title: Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes.
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 hypothesized 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 100 nM. However, l-carnitine uptake was modestly increased at a dose of 150 nM insulin. A range of AMPK activators that increase intracellular calcium content [caffeine (10 mM, 5 mM, 1 mM, 0.5 mM), A23187 (10 μM)], inhibit mitochondrial function [sodium azide (75 μM), rotenone (1 μM), berberine (100 μM), DNP (500 μM)], or directly activate AMPK [AICAR (250 μM)] were assessed for their ability to regulate l-carnitine uptake. All compounds tested significantly inhibited l-carnitine uptake. Inhibition by caffeine was not dantrolene (10 μM) sensitive despite dantrolene inhibiting caffeine-mediated calcium release. Saturation curve analysis suggested that caffeine did not competitively inhibit l-carnitine transport. To assess the potential role of AMPK in this process, we assessed the ability of the AMPK inhibitor Compound C (10 μM) to rescue the effect of caffeine. Compound C offered a partial rescue of l-carnitine uptake with 0.5 mM 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.
authors.affiliation: Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, United Kingdom.
authors.forename: Andy
authors.initials: A
authors.lastname: Shaw
authors.affiliation: Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, United Kingdom.
authors.forename: Stewart
authors.initials: S
authors.lastname: Jeromson
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; and.
authors.forename: Kenneth R
authors.initials: KR
authors.lastname: Watterson
authors.affiliation: Molecular Pharmacology Group, Institute of Molecular, Cell and Systems Biology, College of Medical, Veterinary and Life Sciences, University of Glasgow, Glasgow, United Kingdom; and.
authors.forename: John D
authors.initials: JD
authors.lastname: Pediani
authors.affiliation: Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, United Kingdom.
authors.forename: Iain J
authors.initials: IJ
authors.lastname: Gallagher
authors.affiliation: Biological and Environmental Sciences, School of Natural Sciences, University of Stirling, Stirling, United Kingdom.
authors.forename: Tim
authors.initials: T
authors.lastname: Whalley
authors.affiliation: Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, United Kingdom.
authors.forename: Gillian
authors.initials: G
authors.lastname: Dreczkowski
authors.affiliation: Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, United Kingdom.
authors.forename: Naomi
authors.initials: N
authors.lastname: Brooks
authors.affiliation: Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, United Kingdom.
authors.forename: Stuart D
authors.initials: SD
authors.lastname: Galloway
authors.affiliation: Physiology, Exercise and Nutrition Research Group, Faculty of Health Sciences and Sport, University of Stirling, Stirling, United Kingdom; d.l.hamilton@stir.ac.uk.
authors.forename: D Lee
authors.initials: DL
authors.lastname: Hamilton
mesh_terms: D055372:AMP-Activated Protein Kinases / Q000235:genetics / Q000378:metabolism; D000620:Aminoimidazole Carboxamide / Q000031:analogs & derivatives / Q000494:pharmacology; D000818:Animals; D001599:Berberine / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002110:Caffeine / Q000494:pharmacology; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000378:metabolism; D002331:Carnitine / Q000037:antagonists & inhibitors* / Q000378:metabolism; D002460:Cell Line; D003620:Dantrolene / Q000494:pharmacology; D004789:Enzyme Activation / Q000187:drug effects; D020536:Enzyme Activators / Q000494:pharmacology*; D015870:Gene Expression; D007328:Insulin / Q000494:pharmacology; D051379:Mice; D032446:Myoblasts / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D027701:Organic Cation Transport Proteins / Q000235:genetics / Q000378:metabolism*; D020033:Protein Isoforms / Q000819:agonists / Q000235:genetics / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D012402:Rotenone / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D000074058:Solute Carrier Family 22 Member 5
chemical_list: D020536:Enzyme Activators; D007328:Insulin; D027701:Organic Cation Transport Proteins; D020033:Protein Isoforms; D012265:Ribonucleotides; C492019:Slc22a5 protein, mouse; D000074058:Solute Carrier Family 22 Member 5; D012402:Rotenone; D001599:Berberine; D000620:Aminoimidazole Carboxamide; D000001:Calcimycin; D002110:Caffeine; D019810:Sodium Azide; D055372:AMP-Activated Protein Kinases; C031143:AICA ribonucleotide; D003620:Dantrolene; D002331:Carnitine; D002118:Calcium
keywords: AMPK; carnitine uptake; insulin; kinase assays