Real-time analysis of intracellular glucose and calcium in pancreatic beta cells by fluorescence microscopy

Glucose is the physiological stimulus for insulin secretion in pancreatic beta cells. The uptake and phosphorylation of glucose initiate and control downstream pathways, resulting in insulin secretion. However, the temporal coordination of these events in beta cells is not fully understood. The recent development of the FLII(12)Pglu-700 mu-delta 6 glucose nanosensor facilitates real-time analysis of intracellular glucose within a broad concentration range. Using this fluorescence-based technique, we show the shift in intracellular glucose concentration upon external supply and removal in primary mouse beta cells with high resolution. Glucose influx, efflux, and metabolism rates were calculated from the time-dependent plots. Comparison of insulin-producing cells with different expression levels of glucose transporters and phosphorylating enzymes showed that a high glucose influx rate correlated with GLUT2 expression, but was largely also sustainable by high GLUT1 expression. In contrast, in cells not expressing the glucose sensor enzyme glucokinase glucose metabolism was slow. We found no evidence of oscillations of the intracellular glucose concentration in beta cells. Concomitant real-time analysis of glucose and calcium dynamics using FLII(12)Pglu-700 mu-delta 6 and fura-2-acetoxymethyl-ester determined a glucose threshold of 4 mM for the [Ca2+](i) increase in beta cells. Indeed, a glucose concentration of 7 mM had to be reached to evoke large amplitude [Ca2+](i) oscillations. The K-ATP channel closing agent glibenclamide was not able to induce large amplitude [Ca2+](i) oscillations in the absence of glucose. Our findings suggest that glucose has to reach a threshold to evoke the [Ca2+](i) increase and subsequently initiate [Ca2+](i) oscillations in a K-ATP channel independent manner. (c) 2012 Elsevier B.V. All rights reserved.