Anti-hyperglycaemic effects of the hydroxybenzoic acid salicylate might stem from effects of the drug on mitochondrial uncoupling, activation of AMP-activated protein kinase, and inhibition of NF-κB signalling. Here, we have gauged the contribution of these effects to control of hepatocyte glucose production, comparing salicylate with inactive hydroxybenzoic acid analogues of the drug. In rat H4IIE hepatoma cells, salicylate was the only drug tested that activated AMPK. Salicylate also reduced mTOR signalling, but this property was observed widely among the analogues. In a sub-panel of analogues, salicylate alone reduced promoter activity of the key gluconeogenic enzyme glucose 6-phosphatase and suppressed basal glucose production in mouse primary hepatocytes. Both salicylate and 2,6 dihydroxybenzoic acid suppressed TNFα-induced IκB degradation, and in genetic knockout experiments, we found that the effect of salicylate on IκB degradation was AMPK-independent. Previous data also identified AMPK-independent regulation of glucose but we found that direct inhibition of neither NF-κB nor mTOR signalling suppressed glucose production, suggesting that other factors besides these cell signalling pathways may need to be considered to account for this response to salicylate. We found, for example, that H4IIE cells were exquisitely sensitive to uncoupling with modest doses of salicylate, which occurred on a similar time course to another anti-hyperglycaemic uncoupling agent 2,4-dinitrophenol, while there was no discernible effect at all of two salicylate analogues which are not anti-hyperglycaemic. This finding supports much earlier literature suggesting that salicylates exert anti-hyperglycaemic effects at least in part through uncoupling.
Salicylate; AMPK; mTOR signalling; NF-κB signalling; Gluconeogenesis
Citaion: Amy R. Cameron, Lisa Logie, Kashyap Patel, Sandra Bacon, Calum Forteath, Jean Harthill, Adam Roberts, Calum Sutherland, Derek Stewart, Benoit Viollet, Kei Sakamoto, Gordon McDougall, Marc Foretz, Graham Rena Investigation Of Salicylate Hepatic Responses In Comparison With Chemical Analogues Of The Drug doi:10.1016/j.bbadis.2016.04.015.
Received: 15 January 2016, Revised: 17 April 2016, Accepted: 22 April 2016, Available online: 27 April 2016
Copyright: © 2016 The Authors. Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
In this work, we have investigated responses to SA in hepatocytes. Comparison with a panel of SA analogues suggests that mitochondrial uncoupling and AMPK activation but not other signalling pathways correlate well with published anti-hyperglycaemic effects.
We thank Dr. Craig Beall (Exeter) for assistance on Seahorse experiments. GR gratefully acknowledges support from MRC (MR/K012924/1), the Cunningham Trust, and the Diabetes UK RW & JM Collins studentship (12/0004625), which is supporting CF. SB was supported by a Ph.D. studentship from the Rank Prize Funds, with additional support provided by the University of Dundee. KP was supported by a Wellcome Trust Clinical Ph.D. studentship. The research was also supported by Tenovus Scotland (GR), by the UK Medical Research Council (KS and GR), by the Région Ile de France-CORDDIM (MF), and by the Société Francophone du Diabète (MF). DS and GMcD acknowledge funding from The Scottish Government's Rural and Environment Science and Analytical Services Division.