Study on development of drugs for cerebral nerve by new approach

乙酰乙酰辅酶a （AA-CoA）合成酶（Acetoacetyl-CoA ligase, EC 6.2.1.16）是一种新型的胞质乙酰乙酸激活酶，是乙酰乙酰特异性连接酶。在哺乳动物中，AA-CoA合成酶存在于肝脏、脑、脂肪组织等多种组织中，该酶在大鼠和人脑中均有高表达。在肝脏中，AA-CoA合成酶被认为与胆固醇生物合成和脂肪酸合成有关。然而，除肝脏外，AA-CoA合成酶的生理作用尚不清楚。为了明确AA-CoA合成酶在脑中的生理作用，(1)AA-CoA合成酶蛋白和mRNA在大鼠脑各区域的分布，(2)原位杂交法测定AA-CoA合成酶mRNA在脑中的定位，(3)AA-CoA合成酶mRNA在脑中的分布与其他脂质合成酶的比较，(4)链脲佐菌素（STZ）诱导的糖尿病对AA-CoA合成酶及各种酶的影响。首先，AA-Co - More A合成酶mRNA和蛋白在大鼠脑内广泛表达，且在脑桥、延髓、中脑、大脑皮层和下丘脑的表达量特别高。此外，AA-CoA合成酶mRNA在大鼠脑切片中的定位在小脑白质、海马海马角区和齿状回中尤其高。在包括海马在内的大脑皮层，杂交信号易于在神经细胞中表达。该酶在该区域的神经细胞中高表达，提示该酶可能在记忆和学习功能中起重要作用。此外，AA-CoA合成酶mRNA的表达模式也与HMG-CoA还原酶（胆固醇生物合成的速率决定酶）相似，而与琥珀酰辅酶转移酶（线粒体酮体代谢酶）不相似。这些结果表明，AA-CoA合成酶在大脑中参与胆固醇生物合成而不是代谢能的产生。接下来，为了研究胰岛素耗竭型糖尿病对AA-CoA合成酶的参与，我们检测了链脲佐菌素对大鼠大脑、心脏、肾脏和肝脏中AA-CoA合成酶mRNA表达的影响。结果表明，与正常对照大鼠相比，stz诱导的糖尿病大鼠各组织中AA-CoA合成酶mRNA表达明显降低。此外，我们还比较了链脲佐菌素诱导的糖尿病对脑内脂质合成和酮体代谢酶的AA-CoA合成酶mRNA的影响，因为胰岛素对脂质合成酶的表达也很重要。糖尿病大鼠AA-CoA合成酶、HMG-CoA还原酶、乙酰- coa羧化酶mRNA表达明显降低，琥珀酰coa转移酶mRNA表达变化不大。这些结果表明，AA-CoA合成酶的表达受到胰岛素类似的脂质合成酶的调节。综上所述，AA-CoA合成酶可能通过脑内酮体代谢合成胆固醇，在记忆和学习功能中发挥重要作用。少

Acetoacetyl-CoA(AA-CoA) synthetase (acetoacetyl-CoA ligase, EC 6.2.1.16) is a novel cytosolic acetoacetate-activating enzyme pulified as the acetoacetyl-specific ligase. In mammals, AA-CoA synthetase is present in liver, brain, adipose tissues and other various tissues, and this enzyme was highly expressed in rat and human brain. In the liver, AA-CoA synthetase is considered to be related cholesterol biosynthesis and fatty acid synthesis. However, except in the liver, the physiological role of AA-CoA synthetase is not clear yet. In order to clarify the physiological role of AA-CoA synthetase in the brain, (1)distribution of AA-CoA synthetase protein and mRNA in various regions of rat brain, (2)localization of AA-CoA synthetase mRNA in the brain by in situ hybridization, (3)comparison between AA-CoA synthetase mRNA distribution and other lipid synthesis enzymes' in the brain, and (4)effect of streptozotocin(STZ)-induced diabetes on the AA-CoA synthetase and various enzymes. First, AA-Co … More A synthetase mRNA and protein were widely expressed in rat brain, and these expression were particularly high in pons, medulla oblongata, midbrain, cerebral cortex and hypothalamus. Furthermore, the localization of AA-CoA synthetase mRNA in rat brain slices was found to be especially high in cerebellum white matter, hippocampus cornu ammonis and dentate gyrus. In the cerebral cortex including hippocampus, the hybridization signals are apt to express in neurocyte. The expression of the enzyme was high in neurocyte of the regions suggest that this enzyme may have the important role in memory and learning function. Furthermore, expression pattern of the AA-CoA synthetase mRNA was also similar to that of HMG-CoA reductase (the rate-determining enzyme of cholesterol biosynthesis) and not similar to that of Succinyl-CoA transferase (mitochondrial ketone body metabolic enzyme). These results suggest that AA-CoA synthetase is involved in the roles of cholesterol biosynthesis than the generation of metabolic energy in the brain. Next, in order to investigate the participation of insulin-depleted diabetes on AA-CoA synthetase, effect of streptozotocin administration on expression of this enzyme mRNA was examined in rat brain, heart, kidney and liver. As a result, STZ-induced diabetic rats showed markedly decreased AA-CoA synthetase mRNA expression in all tissues compared with normal control rats. Furthermore, compared effect of streptozotocin-induced diabetes on the AA-CoA synthetase mRNA with lipid synthesis and ketone body metabolic enzymes' in the brain because insulin has also been known to be important for lipogenic enzyme expression. Diabetic rats were markedly decreased AA-CoA synthetase, HMG-CoA reductase, Acetyl-CoA carboxylase mRNA expression but was hardly change Succinyl-CoA transferase mRNA. These results suggest that AA-CoA synthetase expression is regulated by insulin similarly lipid systhesis enzyme. In conclusion, AA-CoA synthetase is likely to play an important role in memory and learning function through ketone body metabolism for synthesis of cholesterol in the brain. Less