Study on development of drugs for cerebral nerve by new approach

乙酰乙酰-辅酶A(AA-CoA)合成酶(乙酰乙酰-辅酶A连接酶，EC 6.2.1.16)是一种新型的胞浆乙酰乙酸酯激活酶，简称为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高度表达。在肝脏中，AA-CoA合成酶被认为与胆固醇的生物合成和脂肪酸的合成有关。然而，除了在肝脏中，AA-CoA合成酶的生理作用尚不清楚。为了阐明AA-CoA合成酶在脑中的生理作用，(1)AA-CoA合成酶蛋白和mRNA在大鼠脑内不同区域的分布；(2)AA-CoA合成酶mRNA在脑内的定位；(3)AA-CoA合成酶mRNA在脑内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合成酶和各种酶的影响。第一，AA-Co…A合成酶基因和蛋白在大鼠脑内广泛表达，以桥脑、延髓、中脑、大脑皮质和下丘脑表达最高。此外，AA-CoA合成酶mRNA在大鼠脑片中的定位以小脑白质、海马角和齿状回最高。在包括海马区在内的大脑皮层，杂交信号更容易在神经细胞中表达。该酶在各脑区神经细胞中均有高表达，提示该酶可能在记忆学习功能中起重要作用。此外，AA-CoA合成酶mRNA的表达模式也与胆固醇生物合成的速率决定酶HMG-CoA还原酶相似，而与线粒体酮体代谢酶琥珀酰辅酶A转移酶的表达模式不同。这些结果表明，AA-CoA合成酶参与了胆固醇的生物合成，而不是大脑中代谢能量的产生。接下来，为了探讨胰岛素耗竭型糖尿病对AA-CoA合成酶的影响，我们观察了链脲佐菌素对大鼠脑、心、肾、肝组织AA-CoA合成酶基因表达的影响。结果表明，与正常对照组大鼠相比，STZ诱导的糖尿病大鼠各组织中AA-CoA合成酶mRNA的表达均显著降低。此外，将链脲佐菌素诱导的糖尿病对AA-CoA合成酶mRNA的影响与大脑中的脂肪合成和酮体代谢酶进行了比较，因为胰岛素也已被认为对造脂酶的表达具有重要作用。糖尿病大鼠AA-CoA合成酶、HMG-CoA还原酶、乙酰辅酶A羧基酶基因表达明显降低，琥珀酰辅酶A转移酶基因表达无明显变化。这些结果表明，AA-CoA合成酶的表达受胰岛素类似的脂合成酶的调节。综上所述，AA-CoA合成酶可能通过酮体代谢在大脑中合成胆固醇而在记忆和学习功能中发挥重要作用。较少

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