REGULATION OF GENE EXPRESSION OF BCKDH AND ITS KINASE

BCKDH及其激酶基因表达的调控

• 批准号: 2684073
• 负责人: SIAMAK A ADIBI
• 金额: $ 22.95万
• 依托单位: UNIVERSITY OF PITTSBURGH AT PITTSBURGH
• 依托单位国家: 美国
• 财政年份: 1976
• 资助国家: 美国
• 起止时间: 1976-06-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2684073
• 关键词: acetaldehyde; alcoholism /alcohol abuse; aldehyde /ketone oxidoreductase; branched chain aminoacid; cyclic AMP; diabetes mellitus; enzyme induction /repression; ethanol; fatty acids; glucose; insulin; laboratory rat; liver cells; liver pharmacology; muscle cells; muscle pharmacology; northern blottings; protein kinase; pyruvates; striated muscles; tissue /cell culture; western blottings

Background: Branched-chain keto acid (BCKA) dehydrogenase is the key enzyme regulating oxidation of branched-chain amino acids (BCAA). It is generally believed that the activity of this enzyme is regulated by phosphorylation-dephosphorylation of the enzyme protein. However, our recent study in primary cultured - hepatocytes showed that dexamethasone and cyclic AMP increase the enzyme activity by increasing the expression of BCKA dehydrogenase subunit genes. These results have prompted questions on the effect of other hormones and nutrients on the expression of these genes. Hypothesis: We hypothesize that a) certain nutrients and hormones alter the activity of BCKA dehydrogenase by regulating the gene expression of the enzyme subunits, and b) the importance of this mechanism versus regulation by modulation of activity of BCKA dehydrogenase kinase (the specific phosphorylation enzyme) varies by different effectors and in different tissues. Specific Aim #1: To determine whether the flux through BCKA dehydrogenase in primary cultured cells or cell lines derived from liver and skeletal muscle is altered by branched-chain amino and keto acids, pyruvate and glucose, fatty acids, ethanol acetaldehyde, insulin, cyclic AMP. Previous studies have shown that these agents cause alterations in oxidation of BCAA in tissues and their concentrations are also altered in diabetes and chronic ethanol consumption. Specific Aims #2 and #3: To investigate whether the effect of each of the above agents includes alteration in gene expression of either BCKA dehydrogenase or BCKA dehydrogenase kinase. Specific Aim #4: To investigate the integrated effect of the above agents by determining the mechanism of alteration in BCAA oxidation in the liver and skeletal muscle of diabetic and ethanol-fed rats. Methods: The measurements will include the flux through BCKA dehydrogenase in cultured cells (liver and muscle) incubated for 6-24 h with a range of concentrations of each of the above agents; basal and total activities of BCKA dehydrogenase; activities of BCKA dehydrogenase kinase and phosphatase; Western analysis of the protein mass of E1alpha, E1beta, and E2 subunits of BCKA dehydrogenase; protein mass of BCKA dehydrogenase kinase; Northern analysis of the relative abundance of mRNAs encoding BCKA dehydrogenase and its specific kinase; rates of gene transcription, and rates of synthesis of BCKA dehydrogenase and its associated kinase. Clinical Significance: Oxidation of BCAA is altered in a wide variety of common clinical conditions, such as diabetes and alcoholism. The up- and down-regulation of BCKA dehydrogenase activity serves to protect the body from toxic effects of BCKA, as seen in Maple Syrup urine Disease, and to conserve body protein as seen during undernutrition.

背景：支链酮酸脱氢酶（BCKA）是 支链氨基酸（BCAA）的氧化调节酶。是 一般认为，这种酶的活性是由 酶蛋白的磷酸化-去磷酸化。但我们的 最近在原代培养的肝细胞中的研究表明，地塞米松 和环AMP通过增加表达来增加酶活性 BCKA脱氢酶亚基基因。这些结果促使 关于其他激素和营养素对表达的影响的问题 这些基因。假设：我们假设a）某些营养素和 激素通过调节基因改变BCKA脱氢酶的活性 酶亚基的表达，和B）这一点的重要性 BCKA活性调节机制与调控 脱氢酶激酶（特异性磷酸化酶）的变化， 不同的效应器和不同的组织 具体目标#1：确定通过BCKA脱氢酶的通量是否 在来源于肝脏和骨骼的原代培养细胞或细胞系中， 肌肉被支链氨基酸和酮酸、丙酮酸和 葡萄糖、脂肪酸、乙醇、乙醛、胰岛素、环腺苷酸。先前 研究表明，这些试剂会导致 组织中的支链氨基酸及其浓度也会在糖尿病患者中发生变化， 慢性酒精消耗 具体目标#2和#3：调查每种方法的效果是否 上述药物包括BCKA基因表达的改变， 脱氢酶或BCKA脱氢酶激酶。 具体目标#4：研究上述药物的综合作用 通过确定肝脏中BCAA氧化的改变机制， 和糖尿病和乙醇喂养的大鼠的骨骼肌。 方法：测量将包括通过BCKA的通量 孵育6-24 h的培养细胞（肝脏和肌肉）中的脱氢酶 使用一系列浓度的上述试剂;基础和 BCKA脱氢酶总活性 激酶和磷酸酶; E1 α蛋白质质量的Western分析， BCKA脱氢酶的E1 β和E2亚基; BCKA的蛋白质量 脱氢酶激酶的相对丰度的北方分析 编码BCKA脱氢酶及其特异性激酶的mRNA;基因表达率 转录和BCKA脱氢酶及其 相关激酶 临床意义：BCAA的氧化在各种各样的 常见的临床症状，如糖尿病和酗酒。上行链路和 BCKA脱氢酶活性的下调有助于保护身体 从BCKA的毒性作用中，如在枫树Sydrophurin病中所见，以及 在营养不良期间保存身体蛋白质。