Mechanisms for Ceramide Mediated Vascular Dysfunction

描述（由申请人提供）：饮食诱导的肥胖和2型糖尿病患者发生心血管并发症的风险更大。一个重要的并发症是血管功能受损。在临床相关的饮食诱导的肥胖症小鼠模型中，我们发现暴露于升高的游离脂肪酸（FFA）降低了一氧化氮（NO）的生物利用度，导致动脉功能障碍。我在2007年的R15试图确定FFA升高和NO生物利用度受损之间的信号联系是否涉及脂质代谢物神经酰胺。用多球壳菌素抑制神经酰胺合成，或杂合缺失二氢神经酰胺去饱和酶（一种催化神经酰胺合成的酶），可预防内皮功能障碍和全身性高血压，并保护脂肪喂养小鼠动脉中的内皮NO合酶（eNOS）磷酸化。使用培养的内皮细胞研究神经酰胺可能降低NO生物利用度的分子机制。棕榈酸诱导的基础和激动剂刺激的eNOS磷酸化，eNOS二聚体的形成，和NO的生产抑制神经酰胺合成后恢复。神经酰胺诱导的eNOS磷酸化或二聚体形成的损害，分别不是受损的激酶介导的eNOS磷酸化或超氧阴离子介导的过氧亚硝酸盐形成的结果。[相反，神经酰胺导致蛋白磷酸酶2A（PP 2A）直接与eNOS/Akt复合物结合，这与基础和激动剂刺激的eNOS磷酸化降低同时发生。自上次提交以来获得的新数据表明，PP 2A通过阻止与eNOS共定位的Akt池的磷酸化和/或通过直接使eNOS去磷酸化来减弱eNOS磷酸化。在这次更新中，目标1将测试PP 2A与Akt-Hsp 90-eNOS复合物相关并破坏其作为神经酰胺从头合成的结果的假设，导致受损的基础和激动剂刺激的eNOS磷酸化。目的2将验证神经酰胺解除PP 2A的抑制剂2（I2 PP 2A）与PP 2A的缔合，使得PP 2A可以移位到膜并与eNOS缔合的假设。目的3将检验神经酰胺诱导的PP 2A介导的血管功能障碍发生在饮食诱导的肥胖小鼠中的假设。这些研究的结果将提供机制的洞察力连接内源性血管神经酰胺生物合成的饮食诱导的肥胖症和2型糖尿病的小鼠模型中的心血管缺陷，并提出新的目标，在这些流行的条件下血管功能障碍的治疗。通过NIH R15机制对我们研究的支持为35名本科研究人员提供了宝贵的经验，每个人都继续攻读生物医学科学的高级学位。 公共卫生相关性：肥胖可导致2型糖尿病和心血管并发症。我们的研究重点是确定肥胖损害血管功能的机制。我们将确定脂肪代谢物神经酰胺损害eNOS酶活性和沉淀动脉功能障碍的机制。

DESCRIPTION (provided by applicant): Patients with diet-induced obesity and type 2 diabetes are at greater risk for developing cardiovascular complications. An important complication is impaired blood vessel function. In a clinically relevant murine model of diet-induced obesity we showed that exposure to elevated free fatty acids (FFAs) reduces nitric oxide (NO) bioavailability leading to arterial dysfunction. My 2007 R15 sought to determine whether the signaling link between elevated FFAs and impaired NO bioavailability involves the lipid metabolite ceramide. Inhibition of ceramide synthesis with myriocin, or heterozygous deletion of dihydroceramide desaturase, an enzyme which catalyzes ceramide synthesis, prevented endothelial dysfunction and systemic hypertension, and preserved endothelial NO synthase (eNOS) phosphorylation in arteries from fat-fed mice. Molecular mechanisms whereby ceramide might lower NO bioavailability were examined using cultured endothelial cells. Palmitate induced repression of basal and agonist-stimulated eNOS phosphorylation, eNOS dimer formation, and NO production were restored following inhibition of ceramide synthesis. The ceramide-induced impairment of eNOS phosphorylation or dimer formation, respectively, was not the result of impaired kinase-mediated eNOS phosphorylation or superoxide anion-mediated peroxynitrite formation. [Instead, ceramide causes protein phosphatase 2A (PP2A) to associate directly with the eNOS/Akt complex, and this is concurrent with decreased basal and agonist-stimulated eNOS phosphorylation. New data obtained since the last submission suggest that PP2A attenuates eNOS phosphorylation by preventing phosphorylation of the pool of Akt that colocalizes with eNOS and / or by directly dephosphorylating eNOS.] In this renewal, Aim 1 will test the hypothesis that PP2A associates with and disrupts the Akt-Hsp90-eNOS complex as a consequence of de novo ceramide synthesis leading to impaired basal and agonist-stimulated eNOS phosphorylation. Aim 2 will test the hypothesis that ceramide relieves the association of inhibitor 2 of PP2A (I2PP2A) with PP2A such that PP2A can translocate to the membrane and associate with eNOS. Aim 3 will test the hypothesis that ceramide-induced, PP2A-mediated vascular dysfunction occurs in mice with diet-induced obesity. Results from these studies will provide mechanistic insight linking endogenous vascular ceramide biosynthesis to cardiovascular defects in a murine model of diet-induced obesity and type 2 diabetes, and present new targets for the treatment of vascular dysfunction in these prevalent conditions. Support for our research via the NIH R15 mechanism has provided > 35 undergraduate researchers with valuable experience and each has continued to pursue an advanced degree in the biomedical sciences. PUBLIC HEALTH RELEVANCE: Obesity can lead to type 2 diabetes and cardiovascular complications. Our research is focused on determining the mechanisms whereby obesity impairs blood vessel function. We will determine the mechanism whereby the fat metabolite ceramide impairs eNOS enzyme activity and precipitates arterial dysfunction.