Interactions of Lipoproteins and Cells in Diabetes Mellitus

糖尿病中脂蛋白和细胞的相互作用

• 批准号: 8240578
• 负责人: RICHARD Louis KLEIN
• 金额: --
• 依托单位: RALPH H JOHNSON VA MEDICAL CENTER
• 依托单位国家: 美国
• 财政年份: 2011
• 资助国家: 美国
• 起止时间: 2011-10-01 至 2015-09-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8240578
• 关键词: Adipocytes; Age; Albumins; Albuminuria; Apolipoprotein A-I; Binding; Binding Proteins; Biological Markers; Bioluminescence; Blood; Blood Proteins; Blood Volume; Cardiovascular Diseases; Caring; Cell physiology; Cells; Ceramides; Cessation of life; Cholesterol Esters; Coagulation Process; Code; Cohort Studies; Complications of Diabetes Mellitus; Confocal Microscopy; Cross-Sectional Studies; Data; Diabetes Mellitus; Diabetic Angiopathies; Diabetic Nephropathy; Energy Transfer; Enrollment; Enzymes; Epidemiology; Excretory function; Exhibits; Fatty acid glycerol esters; Fibrinolysis; Gender; Gene Expression; High Density Lipoproteins; High Pressure Liquid Chromatography; Incubated; Individual; Kidney Diseases; Knowledge; Lipids; Lipoprotein Binding; Lipoproteins; Low-Density Lipoproteins; Measures; Mediating; Metabolism; Methodology; Modeling; Molecular; Monitor; Movement; Myocardial Infarction; Nature; Non-Insulin-Dependent Diabetes Mellitus; Obesity; Patients; Pattern; Phospholipid Transfer Proteins; Phospholipids; Plasma; Plasma Proteins; Plasminogen Activator Inhibitor 1; Plasminogen Inactivators; Process; Production; Race; Recombinants; Recruitment Activity; Relative (related person); Research Design; Risk Factors; Role; SPHK1 enzyme; Sampling; Small Interfering RNA; Source; Sphingolipids; Sphingomyelins; Stroke; Technology; Time; Very low density lipoprotein; Vesicle; Veterans; atheroprotective; clinically relevant; cohort; cooperative study; diabetic patient; enzyme activity; glycemic control; improved; macroalbuminuria; macrovascular disease; plasma protein fraction; programs; receptor; reverse cholesterol transport; sample collection; sphingosine 1-phosphate; sphingosine kinase; tandem mass spectrometry; type I and type II diabetes; type I diabetic

DESCRIPTION (provided by applicant): Plasma levels of HDL, and especially HDL2, are generally considered to be atheroprotective primarily through their role in reverse cholesterol transport. Recent evidence suggests that HDL may contribute additionally through its role as the primary transport lipoprotein for sphingosine-1-phosphate (S1P), a key, bioactive lipid which regulates a variety of beneficial cellular functions. However, there also is emerging evidence that S1P may mediate pro-atherogenic metabolism and in support, we now show that S1P, as well as HDL3, which in normal subjects contains higher concentrations of S1P than HDL2, both stimulate the release of plasminogen activator inhibitor-1 (PAI-1) from adipocytes and, importantly, that circulating S1P concentrations are significantly increased in patients with diabetes and correlate with plasma PAI-1 level. PAI-1 is the negative modulator of fibrinolysis and elevated circulating PAI-1 levels are associated with several risk factors for cardiovascular disease. Recent evidence suggests that adipocytes are a major source of PAI-1, especially in obesity. Although little is known about the mechanisms by which S1P in HDL3 stimulate PAI-1 secretion from adipocytes, we now demonstrate that both the adipocyte S1Pr2 and SR-BI, the HDL binding receptor, are required. These observations have contributed to our hypotheses that the increased total plasma S1P concentration in diabetic patients is increased additionally in patients exhibiting the microvascular or macrovascular complications that can accompany diabetes, and that the increased S1P is transported primarily by HDL. We further postulate that S1P carried by HDL3 leads to increased PAI-1 levels in plasma, that plasma S1P and PAI-1 levels are positively correlated, and that S1Pr2 and SR-BI interact at the molecular level to mediate S1P-HDL3-induced secretion of PAI-1 by adipocytes. We will investigate these hypotheses through the conduct of studies with three Specific Aims. Specific Aim A is epidemiologic in approach and we will analyze samples collected previously as part of our Program Project from Type 1 diabetic patients in the DCCT/EDIC cohort and from Type 2 diabetic patients in the VA Cooperative Study #465 (VADT) to investigate plasma S1P total concentration and relative distribution between the HDL and albumin pools in patients with diabetes complications compared to those without, and determine the association of S1P concentration and distribution with plasma PAI-1 levels. In Specific Aim B, we will employ sophisticated high-performance liquid chromatography-tandem mass spectrometry to quantitate ceramide and sphingolipid subspecies in VLDL, LDL, HDL2, and HDL3 prepared from Type 2 diabetic patients with and without albuminuria. We will determine if the concentration and distribution in lipoproteins of these additional bioactive lipids is altered in diabetic patients with macroalbuminuria and if they correlate with plasma PAI-1 levels. Purified lipoprotein fractions will be incubated with adipocytes and the sphingolipid profile in the lipoproteins will be correlated with PAI-1 secretion into the medium. We also will incubate adipocytes with recombinant HDL models (rHDL) supplemented with C16-sphingomyelin and/or C24-ceramide to determine the effects of other HDL sphingolipids on S1P- stimulated PAI-1 release. In Specific Aim C we will determine if S1P transported in HDL3 can activate S1Pr2 using confocal microscopy to monitor GFP-S1Pr2 metabolism. Using BRET analyses, we will determine if S1Pr2 interacts with SR-BI at the molecular level during this activation. Additionally, we will determine if sphingosine kinase activity is increased by HDL3 binding to adipocytes and if the resulting increase in cellular endogenous S1P level contributes to HDL3-stimulated PAI-1 release from adipocytes. We will determine if cholesteryl ester transfer protein (CETP) and the phospholipid transfer protein (PLTP) mediate S1P movement between lipoproteins. Collectively, these studies will elucidate for the first time the patterns of S1P metabolism in lipoproteins and in diabetes, and define the cellular and plasma mechanisms which mediate this metabolism.

