Avoiding toxicity associated with MTP ablation

高血脂和脂蛋白是各种心血管和代谢紊乱的危险因素。 降低血浆脂质的一种方法是抑制apoB-脂蛋白生物合成，这一过程严重依赖于 一种内质网（ER）驻留伴侣，微粒体甘油三酯转移蛋白（MTP）。MTP 抑制剂减少apoB-脂蛋白分泌并降低血浆胆固醇。但是，它们会增加血浆 转氨酶，如ALT和AST，表明肝损伤。我们假设血浆中的增加 与MTP抑制相关的肝酶是由于微粒体游离胆固醇增加，诱导 内质网应激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，其是一种LXR激动剂，以诱导游离TXR受体。 洛伐他汀，抑制细胞胆固醇生物合成的HMG Co-A还原酶拮抗剂;或 WY 14643，一种增强脂肪酸氧化的PPAR激动剂，持续3或24周。在另一组中，-3 脂肪酸，过氧化物酶体增殖物激活受体/激动剂，将腹腔注射，以减少肝脏甘油三酯和游离 胆固醇此外，将给Alb-Cre-MTPfl/fl小鼠喂食西方饮食，然后用T-0901317处理， 洛伐他汀、WY 14643或-3脂肪酸。然后将在喂食Western Blot的C57 B1/6 J小鼠中进行实验。 饮食，并每天喂食MTP抑制剂。此外，他们将被喂食橄榄油单独或与其他化合物 如上所述，以确定与MTP抑制剂相关的毒性是否可以通过这些治疗来避免。 结果测量将涉及血浆中载脂蛋白B-脂蛋白和肝酶的变化; 甘油三酯、酯化胆固醇和游离胆固醇;定量候选mRNA和蛋白质 参与胆固醇和甘油三酯生物合成、ER应激以及AST/ALT同工型。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 诱导ER应激和凋亡。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，ER应激和诱导之间的联系， 将建立细胞凋亡。第五，ER应激途径的重要性将使用ATF 6-/-证实， 喂食MTP抑制剂的CHOP-/-和Alb-Cre-Ire 1 fl/fl小鼠。第六，我们将确定是否诱导ER应激， 衣霉素增加血浆AST/ALT水平。在这些研究完成后，我们将发现分子 负责与MTP治疗相关的不必要的副作用的机制，并建议避免 这些毒性。这些研究可能会导致新的治疗模式，用于治疗各种 高脂血症，并具有直接的翻译应用潜力。

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 PPARα agonist to enhance β-oxidation of fatty acids, for 3 or 24 weeks. In another group, Ω-3 fatty acids, PPARα/δ 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 Ω-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-Ire1αfl/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.