Evidence for High-Capacity Bidirectional Glucose Transport across the Endoplasmic Reticulum Membrane by Genetically Encoded Fluorescence Resonance Energy Transfer Nanosensors

Abstract

Glucose release from hepatocytes is important for maintenance of blood glucose levels. Glucose-6-phosphate phosphatase, catalyzing the final metabolic step of gluconeogenesis, faces the endoplasmic reticulum (ER) lumen. Thus, glucose produced in the ER has to be either exported from the ER into the cytosol before release into circulation or exported directly by a vesicular pathway. To measure ER transport of glucose, fluorescence resonance energy transfer-based nanosensors were targeted to the cytosol or the ER lumen of HepG2 cells. During perfusion with 5 mM glucose, cytosolic levels were maintained at ϳ80% of the external supply, indicating that plasma membrane transport exceeded the rate of glucose phosphorylation. Glucose levels and kinetics inside the ER were indistinguishable from cytosolic levels, suggesting rapid bidirectional glucose transport across the ER membrane. A dynamic model incorporating rapid bidirectional ER transport yields a very good fit with the observed kinetics. Plasma membrane and ER membrane glucose transport differed regarding sensitivity to cytochalasin B and showed different relative kinetics for galactose uptake and release, suggesting catalysis by distinct activities at the two membranes. The presence of a high-capacity glucose transport system on the ER membrane is consistent with the hypothesis that glucose export from hepatocytes occurs via the cytosol by a yet-to-be-identified set of proteins.

Glucose transporter type 10—lacking in arterial tortuosity syndrome—facilitates dehydroascorbic acid transport

“…However, it is probable that GLUT10 is not a single DAA/ glucose transporter in the endomembranes. Functional studies indicated the heterogeneity of glucose transport in the ER [19,20]. Moreover, it was also demonstrated that various GLUT transporters can sufficiently mediate glucose transport en route from the ER to the plasma membrane [21].…”

“…The presence of GLUT10 in the endomembranes as a DAA and glucose transporter can be the molecular background of the functionally already observed transport activities [19,20,32]. However, it is probable that GLUT10 is not a single DAA/ glucose transporter in the endomembranes.…”

Imaging Cellular Metabolism

“…Fluorescent nanosensors have also been developed for concentration-dependent sensing of maltose in yeast (Fehr et al, 2002), as well as for detecting glucose uptake and subcellular compartmentalization Fehr et al, 2005) or ribose uptake in mammalian cells. Furthermore, bimolecular fluorescence complementation (BiFC) can detect interaction between protein partners in living cells.…”

Molecular tools for cell and systems biology

“…In special cases, a priori knowledge can be used. For example, glucose concentrations in liver cells are known to be equilibrated by reversible GLUT-mediated plasma membrane transport (Fehr et al, 2005). Accordingly, intracellular glucose levels equal those in the extracellular space.…”