Long-chain acylcarnitines activate cell stress and myokine release in C₂C₁₂myotubes: calcium-dependent and -independent effects

Abstract

McCoin CS, Knotts TA, Ono-Moore KD, Oort PJ, Adams SH. Long-chain acylcarnitines activate cell stress and myokine release in C2C12 myotubes: calcium-dependent and -independent effects. Am J Physiol Endocrinol Metab 308: E990 -E1000, 2015. First published April 7, 2015; doi:10.1152/ajpendo.00602.2014.-Acylcarnitines, important lipid biomarkers reflective of acyl-CoA status, are metabolites that possess bioactive and inflammatory properties. This study examined the potential for long-chain acylcarnitines to activate cellular inflammatory, stress, and death pathways in a skeletal muscle model. Differentiated C2C12 myotubes treated with L-C14, C16, C18, and C18:1 carnitine displayed dose-dependent increases in IL-6 production with a concomitant rise in markers of cell permeability and death, which was not observed for shorter chain lengths. L-C16 carnitine, used as a representative long-chain acylcarnitine at initial extracellular concentrations Ն25 M, increased IL-6 production 4.1-, 14.9-, and 31.4-fold over vehicle at 25, 50, and 100 M. Additionally, L-C16 carnitine activated c-Jun NH2-terminal kinase, extracellular signalregulated kinase, and p38 mitogen-activated protein kinase between 2.5-and 11-fold and induced cell injury and death within 6 h with modest activation of the apoptotic caspase-3 protein. L-C16 carnitine rapidly increased intracellular calcium, most clearly by 10 M, implicating calcium as a potential mechanism for some activities of long-chain acylcarnitines. The intracellular calcium chelator BAPTA-AM blunted L-C16 carnitine-mediated IL-6 production by Ͼ65%. However, BAPTA-AM did not attenuate cell permeability and death responses, indicating that these outcomes are calcium independent. The 16-carbon zwitterionic compound amidosulfobetaine-16 qualitatively mimicked the L-C16 carnitineassociated cell stress outcomes, suggesting that the effects of high experimental concentrations of long-chain acylcarnitines are through membrane disruption. Herein, a model is proposed in which acylcarnitine cell membrane interactions take place along a spectrum of cellular concentrations encountered in physiologicalto-pathophysiological conditions, thus regulating function of membrane-based systems and impacting cell biology. cell death; interleukin-6; skeletal muscle PLASMA ACYLCARNITINES HAVE long been used as surrogate readouts reflecting tissue acyl-CoA pools (6). Thus, these indexes are used in newborn screening as diagnostic biomarkers of inherited disorders of metabolism involving enzymatic lesions in lipid and amino acid metabolism, in which there are increases or decreases in specific acyl-CoA tissue pools (7, 37). For example, long-chain fatty acid oxidation disorders (FAOD) are characterized by defects in mitochondrial oxidative enzymes, i.e., long-chain 3-hydroxyacyl-CoA dehydrogenase and carnitine palmitoyltransferase 2 (CPT2) deficiencies, and these lead to increased tissue, blood, and urine long-chain fatty acylcarnitines. Under certain FAOD conditions, plasma longchain acylcarnitines can increase s...

Acylcarnitines—old actors auditioning for new roles in metabolic physiology

“…78 In the mouse myocyte studies outlined above, the IL-6 inflammatory response was concurrent with a rise in markers of cell death and loss of membrane integrity (measured by adenylate kinase leakage), with a blunting of mitochondrial function, but no apparent apoptosis. Treatment with LCACs at concentrations as low as 5–10 μM increased mouse myotube intracellular Ca 2+ levels, which in turn mediated the LCAC-associated production of IL-6 but not cell death and/or permeability 32 . As discussed in detail below, L-C16-carnitine can bind to, 33 and inhibit the ability of, the lipid-sensitive and Ca 2+ -sensitive conventional PKCs(α, β I , β II and γ)) to phosphorylate substrates.…”

“…By contrast, the remainder of this Review will offer new perspectives on the bioactivity of LCACs by highlighting the growing body of evidence to support the concept that these naturally-occurring lipid derivatives directly influence cell metabolism and health outcomes. As discussed below, LCACs can affect several biological processes, including cardiac function 28 , ion balance 29 , insulin signalling in muscle 30 , inflammation 31 , cellular stress 32 , and outcomes related to modulation of protein kinase C. 33 Gaining insight into the physiological and pathophysiological actions of LCACs will fill a knowledge gap that might in turn improve understanding of the underlying causes and symptoms of FAODs and other metabolic diseases.…”

“…32 The amphipathic nature of LCACs promotes micelle formation, with C16-carnitine having a proposed critical micelle concentration of 75–100 μM, depending on the test system used. 80 Thus, our working model (Figure 2) predicts that under pathological conditions, LCACs alter plasma membrane fluidity and/or function, leading to cell stress, and compromised membrane integrity.…”

“…Mouse myotubes treated with C4-carnitine display blunted insulin-stimulated phosphorylation of Akt, similar to the effects of C14-carnitine and C16-carnitine; 30 however, C4-carnitine does not elicit myocyte permeability and/or death, probably owing to short length of the acyl chain and hence a more limited membrane association than is observed with LCACs. 32 Studies on carnitine O-acetyltransferase , a major producer of acetylcarnitine, have shown the importance of this highly-abundant metabolite as a trafficking agent for carbon atoms and for mitochondrial protein acetylation. 87 …”

Developmental origins of NAFLD: a womb with a clue

“…Owing to increased lipid load and lack of an adaptive increase in mitochondrial oxidation, excess fatty acids in the fetus might not be completely oxidized, creating a backup of incomplete acylcoenzyme A species that can impair cell metabolism via increased cell stress, increased inflammation and decreased mitochondrial function and gene expression 84,85 . In this context, excess free fatty acids combined with reduced mitochondrial metabolism could limit mitochondrial biogenesis.…”