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AMPK Activation Affects Glutamate Metabolism in Astrocytes

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Abstract

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-13C]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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Abbreviations

AAT:

Aspartate aminotransferase

AICAR:

5-Aminoimidazole-4-carboxamide 1-β-d-ribofuranoside

ALAT:

Alanine aminotransferase

AMPK:

AMP activated protein kinase

BCA:

Bicinchoninic acid

BSA:

Bovine serum albumin

DMEM:

Dulbecco’s modified Eagle’s medium

FCS:

Foetal calf serum

GDH:

Glutamate dehydrogenase

GC–MS:

Gas chromatography–mass spectrometry

HPLC:

High-performance liquid chromatography

LDH:

Lactate dehydrogenase

ME:

Malic enzyme

OPA:

o-Phthaldialdehyde

PBS:

Phosphate buffered saline

PC:

Pyruvate carboxylase

PDH:

Pyruvate dehydrogenase

TCA:

Tricarboxylic acid

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Acknowledgments

This work was supported by the Novo Nordisk Foundation (2293). Heidi Nielsen and Anna Hansen are acknowledged for their excellent technical assistance. Stud. pharm. Omran Abdel Rahman is acknowledged for contributing to GC–MS analyses.

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Author notes

  1. Kamilla Pajęcka

    Present address: Department of Clinical Medicine, The Department of Endocrinology and Diabetes, University of Aarhus, 8000, Århus, Denmark

Authors and Affiliations

  1. Department of Drug Design and Pharmacology, Faculty of Health and Medical Sciences, University of Copenhagen, Universitetsparken 2, 2100, Copenhagen, Denmark

    Caroline M. Voss, Kamilla Pajęcka, Malin H. Stridh, Jakob D. Nissen, Arne Schousboe & Helle S. Waagepetersen

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  1. Caroline M. Voss
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Correspondence to Helle S. Waagepetersen.

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Special Issue: In honor of Dr. Gerald Dienel.

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Voss, C.M., Pajęcka, K., Stridh, M.H. et al. AMPK Activation Affects Glutamate Metabolism in Astrocytes. Neurochem Res 40, 2431–2442 (2015). https://doi.org/10.1007/s11064-015-1558-5

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