Lynch C J, Hazen S A, Horetsky R L, Carter N D, Dodgson S J
Department of Cellular and Molecular Physiology, Pennsylvania State University College of Medicine, Hershey 17033.
Am J Physiol. 1993 Jul;265(1 Pt 1):C234-43. doi: 10.1152/ajpcell.1993.265.1.C234.
Carbonic anhydrase (CA) was examined in two adipocyte cell lines, 3T3-L1 and 3T3-F442A. Both CA III and non-CA III activities, measured by 18O mass spectrometry, were present in 3T3-L1 and 3T3-F442A adipocytes; however, no CA activity was detected in 3T3 preadipocytes of either line. These observations were supported by immunoblot experiments employing CA III and CA II isoform-specific antisera. CA III, a major protein in rodent and murine adipocytes, and CA II, another isoform known to be present in adipose tissue, were observed only in the differentiated 3T3 adipocytes. The differentiation-dependent expression of these isozymes may imply an adipocyte-related role for CA. Compared with cultures maintained in the absence of insulin, 3T3 adipocytes maintained in the presence of insulin exhibited 65-90% lower concentrations of CA III. CA II was unaffected. This negative effect of insulin on CA III may explain the metabolic regulation of adipose CA III observed in vivo. After media changes, 3T3 adipocyte cultures rapidly lower media pH, which in turn lowers the bicarbonate/CO2 of bicarbonate/CO2-buffered media. Cultures maintained at low pH displayed 50-90% lower concentrations of CA II and CA III. Similarly, cultures maintained in a low bicarbonate/CO2 media (GibCO2-I medium containing 1 mM bicarbonate under an atmosphere of 100% humidified air) displayed 30-50% lower CA II and CA III concentrations. Thus CA II and CA III concentrations are influenced by pH and bicarbonate/CO2. Neither effect, the pH or the GibCO2-I media effect, was associated with changes in the concentration of pyruvate carboxylase or ATP citrate lyase (2 markers of adipocyte differentiation). Because the regulation by pH and bicarbonate/CO2 may be relatively selective for CA in adipocytes, a simple method for reducing the concentration/activity of CA in 3T3 adipocytes is described that may be a useful tool for studies on the physiological role of the enzyme.
在两种脂肪细胞系3T3-L1和3T3-F442A中检测了碳酸酐酶（CA）。通过18O质谱法测定，CA III活性和非CA III活性在3T3-L1和3T3-F442A脂肪细胞中均有存在；然而，在这两种细胞系的3T3前脂肪细胞中均未检测到CA活性。这些观察结果得到了使用CA III和CA II同工型特异性抗血清的免疫印迹实验的支持。CA III是啮齿动物和小鼠脂肪细胞中的主要蛋白质，CA II是已知存在于脂肪组织中的另一种同工型，仅在分化的3T3脂肪细胞中观察到。这些同工酶的分化依赖性表达可能意味着CA在脂肪细胞中具有相关作用。与在无胰岛素条件下培养的细胞相比，在有胰岛素条件下培养的3T3脂肪细胞中CA III的浓度降低了65%-90%。CA II不受影响。胰岛素对CA III的这种负面影响可能解释了体内观察到的脂肪组织CA III的代谢调节。更换培养基后，3T3脂肪细胞培养物会迅速降低培养基的pH值，进而降低碳酸氢盐/CO₂缓冲培养基中的碳酸氢盐/CO₂含量。在低pH值下培养的细胞中，CA II和CA III的浓度降低了50%-90%。同样，在低碳酸氢盐/CO₂培养基（含有1 mM碳酸氢盐的GibCO₂-I培养基，在100%湿度的空气气氛下）中培养的细胞，CA II和CA III的浓度降低了30%-50%。因此，CA II和CA III的浓度受pH值和碳酸氢盐/CO₂的影响。pH值或GibCO₂-I培养基的影响均与丙酮酸羧化酶或ATP柠檬酸裂解酶（脂肪细胞分化的两个标志物）浓度的变化无关。由于pH值和碳酸氢盐/CO₂的调节对脂肪细胞中的CA可能具有相对选择性，因此描述了一种降低3T3脂肪细胞中CA浓度/活性的简单方法，这可能是研究该酶生理作用的有用工具。
