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

TY - JOUR

T1 - AMP-activated protein kinase activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner

AU - Vlachaki Walker, Julia M.

AU - Robb, Josephine L.

AU - Cruz, Ana M.

AU - Malhi, Amrinder

AU - Weightman Potter, Paul G.

AU - Ashford, Michael L. J.

AU - McCrimmon, Rory J.

AU - Ellacott, Kate L. J.

AU - Beall, Craig

N1 - This study was funded by: Diabetes UK RD Lawrence Fellowship (13/0004647 to C.B.), British Society for Neuroendocrinology, Society for Endocrinology, Mary Kinross Charitable Trust and Tenovus Scotland.

PY - 2017/4/21

Y1 - 2017/4/21

N2 - 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.

AB - 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.

KW - Journal article

KW - AMPK

KW - ATP

KW - A-769662

KW - Intracellular calcium

KW - U373

KW - Hypothalamic astrocytes

KW - Cortical astrocytes

U2 - 10.1111/dom.12912

DO - 10.1111/dom.12912

M3 - Article

C2 - 28211632

SN - 1462-8902

VL - 19

SP - 997

EP - 1005

JO - Diabetes, Obesity & Metabolism

JF - Diabetes, Obesity & Metabolism

IS - 7

ER -