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: 4.78). 01/2006; 25(24):11102-12. DOI: 10.1128/MCB.25.24.11102-11112.2005
Source: PubMed


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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    • "Glucose uptake in the mammary glands increases dramatically to meet the requirement for milk synthesis [1], and the increased glucose demand in the mammary gland for lactation is accomplished by increases in the expression of glucose transporters (GLUTs). Facilitative glucose transporters mediate the bidirectional and energy-independent process of glucose transport [2]. Each GLUT has a different transport efficiency, substrate affinity and tissue distribution, indicating that GLUTs have different biological functions in different tissues. "
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    ABSTRACT: Glucose transport, mediated by glucose transporters, is necessary for mammary gland development and lactation. GLUT1 and GLUT12 could both be expressed in the pregnant and lactating mammary gland to participate in the glucose uptake process. In this study, the goat GLUT1 and GLUT12 genes were cloned from Saanen dairy goats and transfected into goat mammary gland epithelial cells to assess their biological functions and distributions. The results showed that both goat GLUT1 and GLUT12 had 12 predicted membrane-spanning helices. Goat GLUT1 and GLUT12 each influenced the mRNA expression of the other transporter and increased the glucose consumption and lactose yield in GLUT1- and GLUT12-transfected goat mammary gland epithelial cells, respectively. The overexpression of GLUT1 or GLUT12 also increased the expression of amino acid transporters SLC1A5, SLC3A2 and SLC7A5 and affected genes expressions in GMGE cells. Using immunofluorescence staining, GLUT1 was detected throughout the cytoplasm and localized to the Golgi apparatus around the nuclear membrane, whereas GLUT12 was mainly distributed in the perinuclear region and cytoplasm. This study contributes to the understanding of how GLUT1 and GLUT12 cooperate in the incorporation of nutrient uptake into mammary gland epithelial cells and the promotion of milk synthesis in the goat mammary gland during lactation.
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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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    • "However, it would be more accurate to use a model that incorporates all contributing fluxes (e.g. Fehr et al., 2005). "
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