The Role of Phosphatidylinositides in Lipid Metabolism

磷脂酰肌醇在脂质代谢中的作用

基本信息

批准号：
10065516
负责人：
Marisa Wong Medina
金额：
$ 42.04万
依托单位：
UNIVERSITY OF CALIFORNIA, SAN FRANCISCO
依托单位国家：
美国
项目类别：
财政年份：
2020
资助国家：
美国
起止时间：
2020-05-12 至 2022-11-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/10065516
关键词：
American Animal Model Animals Autophagocytosis Autophagosome Back Binding Blood Cardiometabolic Disease Cell membrane Cell model Cell surface Cells Cholesterol Defect Development Early Endosome Endosomes Enzymes Eukaryotic Cell Family Fatty acid glycerol esters Female Four Core Genotypes Goals Gonadal Hormones Heart Diseases HepG2 Hepatic Hepatocyte Hormone use Impairment In Vitro Integral Membrane Protein Investigation Knock-out Knockout Mice Lead Lipids Lipoproteins Liver Low Density Lipoprotein Receptor Low-Density Lipoproteins Lysosomes Metabolic Diseases Metabolism Modeling Molecular Mus Nonesterified Fatty Acids Oculocerebrorenal Syndrome Pathway interactions Phenotype Phosphatidylinositols Phosphoric Monoester Hydrolases Plasma Play Ploidies Process Recycling Regulation Risk Factors Role Sex Chromosomes Stains Testing Triglycerides Vesicle cardiometabolic risk cardiovascular disorder prevention disorder risk fatty acid oxidation inhibitor/antagonist inositol-1,4,5-trisphosphate 5-phosphatase knock-down lipid biosynthesis lipid metabolism male mouse model non-alcoholic fatty liver disease novel therapeutics overexpression receptor receptor recycling sex therapeutic target trafficking uptake

项目摘要

SUMMARY As key directors of intracellular trafficking, phosphotidylinsotides (PIs) are a family of low abundance lipids that impact almost every process within the eukaryotic cell. The low-density lipoprotein receptor (LDLR), a major determinant of blood LDL levels, is the most efficacious therapeutic target for the prevention of cardiovascular disease. Upon LDL binding, LDLR at the cell surface is internalized, where it releases the bound LDL and then recycles back to the cell surface or is directed to the lysosome for degradation. Recently, we found that knock- down of transmembrane protein 55B (TMEM55B), a phosphatidylinositol (4,5) bisphosphate [PI(4,5)P] phosphatase, stimulated decay of LDLR protein in cells and raised plasma LDLC in mice. Since PI(4,5)P is involved in lysosome formation, we hypothesize that PI(4,5)P phosphatases (such as TMEM55B) may alter LDLC by regulating LDLR intracellular trafficking. We also found that reduction of PI(4,5)P phosphatases caused severely impaired secretion of triglycerides into plasma. Notably, this occurred without the accumulation of liver fat, and interestingly, was observed only in male mice, with no effect in females. The lack of hepatic fat accumulation may be attributed to a number of different mechanisms including increased lipophagy (a process of selective autophagy in which lipid droplets are targeted for lysosomal decay). PI(4,5)P is an important regulator of lysosome reformation after autophagosome fusion, and we found that TMEM55B knock-down increased lysosomes. These findings suggest that PI(4,5)P phosphatases may modulate hepatic TG secretion and storage through a lysosome-dependent mechanism. Thus, the overall objective of this proposal is to evaluate the role of PI(4,5)P in LDLR recycling and TG secretion. In Aim 1 we will i) Test if PI(4,5)P metabolizing enzymes impact plasma LDL in the absence of LDLR using genetically modified mouse models; ii) Evaluate the effect of modifying PI(4,5)P on Ldlr intracellular levels and localization in mouse hepatocytes; iii) Test if expression of enzymatically inactive Tmem55b rescues the phenotypes observed in the Tmem55b knockout mouse; and iv) Evaluate if PI(4,5)P phosphatase regulation of LDLR recycling is dependent on known regulators of LDLR decay. In Aim 2 we will i) Compare TG secretion in whole body versus liver-specific Tmem55b knockout male mice to confirm that the defect is a hepatic mechanism; ii) Test if lack of hepatic fat accumulation, as would be expected to result from impaired TG secretion, in hepatocytes from Tmem55b knockout mice is due to changes in fatty acid oxidation, synthesis, or uptake, or TG synthesis; iii) Determine PI(4,5)P phosphatases depend on lysosome and/or autophagosomes to impair TG secretion; and iv) Evaluate if the sex-specific effect of PI(4,5)P modulation of TG secretion is due to the sex chromosome complement (XX vs. XY) and/or gonadal hormones. Investigation of the role of PI(4,5)P in lipid and lipoprotein metabolism may lead to the development of new therapeutic options for reducing cardiometabolic disease risk.

