Kinetic validation of 6‐NBDG as a probe for the glucose transporter GLUT1 in astrocytes

Barros, L. Felipe; Bittner, Carla X.; Loaiza, Anitsi; Ruminot, Iván; Larenas, Valeria; Moldenhauer, Hans; Oyarzún, Carolina; Alvarez, M

doi:10.1111/j.1471-4159.2009.05885.x

Cited by 63 publications

(90 citation statements)

References 22 publications

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“…In the presence of glutamate, activation of AMPK had no significant effect on glucose uptake in astrocytes. The main glucose transporter in astrocytes is GLUT1 [33,34]. GLUT1 has been shown to translocate to the plasma membrane in response to AMPK activation in cardiac myocytes [35].…”

Section: Discussionmentioning

confidence: 99%

AMPK Activation Affects Glutamate Metabolism in Astrocytes

et al. 2015

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Mammalian AMP-activated protein kinase (AMPK) functions as a metabolic switch. It is composed of 3 different subunits and its activation depends on phosphorylation of a threonine residue (Thr172) in the α-subunit. This phosphorylation can be brought about by 5-aminoimidazole-4-carboxamide 1-β-D-ribofuranoside (AICAR) which in the cells is converted to a monophosphorylated nucleotide mimicking the effect of AMP. We show that the preparation of cultured astrocytes used for metabolic studies expresses AMPK, which could be phosphorylated by exposure of the cells to AICAR. The effect of AMPK activation on glutamate metabolism in astrocytes was studied using primary cultures of these cells from mouse cerebral cortex during incubation in media containing 2.5 mM glucose and 100 µM [U-(13)C]glutamate. The metabolism of glutamate including a detailed analysis of its metabolic pathways involving the tricarboxylic acid (TCA) cycle was studied using high-performance liquid chromatography analysis supplemented with gas chromatography-mass spectrometry technology. It was found that AMPK activation had profound effects on the pathways involved in glutamate metabolism since the entrance of the glutamate carbon skeleton into the TCA cycle was reduced. On the other hand, glutamate uptake into the astrocytes as well as its conversion to glutamine catalyzed by glutamine synthetase was not affected by AMPK activation. Interestingly, synthesis and release of citrate, which are hallmarks of astrocytic function, were affected by a reduction of the flux of glutamate derived carbon through the malic enzyme and pyruvate carboxylase catalyzed reactions. Finally, it was found that in the presence of glutamate as an additional substrate, glucose metabolism monitored by the use of tritiated deoxyglucose was unaffected by AMPK activation. Accordingly, the effects of AMPK activation appeared to be specific for certain key processes involved in glutamate metabolism.

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Section: Discussionmentioning

confidence: 99%

AMPK Activation Affects Glutamate Metabolism in Astrocytes

et al. 2015

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“…Metabolism may also be investigated at cellular resolution with fluorescent glucose analogs, such as 6-NBDG and 2-NBDG, which probe glucose transport and consumption (Kim et al 2012). Although these large molecules are transported and metabolized much more slowly than glucose, they still serve as tracers for studies in vitro and in vivo (Loaiza et al 2003;Rouach et al 2008;Barros et al 2009a;Chuquet et al 2010).…”

