Lipid Binding Proteins in Obesity/Diabetes Syndromes

描述(申请人提供)：在美国，每三个人中就有一个被认为超重或肥胖。肥胖不仅仅是一个美容问题，它还与心血管疾病、高血压、动脉粥样硬化和糖尿病呈正相关。瘦素作为一种脂肪细胞来源的分泌细胞因子的发现，将我们对脂肪细胞的看法从卡路里过剩的储存库扩展到了一种动态细胞类型，其功能是评估和整合整体能量稳态。该实验室的重点是肥胖、脂肪酸代谢和脂肪分解之间的联系，重点是脂肪细胞和NIDDM中的脂肪酸运输。该实验室研究脂肪细胞的脂肪酸携带者，特别是FABP4和FABP5基因的产物。在脂肪细胞中，这些蛋白质与脂肪酸和其他疏水性单酰基脂形成1：1的非共价复合体。AP2(FABP4)和KLBP(FABP5)分别与激素敏感脂肪酶(HSL)形成物理复合体，HSL是脂肪分解的关键限速酶，将三酰甘油分解为脂肪酸。与aP2与HSL的关联一致，FABP4基因缺失的小鼠表现出基础脂肪分解和激素刺激的脂肪分解减少，这表明脂肪酸结合蛋白通过从脂肪酶反应中去除最终产物脂肪酸并促进它们从细胞中流出来影响这一过程。FABP4基因缺失的小鼠表现出对肥胖相关的胰岛素抵抗的保护，而不会减少脂肪质量或瘦素水平。双FABP4-FABP5基因缺失小鼠的脂肪质量和瘦素水平较低。相反，在aP2启动子下在脂肪细胞中过度表达KLBP的转基因小鼠，脂肪细胞质量增加，比野生型动物更具胰岛素抵抗。我们假设FABP通过与HSL的相互作用刺激脂肪分解，这种相互作用的破坏导致脂肪细胞脂肪分解的脂肪酸分子种类和丰度的数量上的差异。脂肪分解衍生的脂肪酸的这种变化会影响与胰岛素抵抗的发展有关的脂肪酸的供应。为了验证这一假说，我们建议：具体目标A：在体内和原位评估低脂和高脂喂养的含有FABP基因中断或aP2-KLBP转基因的小鼠的脂解作用。在这样的动物模型中，脂肪分解的速度和程度与胰岛素抵抗相关。具体目的B：研究FABP与HSL的体外物理相互作用。评估FABP在调节HSL活性中的作用，并绘制两种蛋白质的相互作用结构域。具体目的C：用荧光共振能量转移法评估FABPs与293细胞和3T3-L1脂肪细胞中HSL的关系。研究HSL的磷酸化/易位及其与FABP关联的关系。

DESCRIPTION (provided by applicant): In the United States, one in every three individuals is considered very overweight or obese. More than a cosmetic issue, obesity is a positive correlate to cardiovascular disease, hypertension, atherosclerosis and diabetes. The discovery of leptin as an adipocyte-derived secreted cytokine has expanded our view of the fat cell beyond being a repository for caloric excess to a dynamic cell type functioning in the assessment and integration of overall energy homeostasis. The focus of the laboratory is on the linkage between obesity, fatty acid metabolism and lipolysis with an emphasis on fatty acid trafficking in the adipocyte and NIDDM. The laboratory studies fatty acid carriers of the adipocyte, specifically the products of the FABP4 and FABP5 genes. In fat cells, these proteins form 1:1 non-covalent complexes with fatty acids and other hydrophobic monoacyl lipids. aP2 (FABP4) and KLBP (FABP5) each form a physical complex with the hormone-sensitive lipase (HSL), the key rate-limiting enzyme of lipolysis that hydrolyzes triacylglycerol to fatty acids. Consistent with the association of aP2 with the HSL, FABP4 null mice exhibit reduced basal and hormone-stimulated lipolysis suggesting fatty acid binding proteins affect the process by removing end-product fatty acids from the lipase reaction and facilitating their efflux from the cell. FABP4 null mice exhibit protection from obesity-linked insulin resistance without reduction in fat mass or leptin levels. Double FABP4-FABP5 null mice have lower fat mass and leptin levels. Conversely, transgenic mice that overexpress KLBP in fat cells under the aP2 promoter, have elevated fat cell mass and are more insulin resistant than are wild type animals. We hypothesize that FABPs stimulate lipolysis via interaction with HSL and that the disruption of the interaction results in a quantitative difference in both the molecular species and abundance of fatty acids that are lipolyzed from the fat cell. Such changes in lipolysis-derived fatty acids affect the availability of fatty acids linked to the development of insulin resistance. To test this hypothesis we propose to: Specific Aim A: Evaluate lipolysis in vivo and in situ from low and high fat fed mice harboring either disrupted FABP genes or the aP2-KLBP transgene. Correlate the rate and extent of lipolysis to insulin resistance in such animal models. Specific Aim B: Examine the physical interaction of FABPs with HSL in vitro. Assess the role of FABPs in regulating the activity of HSL and map the interaction domains of the two proteins. Specific Aim C: Assess the association of FABPs with HSL in 293 cells and 3T3-L1 adipocytes using fluorescence resonance energy transfer. Examine the phosphorylation/translocation of HSL and its relationship to FABP association.