Effect of dietary fat on diet-induced insulin resistance and vascular dysfunction

描述（由申请人提供）： 美国超重和肥胖人群的惊人增长是一个主要的医疗保健问题，因为它导致2型糖尿病，血管疾病和心血管事件的发生率较高。由于膳食饱和脂肪酸与血管功能障碍和胰岛素抵抗（2型糖尿病的前奏）有关，因此膳食脂肪酸操作可能是预防或逆转与肥胖相关的血管异常的重要目标。这项建议的重点是解决膳食脂肪酸诱导的血管功能障碍的机制。我们假设两种潜在的机制：高饱和脂肪饮食可能产生异常的膜脂质组成，1）影响专门的膜室（脂筏）的形成和信号分子的激活或2）促进血管组织内独特的鞘脂信号通路。饱和脂肪酸的这些异常膜效应可以通过用单不饱和（MUFA）或多不饱和（PUFA）脂肪酸丰富饮食来逆转。为了测试我们的假设，我们将使用高饱和脂肪诱导的肥胖症的小鼠模型并操纵膳食脂肪组成以解决以下具体目的：目的1：确定用MUFA或n-3 PUFA替代一部分膳食饱和脂肪是否通过改变关键信号分子的小窝相对于非小窝定位来恢复血管功能至正常。我们的工作假设是，富含MUFA和n-3 PUFA的饮食通过改变膜组成将内皮型一氧化氮合酶（eNOS）和/或环氧合酶（考克斯）靶向膜脂筏，这与高饱和脂肪饮食不同。这种定位的变化定义了它们的激活和血管功能。我们将测量小鼠的小股薄动脉和主动脉对富含饱和脂肪、富含MUFA的橄榄油或富含n-3 PUFA的鲱鱼油的饮食的反应，以及膜脂肪酸组成和eNOS和考克斯在脂筏内的定位。我们将比较饮食对血管功能的影响，使用野生型和小窝蛋白-1（cav-1）缺陷小鼠缺乏血管小窝的信号通路的定位，以分离对小窝与非小窝脂筏的影响。目标二：确定膳食脂肪酸饱和度是否影响鞘脂信号通路-特别是鞘氨醇激酶和鞘氨醇-1-磷酸（S1 P）-以改变血管功能。我们的工作假设是，膳食脂肪酸通过形成影响血管功能的独特鞘脂蛋白质来促进信号传导。我们将测量小股薄肌动脉和主动脉的反应，从小鼠的饮食富含饱和脂肪，MUFA或n-3 PUFA结合鞘氨醇激酶和S1 P血管组织内的水平。我们将通过比较表达水平和使用药理学抑制剂和转基因小鼠来评估膳食脂肪酸对鞘氨醇激酶的影响。小鼠的高脂肪喂养是用于研究饮食的血管并发症的相关模型，因为小鼠变得肥胖，发展类似于2型糖尿病的胰岛素抵抗和异常葡萄糖耐量。高脂肪饮食是人们肥胖的最大原因。这些研究是创新性的，直接测试机制，涉及膳食脂肪对血管功能的影响，使用肥胖症的小鼠模型：异常膜组成和定位的信号蛋白在特定的脂筏或脂肪酸依赖性信号通路。这些研究意义重大，因为膳食脂肪是已知的治疗靶点，但逆转心血管疾病进展的最佳饮食组成及其相关机制尚未明确定义。

DESCRIPTION (provided by applicant): The alarming increase in overweight and obese individuals in the US is a major healthcare issue since it contributes to higher rates of type 2 diabetes, vascular disease and cardiovascular events. Because dietary saturated fatty acids have been linked to vascular dysfunction and insulin resistance (a prelude to type 2 diabetes), dietary fatty acid manipulation may be an important target for preventing or reversing vascular abnormalities associated with obesity. The focus of this proposal is to address the mechanisms underlying dietary fatty acid-induced vascular dysfunction. We hypothesize two potential mechanisms: high saturated fat diets may produce abnormal membrane lipid composition which 1) affects formation of specialized membrane compartments (lipid rafts) and activation of signaling molecules or 2) promotes unique sphingolipid signaling pathways within vascular tissue. These abnormal membrane effects of saturated fatty acids may be reversed by enriching the diet with monounsaturated (MUFAs) or polyunsaturated (PUFAs) fatty acids. To test our hypothesis, we will use a mouse model of high saturated fat-induced obesity and manipulate dietary fat composition to address the following Specific Aims: Aim 1: To determine whether replacement of a portion of dietary saturated fats with MUFA or n-3 PUFAs restores vascular function to normal by modifying caveolar versus non-caveolar localization of key signaling molecules. Our working hypothesis is that diets enriched in MUFAs and n-3 PUFAs target endothelial nitric oxide synthase (eNOS) and/or cyclooxygenase (Cox) to membrane lipid rafts differently than a high saturated fat diet by altering membrane composition. This shift in localization defines their activation and vascular function. We will measure responses of small gracilis arteries and aorta from mice on diets enriched in saturated fats, MUFA enriched olive oil or n-3 PUFA enriched menhaden oil in conjunction with membrane fatty acid composition and localization of eNOS and Cox within lipid rafts. We will compare the effects of diet on vascular function with localization of signaling pathways using wild type and caveolin-1 (cav-1) deficient mice devoid of vascular caveolae to separate effects on caveolar versus non-caveolar lipid rafts. Aim 2: To determine whether dietary fatty acid saturation affects sphingolipid signaling pathways- specifically sphingosine kinase and sphingosine-1-phosphate (S1P)- to modify vascular function. Our working hypothesis is that dietary fatty acids promote signaling through formation of unique sphingolipid-based proteins that impact vascular function. We will measure responses of small gracilis arteries and aorta from mice on diets enriched in saturated fats, MUFAs or n-3 PUFAs in conjunction with levels of sphingosine kinase and S1P within vascular tissue. We will assess the effects of dietary fatty acids on sphingosine kinase by comparing expression levels and using pharmacological inhibitors and genetically modified mice. High fat feeding of mice is a relevant model for study of vascular complications of diet since mice become obese, develop insulin resistance and abnormal glucose tolerance similar to type 2 diabetics. High fat diets are the greatest cause of obesity in people. These studies are innovative in directly testing the mechanisms involved in effects of dietary fats on vascular function using a mouse model of obesity: abnormal membrane composition and localization of signaling proteins within specific lipid rafts or fatty acid-dependent signaling pathways. The studies are significant because dietary fats are a known therapeutic target but the optimal diet composition for reversing the progression of cardiovascular disease and the mechanisms involved are not clearly defined.