MITOCHONDRIAL OXIDATIVE ENZYME EXPRESSION

申请人的近期和长期目标是保持一个积极的 分子生物学和生物化学实验室，专注于基因， 酶和蛋白质产物涉及中间代谢。关注 将针对与人类疾病有关的因素，特别是 包括糖尿病、肥胖和遗传缺陷 在脂肪酸代谢中。启动资金和研究补助金， 美国糖尿病协会已被用来启动研究， 本建议书初步数据部分所述。的 正在寻求NIDDK 15 A，以提供持续的 在成长期提供工资支持。设备齐全的实验室 在MGH东部糖尿病单位内建立。机会 与分子生物学和遗传学的前同事的互动 部门，其他MGH东部部门，以及新同事在 内分泌科的单位很多。申请人将投入>95%的努力 在15 A期间进行研究。 本提案所述研究的目标是 人肝细胞癌的决定因子和调节因子的特征 肉毒碱棕榈酰转移酶I（hCPT-I）基因表达。是 假设编码线粒体脂肪酸的基因 β-氧化和生酮途径受到协调调节; 这种调节部分是由过氧化物酶体增殖剂 活化受体α（PPARalpha），一种核转录因子 其活性受线粒体外脂肪酰基调节 中间体;并且该活性提供了一种机制， 胰岛素和反调节激素可以影响 通过调节脂肪酸流向肝脏的代谢酶基因 从外围。将从原代肝细胞中收获RNA， 大鼠组织用于印迹研究。细胞将在培养基中培养 补充有包括中链或长链 脂肪酸，线粒体的药理学操纵者， β-氧化，噻唑烷二酮类，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 通过已知影响表达的刺激调节hCPT-I表达 或影响脂肪酸代谢 in vivo.补充研究将涉及对 hCPT-15，使用HepG 2共转染的调节区与嵌合的 CPT-I启动子/报告质粒构建体，特别关注 和处理所观察到的现象的机制 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.