Section: Measuring Metabolism Toward Single-cell Readoutsmentioning

confidence: 99%

The Astrocyte: Powerhouse and Recycling Center

Weber

Barros

2015

Cold Spring Harb Perspect Biol

147

121

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Brain metabolism is characterized by fuel monodependence, high-energy expenditure, autonomy from the rest of body, local recycling, and marked division of labor between cell types. Although neurons spend most of the brain's energy on signaling, astrocytes bear the brunt of the metabolic load, controlling the composition of the interstitial fluid, supplying neurons with energy substrates and precursors for biosynthesis, and recycling neurotransmitters, oxidized scavengers, and other waste products. Outstanding questions in this field are the role of oligodendrocytes, the metabolic behavior of the different subtypes of astrocytes during development and disease, and the emerging notion that metabolism may participate directly in information processing. T he energy requirements of the central nervous system (CNS) are very high compared with those of other organs. Although the brain accounts for merely 2% of body weight, it receives 15% of cardiac output, and uses 20% of the oxygen and 25% of the glucose of the total body turnover (Magistretti 2008). A special microarchitecture has evolved to support this extreme need, in which glial cells play a central role (Fig. 1). The microvasculature consists of a complex and dense network of highly interconnected blood vessels (Weber et al. 2008;Blinder et al. 2013) to ensure adequate delivery of oxygen and glucose. Although oxygen diffuses freely into the parenchyma, glucose and other hydrophilic energy substrates are translocated across membranes via specific transporter proteins (Fig. 1). The astrocyte is a polarized cell. One set of astrocytic processes ensheaths the vasculature and a second set reaches toward the synapse, the site of highest energy demand (Harris et al. 2012). Much of the ATP produced in the brain is spent by neurons on the recovery of ion gradients challenged by postsynaptic potentials, with a smaller investment in action potentials and neurotransmitter recycling (Harris et al. 2012). Astrocytes consume considerable energy for their own needs and the cycling of metabolically relevant substances for neurons. These metabolic processes in astrocytes and neurons are the basis for brain mapping using functional magnetic resonance imaging and positron emission tomography (PET)-methods that capture local metabolism either directly or indirectly via hemodynamic changes (Magistretti 2008;Belanger et al. 2011a). Levels of energy consumption in the whole brain are almost constant. However, individual neurons can increase their consumption dramatically. For example, a striatal neuron may Overview of astrocytic metabolism. (Inset) Astrocytes (green) reside between blood vessels (red) and neurons (yellow). Neurons and oligodendrocytes (blue) are not in direct contact with vessels. Astrocytes form metabolic domains and are coupled by gap junctions, through which they exchange metabolites and ions. Energy: The main energy substrate of the brain is glucose (glc), which crosses the endothelium and enters astrocytes via the glucose transporter GLUT1. Glucose is converted b...

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“…We validated this protocol by measuring the glucose uptake in the presence of cytochalasin B (CyB; 10 μM), which inhibits some glucose transporters, including glucose transporter type 1 (GLUT1), in astrocytes [23,24]. CyB largely inhibited the uptake of glucose in astrocytes (n=5, P<0.001 vs. DMSO control; Fig.…”

Section: H-deoxyglucose Uptake In Neuron and Astrocyte Culturesmentioning

confidence: 99%

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain

Lemos

Pinheiro

Beleza

et al. 2015

Purinergic Signalling

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ATP consumption during intense neuronal activity leads to peaks of both extracellular adenosine levels and increased glucose uptake in the brain. Here, we investigated the hypothesis that the activation of the low-affinity adenosine receptor, the A 2B receptor (A 2B R), promotes glucose uptake in neurons and astrocytes, thereby linking brain activity with energy metabolism. To this end, we mapped the spatiotemporal accumulation of the fluorescent-labelled deoxyglucose, 2-(N-(7-nitrobenz-2-oxa-1,3-diazol-4-yl)amino)-2-deoxyglucose (2-NBDG), in superfused acute hippocampal slices of C57Bl/6j mice. Bath application of the A 2B R agonist BAY606583 (300 nM) triggered an immediate and stable (>10 min) increase of the velocity of 2-NBDG accumulation throughout hippocampal slices. This was abolished with the pretreatment with the selective A 2B R antagonist, MRS1754 (200 nM), and was also absent in A 2B R null-mutant mice. In mouse primary astrocytic or neuronal cultures, BAY606583 similarly increased 3 H-deoxyglucose uptake in the following 20 min incubation period, which was again abolished by a pretreatment with MRS1754. Finally, incubation of hippocampal, frontocortical, or striatal slices of C57Bl/6j mice at 37°C, with either MRS1754 (200 nM) or adenosine deaminase (3 U/mL) significantly reduced glucose uptake. Furthermore, A 2B R blockade diminished newly synthesized glycogen content and at least in the striatum, increased lactate release. In conclusion, we report here that A 2B R activation is associated with an instant and tonic increase of glucose transport into neurons and astrocytes in the mouse brain. These prompt further investigations to evaluate the clinical potential of this novel glucoregulator mechanism.

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Kinetic validation of 6‐NBDG as a probe for the glucose transporter GLUT1 in astrocytes

Cited by 63 publications

References 22 publications

AMPK Activation Affects Glutamate Metabolism in Astrocytes

AMPK Activation Affects Glutamate Metabolism in Astrocytes

The Astrocyte: Powerhouse and Recycling Center

Adenosine A2B receptor activation stimulates glucose uptake in the mouse forebrain

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