GLUT1 and GLUT9 as major contributors to glucose influx in HepG2 cells identified by a high sensitivity intramolecular FRET glucose sensor

Carnegie Institution for Science, 260 Panama Street, Stanford CA 94305, USA.
Biochimica et Biophysica Acta (Impact Factor: 4.66). 05/2008; 1778(4):1091-9. DOI: 10.1016/j.bbamem.2007.11.015
Source: PubMed


Genetically encoded FRET glucose nanosensors have proven to be useful for imaging glucose flux in HepG2 cells. However, the dynamic range of the original sensor was limited and thus it did not appear optimal for high throughput screening of siRNA populations for identifying proteins involved in regulation of sugar flux. Here we describe a hybrid approach that combines linker-shortening with fluorophore-insertion to decrease the degrees of freedom for fluorophore positioning leading to improved nanosensor dynamics. We were able to develop a novel highly sensitive FRET nanosensor that shows a 10-fold higher ratio change and dynamic range (0.05-11 mM) in vivo, permitting analyses in the physiologically relevant range. As a proof of concept that this sensor can be used to screen for proteins playing a role in sugar flux and its control, we used siRNA inhibition of GLUT family members and show that GLUT1 is the major glucose transporter in HepG2 cells and that GLUT9 contributes as well, however to a lower extent. GFP fusions suggest that GLUT1 and 9 are preferentially localized to the plasma membrane and thus can account for the transport activity. The improved sensitivity of the novel glucose nanosensor increases the reliability of in vivo glucose flux analyses, and provides a new means for the screening of siRNA collections as well as drugs using high-content screens.

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    • "), using a FRET-based reporter of glucose concentration (Takanaga et al. 2008). Correspondingly, an excitotoxicity-mediated increase in glucose import has been measured in CGN populations (Ward et al. 2007; Bak et al. 2009; Minervini et al. 1997) and in single cortical neurons using 2-NBDG (Ferreira et al. 2011). "
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    ABSTRACT: Excitotoxicity is a pathological process implicated in neuronal death during ischaemia, traumatic brain injuries and neurodegenerative diseases. Excitotoxicity is caused by excess levels of glutamate and over-activation of NMDA or calcium-permeable AMPA receptors on neuronal membranes, leading to ionic influx, energetic stress and potential neuronal death. The metabolic response of neurons to excitotoxicity is complex and plays a key role in the ability of the neuron to adapt and recover from such an insult. Single-cell imaging is a powerful experimental technique that can be used to study the neuronal metabolic response to excitotoxicity in vitro and, increasingly, in vivo. Here, we review some of the knowledge of the neuronal metabolic response to excitotoxicity gained from in vitro single-cell imaging, including calcium and ATP dynamics and their effects on mitochondrial function, along with the contribution of glucose metabolism, oxidative stress and additional neuroprotective signalling mechanisms. Future work will combine knowledge gained from single-cell imaging with data from biochemical and computational techniques to garner holistic information about the metabolic response to excitotoxicity at the whole brain level and transfer this knowledge to a clinical setting.
    Journal of Bioenergetics 09/2014; 47(1-2). DOI:10.1007/s10863-014-9578-4 · 3.21 Impact Factor
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    • "Linkers can have dramatic effects on sensor responses (Deuschle et al., 2005b; Hires et al., 2008; Takanaga et al., 2008). Therefore, five different linkers were inserted between the two PAS domains in dPAS110. "
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    ABSTRACT: Cytosolic hormone levels must be tightly controlled at the level of influx, efflux, synthesis, degradation and compartmentation. To determine ABA dynamics at the single cell level, FRET sensors (ABACUS) covering a range ∼0.2–800 µM were engineered using structure-guided design and a high-throughput screening platform. When expressed in yeast, ABACUS1 detected concentrative ABA uptake mediated by the AIT1/NRT1.2 transporter. Arabidopsis roots expressing ABACUS1-2µ (Kd∼2 µM) and ABACUS1-80µ (Kd∼80 µM) respond to perfusion with ABA in a concentration-dependent manner. The properties of the observed ABA accumulation in roots appear incompatible with the activity of known ABA transporters (AIT1, ABCG40). ABACUS reveals effects of external ABA on homeostasis, that is, ABA-triggered induction of ABA degradation, modification, or compartmentation. ABACUS can be used to study ABA responses in mutants and quantitatively monitor ABA translocation and regulation, and identify missing components. The sensor screening platform promises to enable rapid fine-tuning of the ABA sensors and engineering of plant and animal hormone sensors to advance our understanding of hormone signaling. DOI:
    eLife Sciences 04/2014; 3(3):e01741. DOI:10.7554/eLife.01741 · 9.32 Impact Factor
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    • "The effectiveness of a theta burst to activate neurons was confirmed by a strong calcium transient (Fig. 5A). Measurement with a glucose nanosensor [17] showed no changes in the neuronal glucose pool during or after the theta burst (Fig. 5B), a pool that is known to be rapidly mobilized in response to a pH change [18]. In contrast, the pyruvate sensor revealed that within seconds of the first train of impulses, the rate of mitochondrial metabolism in neurons went up by over 300% (Figs. "
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    ABSTRACT: Mitochondrial flux is currently accessible at low resolution. Here we introduce a genetically-encoded FRET sensor for pyruvate, and methods for quantitative measurement of pyruvate transport, pyruvate production and mitochondrial pyruvate consumption in intact individual cells at high temporal resolution. In HEK293 cells, neurons and astrocytes, mitochondrial pyruvate uptake was saturated at physiological levels, showing that the metabolic rate is determined by intrinsic properties of the organelle and not by substrate availability. The potential of the sensor was further demonstrated in neurons, where mitochondrial flux was found to rise by 300% within seconds of a calcium transient triggered by a short theta burst, while glucose levels remained unaltered. In contrast, astrocytic mitochondria were insensitive to a similar calcium transient elicited by extracellular ATP. We expect the improved resolution provided by the pyruvate sensor will be of practical interest for basic and applied researchers interested in mitochondrial function.
    PLoS ONE 01/2014; 9(1):e85780. DOI:10.1371/journal.pone.0085780 · 3.23 Impact Factor
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