Kamagate Adama, Sener Abdullah, Courtois Philippe, Malaisse Willy J, Herchuelz André
Laboratoire de Pharmacodynamie et de Thérapeutique, Université Libre de Bruxelles (ULB), 808 Route de Lennik, B-1070 Brussels, Belgium.
Biosci Rep. 2008 Oct;28(5):251-8. doi: 10.1042/BSR20080042.
In order to investigate the possible link between PMCA (plasma-membrane Ca(2+)-ATPase) activity and D-glucose catabolism in insulin-producing cells, BRIN-BD11 cells were transfected with two isoforms of PMCA2. Transfection of insulin-producing BRIN-BD11 cells with PMCA2yb and PMCA2wb was documented by RT-PCR (reverse transcription-PCR), Western blot analysis, indirect immunofluorescence microscopy and (45)Ca(2+) uptake by microsomes. In the transfected cells, the overexpression of PMCA coincided with three major anomalies of D-glucose metabolism, namely a lower rate of D-[5-(3)H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-(14)C]oxidation and D-[5-(3)H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of (14)C-labelled acidic metabolites and amino acids and that of (3)H(2)O from D-[5-(3)H]glucose. These anomalies resulted in a decreased estimated rate of ATP generation (linked to the catabolism of the hexose) and a lowered ATP cell content, whether at low or high extracellular D-glucose concentrations. The net uptake of (45)Ca(2+) by intact cells was also decreased in the transfected cells, but to a greater extent than can apparently be attributed to the change in the ATP-generation rate. These findings document the relevance of PMCA activity to both D-glucose metabolism and Ca(2+) handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca(2+) concentrations in the regulation of D-glucose catabolism. They also reveal that overexpression of PMCA leads, in insulin-producing cells, to an imbalance between ATP generation and consumption.
为了研究胰岛素分泌细胞中质膜钙ATP酶（PMCA）活性与D-葡萄糖分解代谢之间可能存在的联系，用两种PMCA2亚型转染了BRIN-BD11细胞。通过逆转录聚合酶链反应（RT-PCR）、蛋白质免疫印迹分析、间接免疫荧光显微镜检查以及微粒体对（45）Ca（2+）的摄取，记录了用PMCA2yb和PMCA2wb转染胰岛素分泌BRIN-BD11细胞的情况。在转染细胞中，PMCA的过表达与D-葡萄糖代谢的三个主要异常情况同时出现，即在低细胞外D-葡萄糖浓度（1.1 mM）时，D-[5-（3）H]葡萄糖利用率较低；在高细胞外葡萄糖浓度（16.7 mM）时，D-[U-（14）C]氧化与D-[5-（3）H]葡萄糖利用率的比率较低；以及从D-[5-（3）H]葡萄糖生成的（14）C标记酸性代谢物和氨基酸的净生成量与（3）H（2）O的净生成量之间的比率较高。这些异常情况导致估计的ATP生成速率（与己糖分解代谢相关）降低，以及ATP细胞含量降低，无论细胞外D-葡萄糖浓度是低还是高。完整细胞对（45）Ca（2+）的净摄取在转染细胞中也有所降低，但降低程度比明显归因于ATP生成速率变化的程度更大。这些发现证明了PMCA活性与胰岛素分泌细胞中D-葡萄糖代谢和Ca（2+）处理的相关性，强调了细胞质和线粒体Ca（2+）浓度在调节D-葡萄糖分解代谢中的关键作用。它们还揭示，在胰岛素分泌细胞中，PMCA的过表达导致ATP生成与消耗之间的失衡。
