Lynch C J, Fox H, Hazen S A, Stanley B A, Dodgson S, Lanoue K F
Department of Cellular and Molecular Physiology, College of Medicine, Milton S. Hershey Medical Center, Pennsylvania State University, Hershey 17033, USA.
Biochem J. 1995 Aug 15;310 ( Pt 1)(Pt 1):197-202. doi: 10.1042/bj3100197.
The role of carbonic anhydrase in de novo lipid synthesis was examined by measuring [1-14C]acetate incorporation into total lipids, fatty acids and non-saponifiable lipids in freshly isolated rat hepatocytes. Two carbonic anhydrase inhibitors, trifluoromethylsulphonamide (TFMS) and ethoxozolamide (ETZ) decreased incorporation of 14C into total lipids. Both fatty acid and non-saponifiable lipid components of the total lipid were inhibited to approximately the same extent by 100 microM TFMS (29 +/- 0.3% and 35 +/- 0.3% of control respectively in replicate studies). However, neither drug significantly affected ATP concentrations or the transport activity of Na+/K(+)-ATPase, two measures of cell viability. To establish the site of this inhibition, water-soluble 14C-labelled metabolites from perchloric acid extracts of the radiolabelled cells were separated by ion-exchange chromatography. TFMS inhibited 14C incorporation into citrate, malate, alpha-oxoglutarate and fumarate, but had no effect on incorporation of 14C into acetoacetate. Since ATP citrate-lyase, the cytosolic enzyme that catalyses the conversion of citrate into acetyl-CoA, catalyses an early rate-limiting step in fatty acid synthesis, levels of cytosolic citrate may be rate controlling for de novo fatty acid and sterol synthesis. Indeed citrate concentrations were significantly reduced to 37 +/- 6% of control in hepatocytes incubated with 100 microM TFMS for 30 min. TFMS also inhibited the incorporation of 14C from [1-14C]pyruvate into malate, citrate and glutamate, but not into lactate. This supports the hypothesis that TFMS inhibits pyruvate carboxylation, i.e. since all of the 14C from [1-14C]pyruvate converted into citric acid cycle intermediates must come via pyruvate carboxylase (i.e. rather than pyruvate dehydrogenase). Our findings indicate a role for carbonic anhydrase in hepatic de novo lipogenesis at the level of pyruvate carboxylation.
通过测量新鲜分离的大鼠肝细胞中[1-14C]乙酸盐掺入总脂质、脂肪酸和非皂化脂质中的量，研究了碳酸酐酶在从头脂质合成中的作用。两种碳酸酐酶抑制剂，三氟甲基磺酰胺（TFMS）和乙氧唑胺（ETZ）降低了14C掺入总脂质中的量。总脂质中的脂肪酸和非皂化脂质成分在100 microM TFMS作用下受到的抑制程度大致相同（重复实验中分别为对照的29±0.3%和35±0.3%）。然而，这两种药物均未显著影响ATP浓度或Na+/K(+)-ATPase的转运活性，这两项指标均用于衡量细胞活力。为确定这种抑制作用的位点，通过离子交换色谱法分离了放射性标记细胞的高氯酸提取物中的水溶性14C标记代谢物。TFMS抑制了14C掺入柠檬酸、苹果酸、α-酮戊二酸和富马酸，但对14C掺入乙酰乙酸没有影响。由于ATP柠檬酸裂解酶（催化柠檬酸转化为乙酰辅酶A的胞质酶）催化脂肪酸合成中的早期限速步骤，因此胞质柠檬酸水平可能是从头脂肪酸和甾醇合成的速率控制因素。事实上，在与100 microM TFMS孵育30分钟的肝细胞中，柠檬酸浓度显著降低至对照的37±6%。TFMS还抑制了[1-14C]丙酮酸中的14C掺入苹果酸、柠檬酸和谷氨酸，但不抑制其掺入乳酸。这支持了TFMS抑制丙酮酸羧化的假设，即因为[1-14C]丙酮酸中所有转化为柠檬酸循环中间体的14C都必须通过丙酮酸羧化酶（而不是丙酮酸脱氢酶）。我们的研究结果表明碳酸酐酶在丙酮酸羧化水平的肝脏从头脂肪生成中发挥作用。