概括作为细胞内运输的关键指导者，磷脂酰肌醇 (PI) 是一个低丰度脂质家族，影响真核细胞内的几乎每个过程。低密度脂蛋白受体（LDLR）是一种重要的受体血液低密度脂蛋白水平的决定因素，是预防心血管疾病最有效的治疗靶点疾病。 LDL 结合后，细胞表面的 LDLR 被内化，释放结合的 LDL，然后循环回到细胞表面或被引导至溶酶体进行降解。最近，我们发现敲跨膜蛋白 55B (TMEM55B) 的下调，一种磷脂酰肌醇 (4,5) 二磷酸 [PI(4,5)P] 磷酸酶，刺激细胞中 LDLR 蛋白的衰变，并提高小鼠血浆中的 LDLC。由于 PI(4,5)P 是参与溶酶体形成，我们假设 PI(4,5)P 磷酸酶（例如 TMEM55B）可能会改变 LDLC 通过调节 LDLR 细胞内运输。我们还发现 PI(4,5)P 磷酸酶的减少导致甘油三酯向血浆的分泌严重受损。值得注意的是，这发生在没有肝脏积聚的情况下。有趣的是，仅在雄性小鼠中观察到脂肪，而对雌性小鼠没有影响。缺乏肝脂肪积累可能归因于许多不同的机制，包括增加脂肪自噬（一个过程）选择性自噬，其中脂滴被靶向溶酶体腐烂）。 PI(4,5)P是重要的调节剂自噬体融合后溶酶体重组的影响，我们发现 TMEM55B 敲低增加溶酶体。这些发现表明 PI(4,5)P 磷酸酶可能调节肝脏 TG 分泌和储存通过溶酶体依赖性机制。因此，该提案的总体目标是评估 PI(4,5)P 在 LDLR 回收和 TG 分泌中的作用。在目标 1 中，我们将 i) 测试 PI(4,5)P 代谢酶是否有影响使用转基因小鼠模型在没有 LDLR 的情况下测定血浆 LDL； ii) 评估修改的效果 PI(4,5)P 对 Ldlr 细胞内水平和小鼠肝细胞定位的影响； iii) 测试是否以酶促方式表达失活的 Tmem55b 可以挽救在 Tmem55b 敲除小鼠中观察到的表型； iv) 评估是否 PI(4,5)P 磷酸酶对 LDLR 循环的调节依赖于已知的 LDLR 衰减调节因子。目标 2 我们将 i) 比较全身和肝脏特异性 Tmem55b 敲除雄性小鼠的 TG 分泌，以确认该缺陷是一种肝脏机制； ii) 测试是否没有预期的肝脏脂肪积累 Tmem55b 敲除小鼠的肝细胞中 TG 分泌受损是由于脂肪酸的变化氧化、合成或摄取，或TG合成； iii) 确定依赖于溶酶体的 PI(4,5)P 磷酸酶和/或自噬体损害TG分泌； iv) 评估 PI(4,5)P 调节是否具有性别特异性效应 TG 分泌的减少归因于性染色体补体（XX 与 XY）和/或性腺激素。调查 PI(4,5)P 在脂质和脂蛋白代谢中的作用可能会导致新治疗方案的开发降低心脏代谢疾病的风险。