Effect of bioactive fatty acid ethanolamides (FAEs) on AMPK activity

Details

Supervisors

Rider, Mark H.

Faculty

Faculté de pharmacie et des sciences biomédicales

Degree label

Master [120] en sciences biomédicales, à finalité approfondie

Abstract

AMPK (5'-adenosine monophosphate-activated protein kinase) is known to play a key regulatory role in cellular and whole-body energy homeostasis by stimulating catabolism and by inhibiting anabolic processes. The hypoglycaemic effects of metformin, the front-line drug for Type 2 Diabetes (T2D), are in part mediated by AMPK and its downstream effects. As a result, AMPK is a well-recognised therapeutic target and its upstream regulators and downstream targets have attracted much attention. A study recently provided evidence that long-chain fatty acyl-CoA (LCFA) thioesters could allosterically stimulate AMPK activity and might represent the “missing” endogenous ligands that bind the Allosteric Drug and Metabolite (ADaM) pocket to induce AMPK activation. Independently, our laboratory identified erucamide, a fatty acid amide, by rapidly immunoprecipitating AMPK α1β1γ1 overexpressed in HEK293T cells followed by elution in organic solvent for small-molecule liquid chromatography-mass spectrometric analysis. The goal of this study is to investigate the effect of several bioactive fatty acid ethanolamides (FAEs), such as oleoylethanolamide and fatty acid amides (FAAs) including erucamide on AMPK activity as potential endogenous regulatory ligands of AMPK. Additionally, we aim to validate the recently reported AMPK-activating effect of palmitoyl-CoA, a LCFA. FAEs and FAAs were found to have no effect on pig liver AMPK activity. Similar results were obtained with E.coli expressed naïve AMPK α1β1γ1, α1β2γ1 and α2β2γ1. Importantly, when assays included oleoylethanolamide, a significant increase in activity of LKB1-activated E.coli expressed AMPK α1β1γ1 and α1β2γ1 but not α2β2γ1 was observed. These results were recapitulated using CaMKKβ-activated E.coli expressed AMPK α1β1γ1 and α2β2γ1. Serendipitously, anandamide was found to significantly stimulate the activity of LKB1- and CaMKKβ-activated AMPK α1β1γ1, but not LKB1-activated AMPK α1β2γ1 or LKB1- and CaMKKβ-activated AMPK α2β2γ1. We found that palmitoyl-CoA led to a significant increase in activity of LKB1- and CaMKKβ-activated AMPK α1β1γ1, but not LKB1-activated AMPK α1β2γ1 or LKB1- and CaMKKβ-activated AMPK α2β2γ1. Ultimately, we validated a method for AMPK α1β1γ1 transfection and immunoprecipitation in HEK293T cells for subsequent kinase assay tests to screen for allosteric AMPK regulators.