Multiple AMPK activators inhibit l-carnitine uptake in C2C12 skeletal muscle myotubes

• Published in: American Journal of Physiology: Cell Physiology 2017-06, Vol.312 (6), p.C689-C696
• Main Authors: Shaw, Andy, Jeromson, Stewart, Watterson, Kenneth R, Pediani, John D, Gallagher, Iain J, Whalley, Tim, Dreczkowski, Gillian, Brooks, Naomi, Galloway, Stuart D, Hamilton, D Lee
• Format: Article
• Language: English
• Subjects: Aminoimidazole Carboxamide - analogs & derivatives; Aminoimidazole Carboxamide - pharmacology; AMP-Activated Protein Kinases - genetics; AMP-Activated Protein Kinases - metabolism; Analysis; Animals; Berberine; Berberine - pharmacology; Biological Transport - drug effects; Caffeine; Caffeine - pharmacology; Calcimycin; Calcimycin - pharmacology; Calcium (intracellular); Calcium - metabolism; Carnitine; Carnitine - antagonists & inhibitors; Carnitine - metabolism; Cell Line; Cells; Dantrolene - pharmacology; Enzyme Activation - drug effects; Enzyme Activators - pharmacology; Fat metabolism; Fatty acids; Gene Expression; Immunohistochemistry; Insulin; Insulin - pharmacology; Intracellular; L-carnitine transporter; Mice; Mitochondria; mRNA; Musculoskeletal system; Myoblasts - cytology; Myoblasts - drug effects; Myoblasts - enzymology; Myotubes; Organic Cation Transport Proteins - genetics; Organic Cation Transport Proteins - metabolism; Physiology; Protein Isoforms - agonists; Protein Isoforms - genetics; Protein Isoforms - metabolism; Protein kinase A; Ribonucleotides - pharmacology; Rotenone; Rotenone - pharmacology; Skeletal muscle; Sodium azide; Sodium Azide - pharmacology; Solute Carrier Family 22 Member 5; Supplements
• ISSN: 0363-6143; 1522-1563
• DOI: 10.1152/ajpcell.00026.2016

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.