AMP-activated protein kinase activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner

Julia M. Vlachaki Walker, Josephine L. Robb, Ana M. Cruz, Amrinder Malhi, Paul G. Weightman Potter, Michael L. J. Ashford, Rory J. McCrimmon, Kate L. J. Ellacott, Craig Beall (Lead / Corresponding author)

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14 Citations (Scopus)
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Abstract

AIM: Astrocytes are the main sources of extracellular ATP (eATP) within the brain, which functions as a gliotransmitter, capable of modulating neuronal and astrocytic activity. These cells play an important role in regulating energy homeostasis partly via astrocyte-derived ATP. Given the role of AMPK in regulating intracellular ATP levels, we hypothesised that AMPK may alter ATP release from astrocytes.

METHODS: Measurements of ATP release were made from human U373 astrocytoma cells, primary mouse hypothalamic (HTAS) and cortical astrocytes (CRTAS) and wild type and AMPK α1/α2 null mouse embryonic fibroblasts (MEFs). Cells were treated with drugs known to modulate AMPK activity: A-769662, AICAR and metformin, for up to 3 hours. Intracellular calcium was measured using Fluo4 and Fura-2 calcium-sensitive fluorescent dyes.

RESULTS: In U373 cells, A-769662 (100 μM) increased AMPK phosphorylation, whereas AICAR and metformin (1 mM) induced a modest increase or had no effect, respectively. Only A-769662 increased extracellular ATP (eATP) levels, which was partially blocked by AMPK inhibitor Compound C. A-769662-induced increases in eATP were preserved in AMPK α1/α2 null MEF cells. A-769662 increased intracellular calcium in U373, HTAS and CRTAS cells and chelation of intracellular calcium using BAPTA-AM reduced A-769662-induced eATP levels. A-769662 also increased ATP release from a number of other central and peripheral endocrine cell types.

CONCLUSIONS: AMPK is required to maintain basal eATP levels but is not required for A-769662-induced increases in eATP. A-769662 (>50 μM) enhanced intracellular calcium levels leading to ATP release in an AMPK and purinergic receptor independent pathway.

Original languageEnglish
Pages (from-to)997-1005
Number of pages9
JournalDiabetes, Obesity & Metabolism
Volume19
Issue number7
Early online date17 Feb 2017
DOIs
Publication statusPublished - 21 Apr 2017

Keywords

  • Journal article
  • AMPK
  • ATP
  • A-769662
  • Intracellular calcium
  • U373
  • Hypothalamic astrocytes
  • Cortical astrocytes

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