Avoiding toxicity associated with MTP ablation

描述（由申请人提供）：高血脂和脂蛋白是各种心血管和代谢疾病的危险因素。降低血脂的一种方法是抑制载脂蛋白生物合成，这一过程严重依赖于内质网（ER）常驻伴侣，微粒体甘油三酯转移蛋白（MTP）。MTP抑制剂可减少载脂蛋白分泌，降低血浆胆固醇。然而，它们增加血浆转氨酶，如ALT和AST，表明肝损伤。我们假设与MTP抑制相关的血浆肝酶的增加是由于微粒体游离胆固醇的增加、内质网应激的诱导和细胞死亡。我们进一步假设，在抑制MTP的同时降低细胞游离胆固醇可能会降低高脂血症，并避免与MTP拮抗剂相关的毒性。在第一个目标中，将Alb-Cre-MTPfl/fl或MTPfl/fl小鼠喂食LXR激动剂T-0901317诱导游离胆固醇外排；洛伐他汀，一种抑制细胞胆固醇生物合成的HMG Co-A还原酶拮抗剂；或WY14643，一种PPAR1激动剂，可增强脂肪酸的2氧化作用，治疗3或24周。另一组腹腔注射& 3脂肪酸，PPAR1/4激动剂，以降低肝脏甘油三酯和游离胆固醇。此外，Alb-Cre-MTPfl/fl小鼠将饲喂西式饮食，然后给予T-0901317、洛伐他汀、WY14643或& 3脂肪酸。实验将在C57Bl/6J小鼠中进行，C57Bl/6J小鼠分别饲喂西餐和每日饲喂MTP抑制剂。此外，他们将被单独喂食橄榄油或与上述其他化合物一起喂食，以确定这些治疗是否可以避免与MTP抑制剂相关的毒性。结果测量包括血浆中载脂蛋白和肝酶的变化；肝甘油三酯、酯化胆固醇和游离胆固醇；参与胆固醇和甘油三酯生物合成、内质网应激以及AST/ALT亚型的候选mrna和蛋白质的量化。这些研究将表明，与MTP抑制相关的毒性可以通过降低肝脏游离胆固醇来避免。第二个目的是验证血浆中肝酶释放是由于内质网应激和细胞凋亡诱导的假设。我们将首先证明MTP抑制增加微粒体游离胆固醇。其次，我们将确定由MTP消融/抑制激活的内质网应激途径。第三，我们将确定MTP抑制诱导细胞凋亡。第四，建立内质网应激与诱导细胞凋亡之间的联系。第五，内质网应激途径的重要性将通过喂食MTP抑制剂的ATF6-/-、CHOP-/-和Alb-Cre-Ire11fl/fl小鼠得到证实。第六，我们将确定tunicamycin诱导内质网应激是否会增加血浆AST/ALT水平。在这些研究完成后，我们将发现与MTP治疗相关的不良副作用的分子机制，并提出避免这些毒性的解决方案。这些研究可能会导致治疗各种高脂血症的新治疗方式，并具有立即转化应用的潜力。

DESCRIPTION (provided by applicant): High plasma lipids and lipoproteins are risk factors for various cardiovascular and metabolic disorders. An approach to lower plasma lipids is to inhibit apoB-lipoprotein biosynthesis, a process critically dependent on an endoplasmic reticulum (ER) resident chaperone, microsomal triglyceride transfer protein (MTP). MTP inhibitors decrease apoB-lipoprotein secretion and lower plasma cholesterol. However, they increase plasma aminotransferases, such as ALT and AST, indicating liver injury. We hypothesize that increases in plasma hepatic enzymes associated with MTP inhibition are due to increases in microsomal free cholesterol, induction of ER stress and cell death. We further hypothesize that reducing cellular free cholesterol along with MTP inhibition might reduce hyperlipidemias and avoiding toxicities associated with MTP antagonists. In the first aim, Alb-Cre-MTPfl/fl or MTPfl/fl mice will be fed T-0901317, a LXR agonist to induce free cholesterol efflux; lovastatin, a HMG Co-A reductase antagonist to inhibit cellular cholesterol biosynthesis; or WY14643, a PPAR1 agonist to enhance 2-oxidation of fatty acids, for 3 or 24 weeks. In another group, &-3 fatty acids, PPAR1/4 agonists, will be injected intraperitoneally to reduce hepatic triglyceride and free cholesterol. In addition, Alb-Cre-MTPfl/fl mice will be fed a western diet and then treated with T-0901317, lovastatin, WY14643, or &-3 fatty acids. Experiments will then be performed in C57Bl/6J mice fed a western diet and fed daily with MTP inhibitors. Additionally, they will be fed olive oil alone or with other compounds described above to determine if toxicities associated with MTP inhibitors can be avoided by these treatments. Outcome measurements will involve changes in apoB-lipoproteins and hepatic enzymes in the plasma; hepatic triglycerides, esterified cholesterol, and free cholesterol; quantification of candidate mRNAs and proteins involved in cholesterol and triglyceride biosynthesis, ER stress, as well as AST/ALT isoforms. These studies will show that toxicities associated with MTP inhibition can be avoided by reducing hepatic free cholesterol. The second aim is to test the hypothesis that release of hepatic enzymes in the plasma is due to the induction of the ER stress and apoptosis. We will first demonstrate that MTP inhibition increases microsomal free cholesterol. Second, we will identify the ER stress pathways activated by MTP ablation/inhibition. Third, we will establish that MTP inhibition induces apoptosis. Fourth, a link between the ER stress and induction of apoptosis will be established. Fifth, importance of the ER stress pathways will be substantiated using ATF6-/-, CHOP-/- and Alb-Cre-Ire11fl/fl mice fed MTP inhibitors. Sixth, we will determine if induction of ER stress by tunicamycin increases plasma AST/ALT levels. At the completion of these studies, we will find out molecular mechanisms responsible for unwanted side effects associated with MTP therapy and suggest solutions to avoid these toxicities. These studies may lead to new therapeutic modalities for the treatment of various hyperlipidemias and have immediate potential for translational use.