Inhibition of Fat Absorption as a Mechanism to Treat Obesity

描述(由申请人提供)： 在美国，退伍军人中的肥胖症已经上升到流行的程度。根据疾病预防控制中心的指南，33%的男性退伍军人和37%的女性退伍军人患有肥胖症。多年来的研究一直试图解决肥胖的原因。虽然我们对这一问题的认识有了很大提高，但没有有效的措施取得成功，肥胖率继续上升。这里提出的方案旨在通过抑制肠道水平的饮食脂肪吸收来治疗肥胖症。目前，已经开发出一种名为四氢脂抑素的化合物来抑制胰腺脂肪酶，胰腺脂肪酶是一种在吸收之前分解膳食脂肪所需的酶。虽然这在减少脂肪吸收方面起到了作用，但它的副作用如腹胀、放气和腹泻限制了它的有效性。拟议的方案在脂肪被肠道吸收之后，但在进入人体之前抑制脂肪的吸收，因此脂肪酶抑制剂的许多症状都得到了缓解。我们的计划是在肠道吸收细胞的水平上阻止吸收的脂肪。这种类型的阻断是在一种罕见的人类疾病中模拟的，称为反脂蛋白血症。在这种情况下，脂肪会被吸收不良，但这种吸收不良的症状会因为这样一个事实而减轻：肠道细胞持有的脂肪只会被释放到肠腔，然后在肠道细胞死亡时释放到结肠。每个细胞存活2至4天。我们的实验室已经确定了大鼠饮食脂肪吸收的限速步骤。TIS位于内质网(ER)水平。接下来，我们发现脂肪以一个小泡的形式离开内质网，即前乳糜粒运输小泡(PCTV)。我们知道选择货运乳胶粒的蛋白质包含在囊泡中，以及启动这个过程的蛋白质。 乳糜粒是一种脂蛋白，通过它将膳食脂肪输送到循环中。我们的建议解决了我们研究中的一个难题。肝脏脂肪酸结合蛋白(FABP1)本身可以在没有ATP提供的蛋白质磷酸化的情况下产生PCTV。相比之下，含有相当多FABP1的肠道细胞胞浆在没有ATP的情况下不能产生PCTV。为什么？我们在初步研究中发现，胞浆中的L-FABP不是以单一蛋白质的形式存在，而是与其他三种蛋白质形成一个蛋白质复合体，我们知道每种蛋白质都是存在的。我们的第一个目标是确定复合体中的哪些蛋白质在脂肪吸收过程中被磷酸化，并证明阻断这种磷酸化事件在防止FABP1附着到ER膜上启动PCTV发芽级联是有效的。如果我们成功了，那么我们就已经确定了这一过程中受到药物攻击的一个步骤。我们的第二个目标是鉴定内质网表面被三磷酸腺苷磷酸化的蛋白质，并通过这样做增加L-FABP从胞浆蛋白复合体到内质网膜的结合。阻断这一磷酸化步骤也可能抑制PCTV的产生。同样，这种蛋白质可能会受到药物的攻击。我们的第三个目标是将内质网或高尔基体中蛋白质磷酸化的变化与由于饮食或向肠道输送磷脂酰胆碱的改变而导致的进入淋巴的乳胶粒输出的变化相关联。这些变化也将与内质网的PCTV生成活动相关。这将给出我们的体外研究结果的生理构象。

DESCRIPTION (provided by applicant): Obesity in the Veteran population has risen to epidemic proportions in the United States. 33% of male Veterans and 37% of female Veterans are obese as classified by CDC guidelines. Studies over many years have tried to address the causes of obesity. While our understanding of this problem has greatly increased, no effective measures have been successful and obesity rates continue to rise. The program proposed here is designed to treat obesity by inhibiting dietary fat absorption at the level of the intestine. Currently, a compound called tetrahydrolipstatin has been developed to inhibit pancreatic lipase, an enzyme required to break down dietary fat prior to its absorption. While this works in reducing fat absorption, its side effects of bloating, gas, nd diarrhea limit its usefulness. The proposed program inhibits fat absorption after the fat is absorbed into the intestine but before it enters the body so that many of the symptoms of the lipase inhibitor are muted. Our plan is to block the absorbed fat at the level of the intestinal absorptive cell. This type of blockade is modeled in a rare human condition called abetalipoproteinemia. In this condition, fat is malabsorbed but the symptoms of this malabsorbtion are lessened by the fact that the intestinal cell holds the fat that is only released into the intestinal lumen and then to the colon when the intestinal cell dies. Each cell lives 2 to4 days. Our laboratory has identified the rate-limiting step in dietary fat absorption in the rat. Tis was found to be at the level of the endoplasmic reticulum (ER). Next we found that the fat exited the ER in a vesicle, the pre-chylomicron transport vesicle (PCTV). We know the proteins that select cargo chylomicrons for inclusion in the vesicle and the protein that initiates this process. Chylomicrons are the lipoprotein by which dietary fat is delivered to the circulation. Our proposal attacks a conundrum in our studies. The liver fatty acid binding protein (FABP1) can, by itself, generate PCTV without the addition of protein phosphorylation supplied by ATP. By contrast, intestinal cell cytosol, which has considerable FABP1, cannot generate PCTV without ATP. Why? We have found in preliminary studies that L-FABP in cytosol is not present as a single protein but is in a protein complex with 3 other proteins, each of which we know. Our first aim is to determine which of the proteins in the complex is phosphorylated during fat absorption and show that blocking this phosphorylation event is effective in preventing FABP1 from attaching to the ER membrane to start the PCTV budding cascade. If we are successful, then we will have identified one step in the process that is subject to pharmaceutical attack. Our second aim is to identify proteins on the ER surface that are phosphorylated by ATP and in so doing increase the binding of L-FABP from the cytosolic protein complex to the ER membrane. Blocking this phosphorylation step may also inhibit PCTV generation. Again, this protein may be open to pharmaceutical attack. Our third aim is to correlate changes in phosphorylation of proteins in the ER or Golgi with changes in chylomicron output into the lymph due to changes in diet or delivery of phosphatidylcholine to the intestine. These changes will also be correlated with PCTV generating activity of the ER. This will give physiological conformation of our in vitro findings.