Authors: Scott A John Michela Ottolia James N Weiss Bernard Ribalet
Publish Date: 2007/12/11
Volume: 456, Issue: 2, Pages: 307-322
Abstract
To study intracellular glucose homeostasis the glucose nanosensor FLIPglu600μM which undergoes changes in fluorescence resonance energy transfer FRET upon interaction with glucose was expressed in four mammalian cell lines COS7 CHO HEK293 and C2C12 Upon addition of extracellular glucose the intracellular FRET ratio decreased rapidly as intracellular glucose increased The kinetics were fast τ = 5 to 15 s in COS and C2C12 cells and slow τ = 20 to 40 s in HEK and CHO cells Upon removal of extracellular glucose the FRET ratio returned to its initial value at similar rates τ = 15 to 40 s in all cell types In all cell types the glucose uptake FRET signal was blocked by the glucose transporter GLUTx inhibitor cytochalasin B and was not affected by the Na/glucose transporter inhibitor phlorizin Glucose clearance was inhibited by the glycolytic inhibitor iodoacetate Using βescin to permeabilize the cell we found that the glucose gradient across the membrane was strongly dependent on the rates of glucose uptake versus glucose clearance With 10 mM extracellular glucose and a high rate of glucose clearance intracellular glucose level fell below 100 μM when glucose uptake rate was low whereas it exceeded 05 mM when glucose uptake was high Cells cultured in high glucose maintained lower basal intracellular glucose levels than cells cultured in low glucose attributed to “reciprocal regulation” of glycolysis and gluconeogenesis Basal glucose level also increased with elevated temperatures Experiments performed with C2C12 cells demonstrated a shift from fast glucose uptake to slow glucose uptake in the absence of insulin during differentiationThe authors thank Dr W F Frommer for his generous gift of the glucose sensor FLIPGlu600μM Dr LH Xie for his constructive comments and Dr P Sdek for helping with C2C12 cell differentiation This work was supported by NIH grants R37HL60025 and P01 HL071870 and Laubisch and Kawata Endowments to JNW
Keywords: