Apolipoprotein AV and intestinal transport

 描述（由申请人提供）：冠心病、糖尿病和肥胖仍然是全球主要的临床问题，血浆甘油三酯（TG）水平升高构成了一个独立的风险因素。载脂蛋白AV（apoAV）是2001年发现的一种由肝脏合成和分泌的蛋白质，与血浆TG水平成反比。缺乏功能性apoAV的小鼠和人具有显著升高的血浆TG，而具有高表达的人apoAV的小鼠具有显著降低的血浆TG。奇怪的是，与其他载脂蛋白如apoB和apoAI相比，apoAV的血浆浓度极低。多年来，研究人员一直着迷于这样一种低循环载脂蛋白可以对血浆TG产生如此深远的影响。我们相信，我们有一些新的见解，这个问题，这涉及到apoAV的作用，形成和分泌乳糜微粒（CM）的肠上皮细胞。我们的初步数据显示：1）apoAV敲除（KO）动物在甘油三酯（TG）和胆固醇的吸收和淋巴转运方面比WT动物显著更有效; 2）apoAV-KO动物比WT动物具有更短的CM出现时间; 3）apoAV-KO动物比WT动物从内质网（ER）释放更多的前CM转运囊泡（PCTV）; 4）apoAV-KO动物比WT动物分泌更多的apoB 48（因此更多的CM颗粒，因为每个颗粒有一个apoB 48）到淋巴中; 5）apoAV在主动脂肪吸收期间与淋巴中的CM相关;和6）apoAV存在于胆汁中。我们假设，由肝脏分泌的apoAV调节细胞内的形成和分泌的乳糜微粒的肠细胞和这种调节是通过胆汁输送和摄取的apoAV从肠腔介导的。我们将在两个具体目标中检验这些假设。目的1：探讨apoAV基因敲除小鼠TG和胆固醇淋巴转运增加的分子机制。本研究从四个方面探讨了apoAV调控小肠CM形成和分泌的分子机制。将确定apoAV在CM和VLDL途径中转运酯化FA的作用。利用我们标记和分离肠CM的能力，还将研究apoAV对CM代谢的作用。目的2：在apoAV-KO动物中使用重组apoAV，我们将确定在循环中恢复apoAV对肠CM转运的影响。我们将确定恢复apoAV的肝生产敲除动物（从而恢复循环和胆汁apoAV）对肠CM转运的影响。最后，我们将直接确定apoAV-KO动物管腔中胆汁apoAV对肠道脂质和载脂蛋白分泌的作用，并研究肠道对35 S甲硫氨酸标记的apoAV的摄取和淋巴转运。

 DESCRIPTION (provided by applicant): Coronary heart disease, diabetes, and obesity remain major clinical problems worldwide and elevated plasma triglyceride (TG) levels constitute an independent risk factor. Discovered in 2001, apolipoprotein AV (apoAV) is a protein synthesized and secreted by the liver and is found to be inversely proportional to plasma TG levels. Mice and humans lacking functional apoAV have markedly elevated plasma TG while mice with high expression of human apoAV have dramatically decreased plasma TG. Curiously, plasma concentrations of apoAV are extremely low compared with other apolipoproteins such as apoB and apoAI. It has fascinated investigators for years that such a low circulating apolipoprotein can exert such a profound effect on plasma TG. We believe we have some novel insight to this question and this involves apoAV's action on the formation and secretion of chylomicrons (CM) by the enterocytes. Our preliminary data show that: 1) apoAV knockout (KO) animals were significantly more efficient in the absorption and lymphatic transport of both triglyceride (TG) an cholesterol than WT animals; 2) apoAV-KO animals have shorter CM appearance time than WT animals; 3) apoAV-KO animals release more pre-CM transport vesicles (PCTV) from endoplasmic reticulum (ER) than WT animals; 4) apoAV-KO animals secrete more apoB48 (therefore more CM particles since there is one apoB48 per particle) into lymph than WT animals; 5) apoAV is associated with CM in lymph during active fat absorption; and 6) apoAV is present in bile. We hypothesize that apoAV secreted by the liver regulates the intracellular formation and secretion of chylomicrons by the enterocytes and this regulation is mediated via biliary delivery and uptake of apoAV from the intestinal lumen. We will test these hypotheses in two specific aims. AIM 1: To determine the molecular mechanisms underlying the increased lymphatic transport of TG and cholesterol by apoAV-KO mice. There are four subaims to investigate the molecular mechanism of how apoAV modulates the formation and secretion of CM by the small intestine. The role of apoAV in CM and VLDL pathways for the transport of esterified FA will be determined. Taking advantage of our ability to label and isolate intestinal CM, the role of apoAV on CM metabolism will also be studied. AIM 2: Using recombinant apoAV in apoAV-KO animals, we will determine the effect of restoring apoAV in the circulation on intestinal CM transport. We will determine the effect of restoring hepatic production of apoAV to knockout animals (thus restoring both circulating and biliary apoAV) on intestinal CM transport. Lastly, we will determine directly the role of biliary apoAV in the lumen of apoAV-KO animals on lipid and apolipoprotein secretion by the gut, and study the uptake and lymphatic transport of 35S methionine labeled apoAV by the intestine will be studied.