Multiple AMPK activators inhibit L-Carnitine uptake in C2C12 skeletal muscle myotubes

Shaw, A., Jeromson, S., Watterson, K. R., Pediani, J. D. , Gallagher, I., Whalley, T., Dreczkowski, G., Brooks, N., Galloway, S. and Hamilton, D. L. (2017) Multiple AMPK activators inhibit L-Carnitine uptake in C2C12 skeletal muscle myotubes. American Journal of Physiology: Cell Physiology, 312(6), C689-C696. (doi:10.1152/ajpcell.00026.2016) (PMID:28298333)

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Abstract

Mutations in the gene that encodes the principal L-Carnitine transporter, OCTN2, can lead to a reduced intracellular L-Carnitine pool and the disease Primary Carnitine Deficiency. L-Carnitine supplementation is used therapeutically to increase intracellular L-Carnitine. As AMPK and insulin regulate fat metabolism and substrate uptake we hypothesised that AMPK activating compounds and insulin would increase L-Carnitine uptake in C2C12 myotubes. The cells express all three OCTN transporters at the mRNA level and immunohistochemistry confirmed expression at the protein level. Contrary to our hypothesis, despite significant activation of PKB and 2DG uptake, insulin did not increase L-Carnitine uptake at 100nM. However, L-Carnitine uptake was modestly increased at a dose of 150nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10mM, 5mM, 1mM, 0.5mM), A23187 (10μM)], inhibit mitochondrial function [Sodium Azide (75μM), Rotenone (1μM), Berberine (100μM), DNP (500μM)] or directly activate AMPK [AICAR (250μM)] were assessed for their ability to regulate L-Carnitine uptake. All compounds tested significantly inhibited L-Carnitine uptake. Inhibition by caffeine was not dantrolene (10μM) sensitive. Saturation curve analysis suggested that caffeine did not competitively inhibit L-Carnitine transport. However, the AMPK inhibitor Compound C (10μM) partially rescued the effect of caffeine suggesting that AMPK may play a role in the inhibitory effects of caffeine. However, caffeine likely inhibits L-Carnitine uptake by alternative mechanisms independently of calcium release. PKA activation or direct interference with transporter function may play a role.

Item Type:Articles
Keywords:AMPK, carnitine, insulin.
Status:Published
Refereed:Yes
Glasgow Author(s) Enlighten ID:Pediani, Dr John and Watterson, Dr Kenneth
Authors: Shaw, A., Jeromson, S., Watterson, K. R., Pediani, J. D., Gallagher, I., Whalley, T., Dreczkowski, G., Brooks, N., Galloway, S., and Hamilton, D. L.
College/School:College of Medical Veterinary and Life Sciences > Institute of Molecular Cell and Systems Biology
Journal Name:American Journal of Physiology: Cell Physiology
Publisher:American Physiological Society
ISSN:0363-6143
ISSN (Online):1522-1563
Published Online:15 March 2017
Copyright Holders:Copyright © 2017 American Physiological Society
First Published:First published in American Journal of Physiology: Cell Physiology 312(6):C689-C696
Publisher Policy:Reproduced in accordance with the publisher copyright policy

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