Hazen S A, Waheed A, Sly W S, LaNoue K F, Lynch C J
Department of Cellular and Molecular Physiology, College of Medicine, The Milton S. Hershey Medical Center, Hershey, Pennsylvania 17033, USA.
FASEB J. 1996 Mar;10(4):481-90. doi: 10.1096/fasebj.10.4.8647347.
The incorporation of radioactivity from 14C-labeled compounds into metabolic intermediates and total lipids was examined in 3T3 adipocytes. The heterocyclic sulfonamide carbonic anhydrase inhibitor (SCAI) 6-ethoxyzolamide (ETZ) caused a decrease (42+/-7% of control, IC50 = 2.2+/-1.1 x 10(-7) M) in the incorporation of [14C] bicarbonate into several Krebs cycle intermediates in 3T3-F442A adipocytes. This decrease in pyruvate carboxylase-mediated [14C] carbon fixation was associated with a reduction in fluorometrically determined [citrate] and [malate]. The ability of ETZ to decrease both the incorporation of radioactivity into and the concentrations of Krebs cycle intermediates was not of sufficient magnitude to lower [ATP], but was associated with a decrease in de novo lipogenesis from [14C]glucose. De novo lipogenesis was also inhibited to a similar extent by trifluormethanesulfonamide, an aliphatic SCAI, which suggests that the effects are mediated by carbonic anhydrase. ETZ did not inhibit de novo lipogenesis from [14C]glutamine (12.38+/-1.068 nmol/mg protein, ETZ; 12.5+/-0.846 nmol/mg protein, DMSO). This suggests that ETZ inhibition of lipogenesis involves an inhibitory effect on pyruvate carboxylase as opposed to acetyl CoA carboxylase, because the incorporation of glutamine into lipids does not involve pyruvate carboxylase. Decreased de novo lipogenesis was also observed by incubating cultures in media that contained 1 mM bicarbonate (atmosphere:100% humidified air) rather than 25 mM bicarbonate (atmosphere: 95% humidified air/5% CO2). This suggests that exogenous CO2/bicarbonate may be required to sustain maximal rates of de novo lipogenesis. Because these results implied that CA V, the mitochondrial isoform of carbonic anhydrase, might be present in adipocytes, CA V levels were measured by immunoblotting. Mitochondrial preparations of adipocytes and liver were found to contain similar concentrations of CA V. Unlike adipocyte CA III, CA V concentrations were not significantly different in lean and obese Zucker rats. However, CA V levels were ninefold higher in differentiated 3T3-F442A adipocytes compared to undifferentiated adipoblasts. Our data indicate that CA V is relatively abundant in adipocyte mitochondria and exhibits differentiation-dependent expression like pyruvate carboxylase and the cytosolic isozymes CA II and CA III. The possible roles of CA II and CA V in pyruvate carboxylation are discussed.
在3T3脂肪细胞中检测了14C标记化合物的放射性掺入代谢中间体和总脂质的情况。杂环磺酰胺碳酸酐酶抑制剂（SCAI）6-乙氧基唑胺（ETZ）导致3T3-F442A脂肪细胞中[14C]碳酸氢盐掺入几种三羧酸循环中间体的量减少（为对照的42±7%，IC50 = 2.2±1.1×10-7 M）。丙酮酸羧化酶介导的[14C]碳固定的这种减少与荧光法测定的[柠檬酸]和[苹果酸]的降低有关。ETZ降低放射性掺入量和三羧酸循环中间体浓度的能力不足以降低[ATP]，但与[14C]葡萄糖的从头脂肪生成减少有关。脂肪族SCAI三氟甲磺酰胺也以类似程度抑制从头脂肪生成，这表明这些作用是由碳酸酐酶介导的。ETZ不抑制[14C]谷氨酰胺的从头脂肪生成（ETZ处理组为12.38±1.068 nmol/mg蛋白质；二甲基亚砜处理组为12.5±0.846 nmol/mg蛋白质）。这表明ETZ对脂肪生成的抑制涉及对丙酮酸羧化酶而非乙酰辅酶A羧化酶的抑制作用，因为谷氨酰胺掺入脂质不涉及丙酮酸羧化酶。在含有1 mM碳酸氢盐（气相：100%湿润空气）而非25 mM碳酸氢盐（气相：95%湿润空气/5%二氧化碳）的培养基中培养细胞时，也观察到从头脂肪生成减少。这表明可能需要外源性二氧化碳/碳酸氢盐来维持最大的从头脂肪生成速率。因为这些结果暗示碳酸酐酶的线粒体同工型CA V可能存在于脂肪细胞中，所以通过免疫印迹法测量了CA V水平。发现脂肪细胞和肝脏的线粒体提取物中CA V的浓度相似。与脂肪细胞CA III不同，在瘦型和肥胖型 Zucker大鼠中，CA V的浓度没有显著差异。然而，与未分化的脂肪母细胞相比，分化的3T3-F442A脂肪细胞中CA V水平高9倍。我们的数据表明，CA V在脂肪细胞线粒体中相对丰富，并且像丙酮酸羧化酶以及胞质同工酶CA II和CA III一样表现出分化依赖性表达。讨论了CA II和CA V在丙酮酸羧化中的可能作用。
