Avoiding toxicity associated with MTP ablation

描述（由申请方提供）：高血脂和脂蛋白是各种心血管和代谢疾病的风险因素。降低血浆脂质的一种方法是抑制apoB-脂蛋白生物合成，这是一个关键依赖于内质网（ER）驻留伴侣，微粒体甘油三酯转移蛋白（MTP）的过程。MTP抑制剂减少apoB-脂蛋白分泌并降低血浆胆固醇。然而，它们会增加血浆转氨酶，如ALT和AST，表明肝损伤。我们推测与MTP抑制相关的血浆肝酶升高是由于微粒体游离胆固醇升高、ER应激诱导和细胞死亡。我们进一步假设，降低细胞游离胆固醇沿着MTP抑制可能减少高血压并避免与MTP拮抗剂相关的毒性。在第一个目标中，将给Alb-Cre-MTPfl/fl或MTPfl/fl小鼠喂食T-0901317（一种诱导游离胆固醇流出的LXR激动剂）、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WY 14643（一种增强脂肪酸2-氧化的PPAR 1激动剂）3周或24周。在另一组中，将腹膜内注射β-3脂肪酸、PPAR 1/4激动剂以降低肝甘油三酯和游离胆固醇。此外，将给Alb-Cre-MTPfl/fl小鼠喂食西方饮食，然后用T-0901317、洛伐他汀、WY 14643或β-3脂肪酸处理。然后在喂食西方饮食并每天喂食MTP抑制剂的C57 Bl/6 J小鼠中进行实验。此外，它们将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。结果测量将涉及血浆中apoB-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的候选mRNA和蛋白质的定量、ER应激以及AST/ALT亚型。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。第二个目的是检验血浆中肝酶的释放是由于ER应激和细胞凋亡的诱导的假设。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ER应激途径。第三，我们将确定MTP抑制诱导细胞凋亡。第四，将建立ER应激和细胞凋亡诱导之间的联系。第五，ER应激途径的重要性将使用喂食MTP抑制剂的ATF 6-/-、CHOP-/-和Alb-Cre-Ire 11 fl/fl小鼠来证实。第六，我们将确定衣霉素诱导的ER应激是否增加血浆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，并提出避免这些毒性的解决方案。这些研究可能会导致新的治疗模式，用于治疗各种高血压，并有直接的潜在转化使用。

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.