MITOCHONDRIAL OXIDATIVE ENZYME EXPRESSION

申请者有近期和长期的目标，即保持积极的 分子生物学和生化实验室，重点研究其基因 酶和蛋白质产品涉及中间代谢。注意 将针对与人类疾病有关的因素，特别是 包括糖尿病、肥胖症和遗传缺陷 在脂肪酸新陈代谢方面。启动资金和来自 美国糖尿病协会已经被用来发起研究，这些研究 在本提案的初步数据一节中所述。这个 正在寻找NIDDK 15A，以便提供持续的 形成期的工资支持。设备齐全的实验室拥有 已在MGH East糖尿病病房内设立。机会： 与分子生物学和遗传学的前同事互动 各部门、东区医院的其他部门和新同事 内分泌学单位很多。申请者将付出95%的努力 在15A时期进行研究。 本提案中描述的研究的目标是 人肝脏决定因素和调节因子的特性研究 肉碱棕榈酰转移酶I(HCPT-I)基因表达。它是 假设编码线粒体脂肪酸元素的基因 β-氧化和生酮途径是协调调节的； 这种调节在一定程度上是由过氧化物酶增殖物引起的。 核转录因子活化受体α(PPARpha) 其活性受线粒体外脂肪酰基调节 中间体；这一活动提供了一种机制，通过它 胰岛素和逆调节激素可影响血管紧张素转换酶的表达 通过调节脂肪酸流向肝脏的代谢酶基因 从外围。RNA将从原代肝细胞中提取并 用于印迹研究的大鼠组织。细胞将在培养液中培养 添加PPARpha激活剂，包括中链或长链 脂肪酸，线粒体的药理操纵者 β-氧化，噻唑二酮，贝特类；或与胰岛素，胰高血糖素， 糖皮质激素、甲状腺激素或维甲酸，以评估 已知影响表达的刺激物对hcpt-I表达的调节 其他代谢酶基因，或影响脂肪酸代谢 在活体内。补充性研究将涉及对 Hcpt-I 5，调节区(S)，利用嵌合的HepG2共转染 CPT-I启动子/报告质粒构建，特别关注 对PPARAlpha：以及对观察到的现象的处理机制 在RNA印迹研究中。

The applicant has immediate and long-term goals of maintaining a vigorous molecular biology and biochemistry laboratory with focus on genes whose enzyme and protein products involve intermediary metabolism. Attention will be directed to factors that relate to human disease, specifically including diabetes mellitus, obesity, and genetically determined defects in fatty acid metabolism. Start-up funds and a research grant from the American Diabetes Association have been used to initiate studies that are described in the Preliminary Data section of the present proposal. The NIDDK 15A is being sought in order to provide a sustained period of salary support during a formative period. A well-equipped laboratory has been established within the MGH East Diabetes Unit. Opportunities for interaction with former colleagues in the Molecular Biology and Genetics Departments, other MGH East departments, and new colleagues in the Endocrinology units are abundant. The applicant will devote >95% effort to research during the period of the 15A. The objective of studies described in this proposal is the characterization of determinants and regulators of human hepatic carnitine palmitoyltransferase I (hCPT-I) gene expression. It is hypothesized that genes encoding elements of the mitochondrial fatty acid Beta-oxidation and ketogenic pathways are coordinately regulated; that this regulation is conferred in part by the peroxisome proliferator activated receptor alpha (PPARalpha), a nuclear transcription factor whose activity is regulated by extramitochondrial fatty acyl intermediates; and that this activity provides a mechanism by which insulin and counterregulatory hormones can influence the expression of metabolic enzyme genes through regulation of fatty acid flux to liver from the periphery. RNA will be harvested from primary hepatocytes and rat tissues for blotting studies. Cells will be cultured in medium supplemented with PPARalpha activators including medium- or long-chain fatty acids, pharmacological manipulators of mitochondrial Beta-oxidation, thiazolidinediones, fibrates; or with insulin, glucagon, glucocorticoids, thyroid hormone, or retinoic acid in order to assess regulation of hCPT-I expression by stimuli known to influence expression of other metabolic enzyme genes, or to influence fatty acid metabolism in vivo. Complementary studies will involve characterization of the hCPT-I 5, regulatory region(s) using HepG2 cotransfections with chimeric CPT-I promoter/reporter plasmid constructs, devoting particular attention to the PPARalpha: and to addressing mechanisms for phenomena observed in the RNA blotting studies.