描述(由申请人提供)： 通常认为，血浆高密度脂蛋白，尤其是高密度脂蛋白2的水平主要通过其在反向胆固醇运输中的作用而起到动脉粥样硬化的保护作用。最近的证据表明，高密度脂蛋白可能通过其作为鞘氨醇-1-磷酸(S1P)主要转运脂蛋白的作用而发挥额外的作用，S1P是一种关键的生物活性脂质，调节着多种有益的细胞功能。然而，也有新的证据表明S1P可能介导了促动脉粥样硬化的代谢，我们现在发现，S1P和HDL3在正常人中含有比HDL2更高的S1P浓度，两者都能刺激脂肪细胞释放纤溶酶原激活物抑制物-1(PAI-1)，重要的是，糖尿病患者循环中S1P浓度显著增加，并与血浆PAI-1水平相关。PAI-1是纤溶的负性调节剂，循环PAI-1水平升高与心血管疾病的几个危险因素相关。最近的证据表明，脂肪细胞是PAI-1的主要来源，特别是在肥胖症中。尽管对HDL3中的S1P刺激脂肪细胞分泌PAI-1的机制知之甚少，但我们现在证明，脂肪细胞S1PR2和SR-BI-高密度脂蛋白结合受体都是必需的。这些观察结果支持了我们的假设，即糖尿病患者血浆总S1P浓度的增加在出现可伴随糖尿病的微血管或大血管并发症的患者中额外增加，并且增加的S1P主要由高密度脂蛋白运输。我们进一步推测，HDL3携带的S1P导致血浆PAI-1水平升高，血浆S1P和PAI-1水平呈正相关，S1PR2和SR-BI在分子水平上相互作用，介导S1P-HDL3诱导脂肪细胞分泌PAI-1。我们将通过三个具体目标的研究来研究这些假设。我们将分析之前作为我们计划项目的一部分从DCCT/EDIC队列中的1型糖尿病患者和退伍军人管理局合作研究#465(VADT)中的2型糖尿病患者中收集的样本，以比较有并发症和无并发症的糖尿病患者的血浆S1P总浓度和高密度脂蛋白与白蛋白之间的相对分布，并确定S1P浓度和分布与血浆PAI-1水平的关系。在特定的目标B中，我们将使用复杂的高效液相色谱-串联质谱仪来定量检测有或没有蛋白尿的2型糖尿病患者制备的极低密度脂蛋白、低密度脂蛋白、高密度脂蛋白2和高密度脂蛋白3中的神经酰胺和鞘磷脂亚类。我们将确定这些额外的生物活性脂类在糖尿病患者大量蛋白尿中的浓度和分布是否发生改变，以及它们是否与血浆PAI-1水平相关。纯化的脂蛋白组份将与脂肪细胞孵育，脂蛋白中的鞘磷脂分布将与PAI-1分泌到介质中相关。我们还将用添加C16-鞘磷脂和/或C24-神经酰胺的重组高密度脂蛋白模型(RHDL)孵育脂肪细胞，以确定其他高密度脂蛋白鞘磷脂对S1P刺激的PAI-1释放的影响。在特定的目标C中，我们将利用共聚焦显微镜监测GFP-S1PR2的代谢来确定在HDL3中运输的S1P是否能够激活S1PR2。利用Bret分析，我们将确定S1PR2在激活过程中是否在分子水平上与SR-BI相互作用。此外，我们将确定HDL3与脂肪细胞结合是否会增加鞘氨醇激酶活性，以及由此导致的细胞内源性S1P水平的增加是否有助于HDL3刺激脂肪细胞释放PAI-1。我们将确定胆固醇酯转移蛋白(CETP)和磷脂转移蛋白(PLTP)是否介导脂蛋白之间的S1P移动。总而言之，这些研究将首次阐明脂蛋白和糖尿病中S1P代谢的模式，并确定调节这种代谢的细胞和血浆机制。