The role of ceramide in obesity-related vascular dysfunction

描述（由申请人提供）：胰岛素抵抗是饮食性肥胖和2型糖尿病中至关重要的代谢异常。内皮功能障碍存在于胰岛素抵抗状态，可能是动脉粥样硬化发展的一个重要早期事件。减少内皮源性一氧化氮（NO）有助于内皮功能障碍。NO的生物利用度取决于内皮NO合成酶（eNOS）产生NO和活性氧（如超氧阴离子（O2-））使NO失活之间的良好平衡。内皮细胞产生NO需要Akt/蛋白激酶B （Akt）和其他上游激酶磷酸化特定的eNOS残基，并形成eNOS同型二聚体。根据我们的初步结果和其他人的数据，存在于饮食诱导肥胖小鼠的内皮功能障碍可能是由akt介导的血管中eNOS磷酸化受损和eNOS同源二聚体的破坏引起的超氧化物（O2-）产生增加。我们将验证鞘脂神经酰胺的积累通过这两种机制减少NO的产生，对胰岛素抵抗状态下血管功能障碍的发病机制起重要作用的整体假设。在目的1中，我们将确定牛主动脉内皮细胞（BAECs）中神经酰胺浓度的增加是否会损害胰岛素信号并降低eNOS功能。两种假设将被验证：1)神经酰胺拮抗胰岛素介导的Akt和eNOS磷酸化，刺激MAPK信号，导致baec中NO的产生减少，内皮素-1的产生增加；2)神经酰胺使O2-增加到一定程度，过氧亚硝酸盐的形成破坏了eNOS同二聚体，导致baec产生的NO减少。在Aim 2中，我们将定义神经酰胺对血管中胰岛素介导的信号转导受损的贡献，内皮依赖性血管松弛减少，以及我们在饮食诱导的肥胖小鼠中观察到的全体性高血压。两种假设将在饮食诱导的肥胖小鼠中进行验证：1)神经酰胺的积累通过损害胰岛素介导的Akt磷酸化和eNOS磷酸化来降低NO的生物利用度；2)神经酰胺的积累通过增强O2诱导的过氧亚硝酸盐的形成，最终破坏血管中eNOS同型二聚体的形成，从而降低NO的生物利用度。两种策略将用于限制神经酰胺在饮食引起的肥胖小鼠中的积累：药理抑制负责神经酰胺合成的限速酶；一种新型敲除模型，其中神经酰胺合成被阻止。通过测试我们的假设产生的信息将为理解神经酰胺对饮食性肥胖小鼠心血管缺陷的贡献提供重要的见解。

DESCRIPTION (provided by applicant): Insulin resistance is a crucially important metabolic abnormality in diet-induced obesity and type 2 diabetes. Endothelial dysfunction exists in insulin-resistant states and may represent an important early event in the development of atherosclerosis. Reduced endothelium-derived nitric oxide (NO) contributes to endothelial dysfunction. The bioavailability of NO depends on a fine balance between NO production via endothelial NO synthase (eNOS) and inactivation of NO by reactive oxygen species such as superoxide anion (O2-). Endothelial production of NO requires the phosphorylation of specific eNOS residues by Akt/protein kinase B (Akt) and other upstream kinases, as well as the formation of eNOS homodimers. Based on our preliminary results and data from others, endothelial dysfunction that exists in mice with diet-induced obesity might be precipitated by impaired Akt-mediated eNOS phosphorylation in the vasculature and by the disruption of eNOS homodimers secondary to increased superoxide (O2-) production. We will test the overall hypothesis that accumulation of the sphingolipid ceramide contributes importantly to the pathogenesis of vascular dysfunction in insulin resistant states by reducing NO production via both of these mechanisms. In Aim 1 we will determine if increasing ceramide concentrations in bovine aortic endothelial cells (BAECs) impairs insulin signaling and decreases eNOS function. Two hypotheses will be tested: 1) Ceramide antagonizes insulin-mediated Akt and eNOS phosphorylation and stimulates MAPK signaling resulting in reduced NO production and increased endothelin-1 production by BAECs; and 2) Ceramide increases O2- to an extent that peroxynitrite formation disrupts eNOS homodimers and leads to reduced NO production by BAECs. In Aim 2 we will define the contribution from ceramide to impaired insulin-mediated signal transduction in the vasculature, reduced endothelium-dependent vasorelaxation, and systemic hypertension that we have observed in mice with diet- induced obesity. Two hypotheses will be tested in mice with diet-induced obesity: 1) Ceramide accumulation reduces NO bioavailability by impairing insulin-mediated Akt phosphorylation and eNOS phosphorylation in the vasculature; and 2) Ceramide accumulation reduces NO bioavailability by potentiating O2- -induced peroxynitrite formation which ultimately disrupts eNOS homodimer formation in the vasculature. Two strategies will be used to limit ceramide accumulation in mice with diet-induced obesity: pharmacological inhibition of the rate-limiting enzyme responsible for ceramide synthesis; and a novel knockout model wherein ceramide synthesis is prevented. Information generated by testing our hypotheses will provide important insight into understanding the contribution from ceramide to cardiovascular defects that exist in mice with diet-induced obesity.