TMEM55B as a molecular determinant of NAFLD

摘要 非酒精性脂肪性肝病(NAFLD)是由脂肪变性引发的一系列疾病，可发展为 非酒精性脂肪性肝炎(NASH)和肝硬变。尽管它是美国肝病的主要原因， 没有针对性的治疗干预措施，突显了确定分子过程的迫切需要 在它的病理基础上。跨膜蛋白55B(TMEM55B)是一种位于细胞表面的PI(4，5)P2磷酸酶。 已被证明影响溶酶体水平和定位的溶酶体。在初步研究中，我们发现 TMEM55B反义寡核苷酸治疗C57BL/6J小鼠6周后引起肝脏脂肪变性，而 TMEM55B基因敲除小鼠在补充高脂肪、胆固醇和果糖的12周后患上NASH (GAN)节食。我们已经确定了TMEM55B基因敲除可以促进NAFLD的几个效应：1) 脂类在溶酶体内堆积，表明脂肪酸(FA)动员受损，2)受损 线粒体FA氧化，线粒体体积减小，线粒体碎裂，以及 氧化应激增加；以及3)体内抑制肝脏甘油三酯的分泌。溶酶体扮演着关键的角色 在分类和处理脂类中的作用，其中适当的溶酶体运动对于其动员FA的能力是必不可少的 从脂滴中分离出来，维持线粒体的动态平衡。TMEM55B在调节溶酶体中的关键作用 定位，因此我们的中心假设是TMEM55B的缺失促进了NAFLD的发生和发展 通过溶酶体对肝脏FA动员和转运、线粒体FA氧化和甘油三酯的影响 分泌物。在目标1中，我们将确定TMEM55B是否影响FA动员和线粒体功能 通过其影响溶酶体定位的能力。我们还将研究TMEM55B对 线粒体形态和功能障碍。肝甘油三酯(TG)主要在极低密度脂蛋白(VLDL)中分泌，后者是 通过多步骤使新生的载脂蛋白脂化，并从脂滴中动员FA产生。在AIM 我们将测试TMEM55B基因敲除是否能防止新生极低密度脂蛋白的脂化，并测试对 这些对溶酶体的影响。众所周知，男性患非酒精性脂肪肝的风险高于绝经前的女性， 然而，这种性别差异的基础却鲜为人知。我们发现TMEM55B基因敲除导致了 雄性而不是雌性小鼠脂肪变性，肝脏TMEM55B缺失在很大程度上抑制了甘油三酯的分泌 雄性小鼠比雌性小鼠程度更高。TG分泌缺陷的性别差异是 归因于性激素的差异。在目标2B中，我们将评估TMEM55B与性别之间的相互作用 激素对雄性和雌性小鼠以及雌激素受体基因敲除小鼠甘油三酯分泌的影响。最后，在目标3中 我们将评估TMEM55B的药理和遗传抑制对发育和发育的影响。 非酒精性脂肪肝的进展。理解TMEM55B丢失对多个临界影响的机理(S) 通过溶酶体的FA代谢轴可以为NAFLD的致病因素提供新的见解。

SUMMARY Non-alcoholic fatty liver disease (NAFLD) is a spectrum of disorders initiated by steatosis that can progress to nonalcoholic steatohepatitis (NASH) and cirrhosis. Although it is the leading cause of liver disease in the US, there are no targeted therapeutic interventions, highlighting the critical need for identifying molecular processes underlying its pathology. Transmembrane protein 55B (TMEM55B) is a PI(4,5)P2 phosphatase located on the lysosome that has been shown to impact lysosome levels and localization. In preliminary studies, we found that TMEM55B ASO treatment caused hepatic steatosis in C57BL/6J mice after 6 weeks on western diet, while TMEM55B knockout mice developed NASH after 12 weeks on a high-fat, cholesterol and fructose supplemented (GAN) diet. We have identified several effects of TMEM55B knockdown that can promote NAFLD: 1) accumulation of lipids within lysosomes, indicative of impaired fatty acid (FA) mobilization, 2) impaired mitochondrial FA oxidation together with reduced mitochondrial volume, mitochondrial fragmentation, and increased oxidative stress; and 3) in vivo inhibition of hepatic triglyceride secretion. The lysosome plays a critical role in sorting and processing lipids, where proper lysosome motility is essential for their ability to mobilize FA from lipid droplets and maintain mitochondrial homeostasis. TMEM55B plays a key role in regulating lysosomal positioning, thus our central hypothesis is that loss of TMEM55B promotes NAFLD onset and progression through lysosomal effects on hepatic FA mobilization and trafficking, mitochondrial FA oxidation, and triglyceride secretion. In Aim 1, we will determine whether TMEM55B impacts FA mobilization and mitochondrial function through its ability to impact lysosomal localization. We will also examine the impact of TMEM55B on mitochondrial morphology and dysfunction. Hepatic triglyceride (TG) is primary secreted within VLDL, which is generated through a multi-step process of lipidating nascent APOB with FA mobilized from lipid droplets. In Aim 2A we will test whether TMEM55B knockdown prevents lipidation of nascent VLDL and test the dependency of these effects on the lysosome. Men are well-known to have higher NAFLD risk than pre-menopausal women, however the basis for this sex difference is poorly understood. We found that TMEM55B knockdown caused steatosis in male, but not female, mice, and that loss of hepatic TMEM55B inhibited TG secretion to a much greater degree in male versus female mice. The sex-dependent differences in TG secretory defects were attributed to differences in sex hormones. In Aim 2B we will evaluate the interaction between TMEM55B and sex hormones on TG secretion in male and female mice, and in estrogen receptor knockout mice. Finally, in Aim 3 we will evaluate the effect pharmacologic and genetic inhibition of TMEM55B on the development and progression of NAFLD. Understanding the mechanism(s) by which loss of TMEM55B impacts multiple critical axes of FA metabolism through the lysosome can yield novel insight into factors contributing to NAFLD.