Blázquez C, Sánchez C, Velasco G, Guzmán M
Department of Biochemistry and Molecular Biology I, School of Biology, Complutense University, Madrid, Spain.
J Neurochem. 1998 Oct;71(4):1597-606. doi: 10.1046/j.1471-4159.1998.71041597.x.
The role of carnitine palmitoyltransferase I (CPT-I) in the control of ketogenesis was studied in primary cultures of rat astrocytes. Ketone bodies were the major product of [14C]palmitate oxidation by cultured astrocytes, whereas CO2 made a minor contribution to the total oxidation products. Using tetradecylglycidate as a specific, cell-permeable inhibitor of CPT-I, a flux control coefficient of 0.77 +/- 0.07 was calculated for CPT-I over the flux of [14C]palmitate to ketone bodies. CPT-I from astrocytes was sensitive to malonyl-CoA (IC50 = 3.4 +/- 0.8 microM) and cross-reacted on western blots with an antibody raised against liver CPT-I. On the other hand, astrocytes expressed significant acetyl-CoA carboxylase (ACC) activity, and consequently they contained considerable amounts of malonyl-CoA. Western blot analysis of ACC isoforms showed that ACC in astrocytes--like in neurons, liver, and white adipose tissue--mostly comprised the 265-kDa isoform, whereas the 280-kDa isoform--which was highly expressed in skeletal muscle--showed much lower abundance. Forskolin was used as a tool to study the modulation of the ketogenic pathway in astrocytes. Thus, forskolin decreased in parallel ACC activity and intracellular malonyl-CoA levels, whereas it stimulated CPT-I activity and [14C]palmitate oxidation to both ketone bodies and CO2. Results show that in cultured astrocytes (a) CPT-I exerts a very high degree of control over ketogenesis from palmitate, (b) the ACC/malonyl-CoA/CPT-I system is similar to that of liver, and (c) the ACC/malonyl-CoA/CPT-I system is subject to regulation by cyclic AMP.
在大鼠星形胶质细胞原代培养物中研究了肉碱棕榈酰转移酶I（CPT-I）在酮体生成控制中的作用。酮体是培养的星形胶质细胞氧化[14C]棕榈酸酯的主要产物，而二氧化碳对总氧化产物的贡献较小。使用十四烷基缩水甘油酸作为CPT-I的特异性、细胞可渗透抑制剂，计算出CPT-I对[14C]棕榈酸酯生成酮体通量的流量控制系数为0.77±0.07。星形胶质细胞的CPT-I对丙二酰辅酶A敏感（IC50 = 3.4±0.8 microM），并且在蛋白质免疫印迹中与针对肝脏CPT-I产生的抗体发生交叉反应。另一方面，星形胶质细胞表达显著的乙酰辅酶A羧化酶（ACC）活性，因此它们含有相当数量的丙二酰辅酶A。ACC同工型的蛋白质免疫印迹分析表明，星形胶质细胞中的ACC——与神经元、肝脏和白色脂肪组织中的情况一样——主要由265 kDa的同工型组成，而在骨骼肌中高表达的280 kDa同工型丰度则低得多。使用福斯可林作为研究星形胶质细胞中酮生成途径调节的工具。因此，福斯可林使ACC活性和细胞内丙二酰辅酶A水平同时降低，而它刺激CPT-I活性以及[14C]棕榈酸酯氧化生成酮体和二氧化碳。结果表明，在培养的星形胶质细胞中：（a）CPT-I对棕榈酸酯生成酮体具有非常高的控制程度；（b）ACC/丙二酰辅酶A/CPT-I系统与肝脏的相似；（c）ACC/丙二酰辅酶A/CPT-I系统受环磷酸腺苷调节。
