Avoiding toxicity associated with MTP ablation

描述(申请人提供)：高血脂和脂蛋白是各种心血管和代谢紊乱的危险因素。降低血脂的一种方法是抑制载脂蛋白的生物合成，这一过程严重依赖于内质网(ER)驻留的伴侣微粒体甘油三酯转移蛋白(MTP)。MTP抑制剂减少载脂蛋白B的分泌，降低血浆胆固醇。然而，它们会升高血浆中的转氨酶，如ALT和AST，表明肝脏受损。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，诱导内质网应激和细胞死亡。我们进一步假设，减少细胞内游离胆固醇的同时抑制MTP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，给Alb-Cre-MTPfl/fl或MTPfl/fl小鼠饲喂T-0901317，LXR激动剂，诱导自由胆固醇外流；洛伐他汀，HMG Co-A还原酶拮抗剂，抑制细胞胆固醇生物合成；或WY14643，PPAR1激动剂，促进脂肪酸的2-氧化，为期3或24周。在另一组，-3脂肪酸，PPAR1/4激动剂，将被注射到腹膜内，以降低肝脏甘油三酯和游离胆固醇。此外，Alb-Cre-MTPfl/F1小鼠将被喂以西式饮食，然后用T-0901317、洛伐他汀、WY14643或&-3脂肪酸治疗。然后，将在喂食西方饮食并每天喂食MTP抑制剂的C57BL/6J小鼠身上进行实验。此外，他们将单独或与上述其他化合物一起喂养橄榄油，以确定这些治疗是否可以避免与MTP抑制剂相关的毒性。结果测量将包括血浆中载脂蛋白和肝酶的变化；肝脏甘油三酯、酯化胆固醇和游离胆固醇的变化；参与胆固醇和甘油三酯生物合成、内质网应激以及AST/ALT异构体的候选mRNAs和蛋白质的定量。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。第二个目的是验证肝酶在血浆中的释放是由于内质网应激和细胞凋亡诱导的假说。我们将首先证明，MTP抑制会增加微粒体中的游离胆固醇。其次，我们将确定MTP消融/抑制激活的内质网应激通路。第三，我们将建立抑制MTP诱导细胞凋亡的机制。第四，将在内质网应激和诱导细胞凋亡之间建立联系。第五，内质网应激通路的重要性将通过喂养MTP抑制剂的ATF6-/-、CHOP-/-和Alb-Cre-Ire11fl/fl小鼠得到证实。第六，我们将确定衣霉素诱导的内质网应激是否会增加血浆AST/ALT水平。在这些研究完成后，我们将找出与MTP治疗相关的不良副作用的分子机制，并提出避免这些副作用的解决方案。这些研究可能导致治疗各种高脂血症的新的治疗方式，并立即具有翻译使用的潜力。

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.