LIPID METABOLISM IN FAT CELLS

摄入中链脂肪酸(MCFA)会导致动物和人类脂肪储存减少，而摄入类似的长链脂肪酸(LCFA)会导致体重增加。我们项目的目标是研究这种现象背后的细胞机制。我们的假设是，MCFA替代LCFA将下调成脂蛋白的表达，降低脂肪细胞的脂肪储存能力，促进储存脂肪的脂肪酸释放，并可能也降低脂肪细胞前体的分化能力。这项研究的具体目的集中在：1)MCFA的代谢命运以及LCFA、葡萄糖和胰岛素如何影响MCFA的代谢；2)MCFA对脂肪储存和脂解的影响，以及对亚细胞成分(质膜、线粒体和内质网)膜脂组成的影响；3)MCFA在分化过程中对成脂蛋白表达的影响；以及4)长期MCFA饮食适应导致的脂肪细胞功能的变化。将使用一种综合的方法来表征MCFA的代谢最终产物，寻找未确定的最终产品，并解释MCFA治疗导致的额外能量消耗。我们将通过核磁共振鉴定和量化脂肪酸(脂类和二氧化碳)的主要代谢最终产物。13C同位素标记的底物将用于适当的实验。这大大提高了核磁共振分析的选择性和灵敏度。我们将通过测量氧耗和氧化还原状态来比较适应MCFA和LCFA的细胞的产热；用高效液相色谱法对重要的代谢物(乙酰辅酶A和乙酰肉碱)进行定量，并用GLC分析脂肪酸组成。如果MCFA处理导致亚细胞膜成分发生显著变化，将用固体核磁共振分析随后对膜结构和流动性的影响。其他标准生化分析将用于测量酮体、总甘油三酯、胆固醇、DNA、蛋白质等。分化相关成脂基因的mRNA产物将通过Northern分析确定。脂肪细胞的形态将通过相差显微镜进行表征。使用这一综合方法，我们预计将开发重要的新信息，以帮助阐明通过MCFA管理提供的控制肥胖和肥胖相关健康障碍的分子机制。

Medium chain fatty acid ingestion (MCFA) results in diminished fat storage in animals and humans, while similar intake of long chain fatty acids (LCFA) results in weight-gain. The goal of our project is to examine the cellular mechanisms underlying this phenomenon. Our hypothesis is that substitution of MCFA for LCFA will down-regulate the expression of adipogenic proteins, lower the lipid storage capacity of adipose cells, facilitate fatty acid release from stored fat, and probably also reduce the capacity for differentiation of adipose cell precursors. The specific aims of this study focus on: 1) the metabolic fate of MCFA and how this is influenced by LCFA, glucose and insulin, 2) the effects of MCFA on fat storage and lipolysis, and on membrane lipid composition of sub-cellular components (plasma membrane, mitochondria, and endoplasmic reticulum); 3) the effects of MCFA on the expression of adipogenic proteins during the differentiation process; and 4) changes in fat cell function as a result of long-term MCFA dietary adaptation. An integrated approach will be used to characterize the metabolic end products of MCFA, to search for unidentified end products, and to explain the excess energy expenditure that results from MCFA treatment. We will identify and quantify, the major metabolic end products of fatty acids (lipids and CO2) by NMR. 13C isotope labeled substrates will be used in appropriate experiments. This greatly enhances the selectivity and sensitivity of NMR analysis. We will compare heat generation by cells adapted to MCFA or LCFA by measuring oxygen consumption and redox state; quantify the important metabolites (acetylCoA and acetylcarnitine) by HPLC, and analyze the fatty acid compositions by GLC. If significant changes in sub-cellular membrane composition are found as a result of MCFA treatment, subsequent effects on membrane structure and fluidity will be analyzed by solid state NMR. Other standard biochemical assays will be used for the measurement of ketone bodies, total triglycerides, cholesterol, DNA, protein, etc. The mRNA products of differentiation- dependent adipogenic genes will be determined by Northern analysis. Fat cell morphology will be characterized by phase- contrast microscopy. Using this integrated approach, we anticipate developing important new information to help shed light on the molecular mechanisms of the control of obesity and obesity-related health disorders afforded through MCFA administration.