Effects of plasma membrane Ca2+-ATPase overexpression upon D-glucose metabolism in insulin-producing BRIN-BD11 cells

Abstract

In order to investigate the possible link between PMCA (plasma-membrane Ca2+-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 45Ca2+ 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-[5-3H]glucose utilization prevailing at a low extracellular concentration of D-glucose (1.1 mM), a low ratio between D-[U-14C]oxidation and D-[5-3H]glucose utilization prevailing at a high extracellular glucose concentration (16.7 mM), and a high ratio between the net generation of 14C-labelled acidic metabolites and amino acids and that of 3H2O from D-[5-3H]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 45Ca2+ 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 Ca2+ handling in insulin-producing cells, with emphasis on the key role of both cytosolic and mitochondrial Ca2+ 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.