The role of AMPK and purinergic signaling in astrocytic hypoglycaemia detection
Vlachaki Walker, Julia Melanie
Thesis or dissertation
University of Exeter
Following recurrent hypoglycaemia (RH) patients with type 1 diabetes (T1D) may develop an impaired counter-regulatory response (CRR) to future hypoglycaemic episodes. This is a result of maladaptive responses to hypoglycaemia, involving AMP-activated protein kinase (AMPK) activity in the ventromedial hypothalamus (VMH). Research has focused mainly on glucosensing neurones, with little emphasis on the potential role of astrocytes. This study aims to examine the response of astrocytes to a hypoglycaemic stimulus and changes to this following RH. The U373 human astrocytoma cell line and mouse primary cortical astrocytes (CRTAS), as well as wild type (WT) and α1/α2 AMPK knockout (KO) mouse embryonic fibroblasts (MEF), were used. Astrocytes were maintained at 25mM glucose, and stepped down to physiological (2.5mM) glucose for the experiments. Western blotting was used to measure total and phosphorylated protein expression. Luminescence and absorbance plate-based assays were used to measure ATP and lactate levels, respectively. A plate-based assay was used to measure intracellular calcium ([Ca2+]i) using Fluo4. Our study demonstrates that astrocytes intrinsically respond to a hypoglycaemic stimulus by increasing AMPK activation. Following RH there was a blunted AMPK activation, accompanied by a reduction in extracellular ATP (eATP). Using the AMPK activator, A-769662, we saw a concentration-dependent increase in AMPK activation and eATP. However, enhancing AMPK activation by co-application of A-769662 and 5-aminoimidazole-4-carboxamide riboside (AICAR), or by exposing astrocytes to a hypoglycaemic stimulus in the presence of A-769662, did not enhance ATP release. A-769662 also caused an increase in [Ca2+]i. Both the A-769662-mediated increase in eATP and [Ca2+]i persisted in MEF α1/α2 AMPK KO cells. The effect of A-769662 on eATP was not attenuated by pre-treating cells with the gap junction blocker carbenoxolone, or by removing extracellular Ca2+, however there was a small attenuation following pre-treatment with the [Ca2+]i chelator, BAPTA-AM. In our study we show that astrocytes intrinsically respond to a hypoglycaemic stimulus by increasing phospho AMPK (pAMPK). Following RH, the AMPK response to hypoglycaemia is blunted, alongside a significant reduction to eATP. We also demonstrate that the changes in eATP observed are likely independent of changes in AMPK activity. Importantly, the AMPK activator, A-769662, has AMPK-independent effects on eATP and [Ca2+]i, and these changes in eATP are mediated by changes in [Ca2+]i.
University of Exeter Medical School
Vlachaki Walker, J.M., Robb, J.L., Cruz, A.M., Malhi, A., Weightman Potter, P.G., Ashford, M.L.J., McCrimmon, R.J., Ellacott, K.L.J., and Beall, C. (2017). AMP-activated protein kinase (AMPK) activator A-769662 increases intracellular calcium and ATP release from astrocytes in an AMPK-independent manner. Diabetes, obesity & metabolism 19, 997-1005.
MPhil in Medical Studies