Postprandial insulin regulation of B100 and importance to VLDL1 secretion

描述(由申请人提供)：该提案的目标是确定胰岛素抑制含有载脂蛋白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的降解，减少B100的合成，限制极低密度脂蛋白的分泌。对胰岛素的抵抗会导致持续分泌大量的VLDL1，导致高甘油三酯血症。我们首先观察了胰岛素对B100的影响，最近证明了降解是由山梨素介导的，并与自噬有关。我们计划进一步确定这一新的胰岛素作用的机制。胰岛素依赖的载脂蛋白B降解通过促进内质网(ER)储存前B100蛋白的降解来降低B100的利用率。关于胰岛素是如何降低载脂蛋白B合成的，人们知之甚少。我们以前的研究证实，胰岛素抑制需要激活内质网中IA类磷脂酰肌醇3-激酶(PI3K)和产物PI(3，4，5)三磷酸(PIP3)。目的1是基于有证据表明胰岛素增加了山梨素与B100的相互作用，以确定山梨素在胰岛素介导的B100降解中的作用。我们认为，这一过程包括PIP3与B100的结合，移动到高尔基体，与山梨素的相互作用，以及通过胰岛素引发的p110诱导的自噬刺激自噬导致山梨素和B100的降解。在目标2中，我们提出了关于PIP3的产生如何对B100产生下游影响的研究。这些研究是新颖的，因为他们提出了与PIP3相关的B100从ER膜的细胞质表面移位，在那里PIP3是在B100的翻译过程中产生的。B100的瞬时细胞质定位是暂停转移序列的结果，该序列允许B100暴露于PIP3并并入原始脂蛋白。提出了一个模型，其中未成熟的极低密度脂蛋白上的B100-PIP3复合体通过特定的囊泡运输和释放到高尔基体中，随后B100与山梨素的相互作用增加。目的3是进一步确定胰岛素对B100合成的影响。正如我们的合作者Khosrow Adeli博士所展示的那样，胰岛素依赖的B100沉默涉及到B100 mRNA进入加工体(P-小体)。我们的研究将定义在P小体形成之前可逆的启动事件。我们假设，与编码区相互作用的B100 mRNA结合蛋白阻止B100转录本进入非活性mRNPs。初步数据支持胰岛素依赖的APOBEC-1互补因子(A1CF)磷酸化导致核滞留，从而降低A1CF对稳定B100 mRNA翻译的细胞质可获得性，从而有利于非活性B100 mRNP的形成。胰岛素调节的肝脏B100分泌抵抗是代谢综合征发展的最早事件之一，并导致餐后高甘油三酯血症，从而促进小的、易氧化的低密度脂蛋白的形成和高密度脂蛋白的不稳定。明确胰岛素调节肝脏极低密度脂蛋白分泌的机制将使人们能够开发出有针对性的治疗策略，以恢复脂蛋白平衡并减少餐后过渡期间导致动脉粥样硬化的脂蛋白分布。

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.