Postprandial insulin regulation of B100 and importance to VLDL1 secretion

描述（由申请方提供）：该提案的目的是确定胰岛素抑制含载脂蛋白B100（B100）的极低密度脂蛋白（VLDL）肝脏分泌的机制。餐后胰岛素增加B100降解并减少B100合成，限制VLDL分泌。对胰岛素的抵抗导致大VLDL 1的持续分泌，从而引起高胆固醇血症。我们首先观察到胰岛素对B100的影响，最近证明了降解是由分拣蛋白介导的，并指向自噬。我们计划进一步确定这种新型胰岛素作用的机制。胰岛素依赖性apo B降解通过增强内质网（ER）分泌前B100蛋白水解降低B100的可用性。关于胰岛素如何减少载脂蛋白B的合成知之甚少。我们之前的研究表明，胰岛素抑制需要激活ER中的IA类磷脂酰肌醇3-激酶（PI 3 K）和产物PI（3，4，5）三磷酸（PIP 3）。目的1是确定分拣蛋白在胰岛素介导的B100降解中的作用，基于显示胰岛素增加分拣蛋白与B100相互作用的证据。我们认为这个过程涉及PIP 3与B100的结合，向高尔基体的移动，与分拣蛋白的相互作用，以及通过胰岛素启动的p110诱导的自噬刺激导致分拣蛋白和B100的降解。在目标2中，我们提出研究PIP 3的产生如何对B100产生下游影响。这些研究是新颖的，因为它们提出了与PIP 3相关的B100从ER膜的细胞质面易位，其中PIP 3在B100的翻译期间产生。B100的瞬时细胞质定向是由于暂停转移序列允许B100暴露于PIP 3并掺入原始脂蛋白而发生的。提出了一个模型，其中B100-PIP 3复合物对未成熟的极低密度脂蛋白的运输和释放到高尔基体的特定囊泡，然后增加B100与分拣蛋白的相互作用。目的3是进一步确定胰岛素对B100合成的影响。胰岛素依赖性B100沉默涉及B100 mRNA进入加工体（P体）的运动，如我们的合作者Khosrow Adeli博士所示。我们的研究将定义在P体形成之前可逆的起始事件。我们假设B100 mRNA结合蛋白与编码区相互作用，抵抗B100转录物进入无活性mRNP的运动。初步数据支持apobec-1互补因子（A1 CF）诱导的细胞核滞留的胰岛素依赖性磷酸化的作用，其降低了A1 CF用于稳定B100 mRNA翻译的细胞质可用性，从而有利于形成无活性的B100 mRNP。对胰岛素调节的肝脏B100分泌的抵抗是代谢综合征发展中的最早事件之一，并导致餐后高胆固醇血症，其促进小的易氧化的LDL的形成和HDL的不稳定。明确胰岛素调节肝脏VLDL分泌的机制，将有助于制定重点治疗策略，以恢复脂蛋白平衡，降低餐后过渡期致动脉粥样硬化脂蛋白谱。

DESCRIPTION (provided by applicant): The goal of the proposal is to define mechanisms responsible for insulin suppression of hepatic secretion of very low density lipoproteins (VLDL) containing apolipoprotein B100 (B100). Postprandial insulin increases B100 degradation and reduces B100 synthesis limiting VLDL secretion. Resistance to insulin results in continuous secretion of large VLDL1 causing hypertriglyceridemia. We were first to observe insulin effects on B100 and recently demonstrated that degradation is mediated by sortilin and directed to autophagy. We plan to further define mechanisms for this novel insulin action. Insulin dependent apo B degradation decreases B100 availability by enhancing post endoplasmic reticulum (ER) presecretory B100 proteolysis. Little is known about how insulin reduces apo B synthesis. Our previous studies established that insulin suppression requires activation of Class IA phosphatidylinositide 3-kinase (PI3K) and product PI (3,4,5) triphosphate (PIP3) in the ER. Aim 1 is to determine the role of sortilin in insulin-mediated B100 degradation based on evidence showing insulin increases sortilin interaction with B100. We propose that this process involves binding of PIP3 to B100, movement to the Golgi, interaction with sortilin, and stimulation of autophagy leading to degradation of both sortilin and B100 through insulin initiated p110¿ induced autophagy. In Aim 2, we propose studies on how PIP3 generation causes downstream effects on B100. These studies are novel as they propose translocation of B100 associated with PIP3 from the cytoplasmic face of the ER membrane where PIP3 is generated during translation of B100. Transient cytoplasmic orientation of B100 occurs as a result of pause-transfer sequences allowing exposure of B100 to PIP3 and incorporation into primordial lipoproteins. A model is presented where B100-PIP3 complexes on immature VLDL are transported and released into Golgi by specific vesicles followed by increased B100 interaction with sortilin. Aim 3 is to further define insulin effects on B100 synthesis. Insulin dependent B100 silencing involves movement of B100 mRNA into processing bodies (P-bodies) as shown by our collaborator, Dr. Khosrow Adeli. Our studies will define initiating events that are reversible preceding formation of P-bodies. We hypothesize that B100 mRNA binding proteins that interact with coding regions resist movement of B100 transcripts into inactive mRNPs. Preliminary data support a role for insulin-dependent phosphorylation of apobec-1 complementation factor (A1CF) induced nuclear retention which reduces cytoplasmic availability of A1CF for stabilization of B100 mRNA translation thereby favoring formation of inactive B100 mRNPs. Resistance to insulin-regulated hepatic B100 secretion is one of the earliest events in development of metabolic syndrome and results in postprandial hypertriglyceridemia which enhances formation of small easily oxidizable LDL and destabilization of HDL. Defining mechanisms involved in insulin regulation of hepatic VLDL secretion will allow focused therapeutic strategies to be developed to restore lipoprotein balance and reduce atherogenic lipoprotein profile during the postprandial transition.