Evidence for high-capacity bidirectional glucose transport across the endoplasmic reticulum membrane by genetically encoded fluorescence resonance energy transfer nanosensors

Carnegie Institution, Stanford, CA 94305, USA.
Molecular and Cellular Biology (Impact Factor: 5.04). 01/2006; 25(24):11102-12. DOI: 10.1128/MCB.25.24.11102-11112.2005
Source: PubMed

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 approximately 80% 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.

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    • "4a–c). A possible explanation for this is that the concentration of 5mM glucose in the basal medium is sufficient to saturate the cellular secretory response, as HepG2 cells express the high affinity glucose transporter GLUT1 (K m ~ 1.9mM) and the high affinity glucose phosphorylating enzyme hexokinase (K m ~ 0.05mM) (Fehr et al, 2005). Therefore, incubation with glucose-only medium containing 20mM glucose had no further stimulatory effect relative to medium with 5 mM glucose. "
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