance of the control of postprandial hypertriglyceridaemia. During the postprandial period, TAGs are also transiently stored as CLDs (cytosolic lipid droplets) in enterocytes. As a first step for determining whether CLDs could play a role in the control of enterocyte TRL secretion, we analysed the protein endowment of CLDs isolated by sucrose-gradient centrifugation from differentiated Caco-2/TC7 enterocytes, the only human model able to secrete TRL in culture and to store transiently TAGs as CLDs when supplied with lipids. Cells were analysed after a 24 h incubation with lipid micelles and thus in a state of CLD-associated TAG mobilization.
Among the 105 proteins identified in the CLD fraction by LC-MS/MS (liquid chromatography coupled with tandem MS), 27 were directly involved in lipid metabolism pathways potentially relevant to enterocyte-specific functions. The transient feature of CLDs was consistent with the presence of proteins necessary for fatty acid activation (acyl-CoA synthetases) and for TAG hydrolysis. In differentiated Caco-2/TC7 enterocytes, we identified for the first time LPCAT2 (lysophosphatidylcholine acyltransferase 2), involved in PC (phosphatidylcholine) synthesis, and 3BHS1 (3-β-hydroxysteroid dehydrogenase 1), involved in steroid metabolism, and confirmed their partial CLD localization by immunofluorescence. In enterocytes, LPCAT2 may provide an economical source of PC, necessary for membrane synthesis and lipoprotein assembly, from the lysoPC present in the intestinal lumen. We also identified proteins involved in lipoprotein metabolism, such as ApoA-IV (apolipoprotein A-IV), which is specifically expressed by enterocytes and has been proposed to play many functions in vivo, including the formation of lipoproteins and the control of their size. The association of ApoA-IV with CLD was confirmed by confocal and immunoelectron microscopy and validated in vivo in the jejunum of mice fed with a high-fat diet.
We report for the first time the protein endowment of Caco-2/TC7 enterocyte CLDs. Our results suggest that their formation and mobilization may participate in the control of enterocyte TRL secretion in a cell-specific manner.
肠道对食物脂质的吸收是一个复杂的过程，由肠细胞来保证，这个过程最终导致 TRL（TAG [三酰甘油]丰富的脂蛋白）的组装和分泌。循环中肠道衍生的 TRL 的积累与动脉粥样硬化有关，这强调了控制餐后高甘油三酯血症的重要性。在餐后期间，TAG 也会作为 CLD（胞质脂质滴）短暂储存在肠细胞中。作为确定 CLD 是否可以在控制肠细胞 TRL 分泌中发挥作用的第一步，我们分析了用蔗糖梯度离心从分化的 Caco-2/TC7 肠细胞中分离的 CLD 所富含的蛋白质，这些细胞是唯一能够在培养中分泌 TRL 并在供应脂质时作为 CLD 短暂储存 TAG 的人类模型。细胞在与脂质微滴孵育 24 小时后进行分析，此时 CLD 相关的 TAG 动员状态。
通过 LC-MS/MS（液相色谱与串联质谱）在 CLD 部分鉴定出的 105 种蛋白质中，有 27 种直接参与潜在与肠细胞特异性功能相关的脂质代谢途径。CLD 的短暂特征与参与脂肪酸激活（酰基辅酶 A 合成酶）和 TAG 水解所需的蛋白质一致。在分化的 Caco-2/TC7 肠细胞中，我们首次鉴定出 LPCAT2（溶血磷脂酰胆碱酰基转移酶 2），其参与 PC（磷脂酰胆碱）的合成，和 3BHS1（3-β-羟类固醇脱氢酶 1），参与类固醇代谢，并通过免疫荧光法证实其部分 CLD 定位。在肠细胞中，LPCAT2 可能从肠腔中的溶血磷脂酰胆碱中提供经济来源的 PC，这是膜合成和脂蛋白组装所必需的。我们还鉴定出参与脂蛋白代谢的蛋白质，如 ApoA-IV（载脂蛋白 A-IV），它是肠细胞特异性表达的，并且被提出在体内发挥许多功能，包括脂蛋白的形成和控制其大小。ApoA-IV 与 CLD 的关联通过共聚焦和免疫电子显微镜证实，并在高脂肪饮食喂养的小鼠空肠中进行了体内验证。
我们首次报道了 Caco-2/TC7 肠细胞 CLD 的蛋白质组成。我们的结果表明，它们的形成和动员可能以细胞特异性的方式参与控制肠细胞 TRL 的分泌。
