MUTATIONS AFFECTING LIPOPROTEIN METABOLISM

在小鼠身上自然发生的突变为识别影响脂代谢的新基因提供了丰富的信息。在这个项目中，我们将分离影响甘油三酯代谢的两个小鼠突变的基因，脂肪肝营养不良(FLD)和混合性脂肪酶缺乏症(CLD)。FLD/FLD突变小鼠在新生儿发育过程中表现出一过性高甘油三酯血症和脂肪肝。我们最近确定，这些动物随后会出现胰岛素抵抗，并严重缺乏白色脂肪组织的储存，并假设FLD突变影响胰岛素信号转导途径的一个组成部分。Cld/Cld小鼠的显著特征是高甘油三酯血症，在出生后2-3天内致命，这是由于脂蛋白脂肪酶和肝脂酶活性的虚拟缺失引起的。最近，我们已经确定这种缺陷的基础是内质网中新合成的脂肪酶分子的错误折叠。我们的两个目标将集中在分离fld和cld基因，以及表征被突变扰乱的细胞过程。在目标1中，我们将使用定位和功能克隆策略来鉴定这两个基因。在FLD的情况下，我们使用遗传和物理策略将临界区域缩小到大约kb。在FLD的情况下，我们已经使用遗传和物理图谱将关键区域缩小到大约kb；通过外显子捕获对该区域的分析发现了一个似乎在FLD基因组中重排的候选FLD基因，该候选基因在突变小鼠的组织中不表达。我们现在将通过序列分析来确认这个候选基因；如果需要，我们将分析通过外显子捕获从该区域分离的其他候选基因。在CLD的情况下，基因临界区已经被分离为一系列重叠的酵母和细菌人工染色体克隆，跨越大约2兆碱基的距离。我们现在准备通过在组织培养细胞系中互补的方法来鉴定含有cld基因的克隆，然后使用已被证明成功的fld突变的方法来鉴定基因。在目标2中，将研究有关突变表型的分子基础的关键问题。对于FLD，这些工作包括确定FLD组织葡萄糖摄取受损的潜在缺陷，利用分离的基因探索蛋白质功能，以及评估该基因在以胰岛素抵抗为特征的人类疾病(如FCHL、糖尿病、动脉粥样硬化)中的作用。对于CLD，我们将调查特定伴侣蛋白Calnexin水平降低是否导致脂肪酶缺陷，这是该突变的标志。一旦该基因被分离出来，研究将集中在Cld蛋白的功能及其在人类合并脂肪酶缺乏症患者中的潜在作用。

Naturally occurring mutations in the mouse represents a rich for identification of novel genes affecting lipid metabolism. In this project, we will isolate the genes for two mouse mutations affecting triglyceride metabolism, fatty liver dystrophy (fld) and combined lipase deficiency (cld). The fld/fld mutant mice exhibit a transient hypertriglyceridemia and fatty liver during neonatal development. We recently determined that these animals subsequently develop insulin resistance and are grossly deficient in stores of white adipose tissue, and hypothesize that the fld mutation affects a component of the insulin signal transduction pathway. The hallmark feature of cld/cld mice is hypertriglyceridemia that is fatal within 2-3 days of birth, caused by the virtual absence of lipoprotein lipase and hepatic lipase activities. Recently we have determined that the basis for the defect is misfolding of newly synthesized lipase molecules in the endoplasmic reticulum. Our two aims will focus on isolation of the fld and cld genes and characterization of the cellular processes disrupted by the mutations. In aim 1 we will identify the two genes using positional and functional cloning strategies. In the case of fld, we have narrowed the critical region to approximately kb using genetic and physical strategies. In the case of fld, we have narrowed the critical region to approximately kb using genetic and physical mapping; analysis of this region via exon trapping has identified an fld gene candidate that appears to be rearranged in the fld genome and is not expressed in tissues from the mutant mouse. We will now confirm this candidate by sequence analysis; additional candidate genes that we have isolated from this region by exon trapping will be analyzed if needed. In the case of cld, the gene critical region has been isolated as a series of over-lapping yeast and bacterial artificial chromosome clones spanning a distance of approximately 2 megabases. We are now poised to identify the clone containing the cld gene by complementation in tissue culture cell line, followed by gene identification using methods that have proved successful for the fld mutation. In Aim 2, key questions concerning the molecular basis of the mutant phenotypes will be investigated. For fld these include determining the underlying defect in impaired glucose uptake by fld tissues, utilizing the isolated gene to explore protein function and evaluating the role of this gene in human diseases characterized by insulin resistance (e.g., FCHL, diabetes, atherosclerosis). For cld, we will investigate whether reduced levels of a specific chaperone, calnexin, contribute to the lipase deficiency that is the hallmark of this mutation. Once the gene is isolated, studies will focus on cld protein function and its potential role in human subjects exhibit combined lipase deficiency.