REGULATION OF GENE EXPRESSION OF BCKDH AND ITS KINASE

背景：支链酮酸脱氢酶是关键 调节支链氨基酸(BCAA)氧化的酶。它是 普遍认为，这种酶的活性是由 磷酸化-酶蛋白的去磷酸化。然而，我们的 最近对原代培养肝细胞的研究表明，地塞米松 环磷酸腺苷通过增加酶的表达来提高酶的活性 BCKA脱氢酶亚单位基因。这些结果促使人们 关于其他激素和营养物质对表达的影响的质疑 这些基因。假设：我们假设a)某些营养物质和 激素通过调节基因来改变BCKA脱氢酶的活性 酶亚基的表达，以及b)这一点的重要性 BCKA活性调节的机制与调控 脱氢酶激酶(特定的磷酸化酶)因不同而不同 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 在原代培养的细胞或来源于肝脏和骨骼的细胞系中 肌肉由支链氨基酸和酮酸、丙酮酸和 葡萄糖、脂肪酸、乙醇、乙醛、胰岛素、环磷酸腺苷。上一首 研究表明，这些试剂会引起氧化反应的改变 糖尿病和糖尿病患者体内的支链氨基酸及其浓度也会发生变化 长期的酒精消费。 具体目标#2和#3：调查每一种药物的效果 上述因素包括BCKA基因表达的改变 脱氢酶或BCKA脱氢酶激酶。 具体目标#4：调查上述药物的综合作用 通过确定肝脏支链氨基酸氧化的改变机制 以及糖尿病和酒精喂养的大鼠的骨骼肌。 方法：测量将包括通过BCKA的流量 培养细胞(肝脏和肌肉)孵育6-24小时的脱氢酶 具有上述每种试剂的浓度范围；基础和 BCKA脱氢酶总活力；BCKA脱氢酶活力 激酶和磷酸酶；E1pha蛋白质量的Western分析， BCKA脱氢酶的E1beta和E2亚基；BCKA的蛋白质量 脱氢酶激酶；相对丰度的Northern分析 编码BCKA脱氢酶及其特异性激酶的mRNAs；基因率 BCKA脱氢酶及其蛋白的转录和合成速率 相关的激酶。 临床意义：支链氨基酸氧化在多种疾病中发生改变 常见的临床症状，如糖尿病和酗酒。向上--和 下调BCKA脱氢酶活性可保护身体 从BCKA的毒性作用，如枫糖浆尿病，和 在营养不良的情况下，保存身体蛋白质。

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.