PURIFICATION OF HUMAN LIVER-DERIVED ADIPOGENIC FACTOR

肥胖已被宣布为一级公共健康危害，因为它是 并伴有高血压、糖尿病和心血管疾病。肥胖 总是伴随着脂肪细胞的肥大和/或增生。 有证据表明，激素和生长因子控制着 前脂肪细胞的确定和分化， 这些因素的质和量的变化会造成异常 导致病理状态如肥胖的病症。现 目前，人们对分化的正调节因子知之甚少。 血清或血清组分中存在尚未鉴定的因子 在脂肪分化中起着重要作用， 包括我们在内的许多实验室反复暗示。 的 成脂因子的特征尚未进行 因为它的难度。然而，对其性质的调查是 这是明显重要的，因为已经表明来自肥胖患者的血清 具有比血清更高的刺激脂肪细胞分化的能力， 名正常人 最近，我们发现，正常肝细胞中， 原代培养和人肝癌细胞系HepG 2合成， 在其培养基中分泌脂肪形成活性。比较 从血清中纯化的脂肪形成因子的生化特性 HepG 2-CM表明它们是相似的。我们还首次展示了 肥胖小鼠肝脏提取物中脂肪形成活性较高的时间 比正常的同窝出生的孩子多。因为HepG 2细胞可以在血清中生长- HepG 2条件培养基（HepG 2CM）是一种理想的无细胞因子的培养基， 用于纯化称为L-的脂肪形成因子的起始材料 ADF通过4步纯化程序将L-ADF纯化146，800倍 回收率为26%。 我们已经获得了氨基酸序列信息 对应于L-ADF的N-末端12个氨基酸。蛋白质搜索 数据库表明L-ADF不同源的任何序列中， 数据库表明它是一种新的蛋白质。 这些新获得的数据 表明了研究分子生物学的重要性， L-ADF的特征。具体而言，建议追求 L-ADF结构研究，获得额外的氨基酸序列 为了克隆L-ADF cDNA并开发抗L-ADF， 抗体的这两种分子探针也将用于调查水平 L-ADF在正常和病理生理状态下的表达 在动物模型和人类受试者中的组织发育。这些研究 将进一步加深我们对脂肪分化调节的理解， 并为人类肥胖症治疗的发展提供工具。

Obesity has been declared a first degree public health hazard since it is accompanied by hypertension, diabetes and cardiovascular diseases. Obesity is always accompanied by hypertrophy and/or hyperplasia of adipocytes. There are evidence to suggest that hormones and growth factors control the determination and differentiation of pre-adipose cells and that any qualitative and quantitative changes of these factors will create abnormal conditions leading to pathological states such as obesity. At the present time, very little is known about positive regulators of differentiation. The existence of yet unidentified factors in serum or in serum fraction playing an important role in the adipose differentiation has been repeatedly implied by many laboratories, including ours. The characterization of the adipogenic factor has not yet been carried out because of its difficulty. However, the investigation of its nature is of obvious importance since it has been shown that serum from obese patients has a higher ability to stimulate adipocyte differentiation than serum from normal subjects. Recently, we have found that normal hepatocytes in primary culture and the human hepatoma cell line HepG2 synthesize and secrete adipogenic activity in their culture medium. Comparison of the biochemical properties of adipogenic factor purified from serum and from HepG2-CM indicate that they are similar. We have also shown for the first time that adipogenic activity in liver extract was higher in obese mice than in their normal littermates. Since HepG2 cells can be grown in serum- free and factor-free medium, HepG2 conditioned medium (HepG2CM) is an ideal starting material for the purification of the adipogenic factor called L- ADF. L-ADF was purified 146,800 fold by a 4 step purification procedure with a recovery of 26%. We have obtained amino-acid sequence information corresponding to N-terminal 12 amino-acids of L-ADF. Search of protein database indicates that L-ADF is not homologous to any sequence in the database suggesting that it is a novel protein. These newly obtained data indicate that it is very important to pursue the molecular and biological characterization of L-ADF. Specifically, it is proposed to pursue structural study of L-ADF, obtain additional amino-acid sequence information in order to clone the L-ADF cDNA and develop anti-L-ADF antibody. Both molecular probes will also be used to investigate the level of L-ADF expression in normal and pathophysiological states of adipose tissue development in animal models and in human subjects. These studies will further our understanding of the regulation of adipose differentiation and provide tools towards the development of treatment of human obesity.