TY - JOUR
T1 - Etomoxir-carnitine, a novel pharmaco-metabolite of etomoxir, inhibits phospholipases A2 and mitochondrial respiration
AU - Ho Moon, Sung
AU - Liu, Xinping
AU - Jenkins, Christopher M.
AU - Dilthey, Beverly Gibson
AU - Patti, Gary J.
AU - Gross, Richard W.
N1 - Publisher Copyright:
© 2024 THE AUTHORS.
PY - 2024/9
Y1 - 2024/9
N2 - Mitochondrial fatty acid oxidation serves as an essential process for cellular survival, differentiation, proliferation, and energy metabolism. Numerous studies have utilized etomoxir (ETO) for the irreversible inhibition of carnitine palmitoylcarnitine transferase 1 (CPT1), which catalyzes the rate-limiting step for mitochondrial long-chain fatty acid β-oxidation to examine the bioenergetic roles of mitochondrial fatty acid metabolism in many tissues in multiple diverse disease states. Herein, we demonstrate that intact mitochondria robustly metabolize ETO to etomoxir-carnitine (ETO-carnitine) prior to nearly complete ETO-mediated inhibition of CPT1. The novel pharmaco-metabolite, ETOcarnitine, was conclusively identified by accurate mass, fragmentation patterns, and isotopic fine structure. On the basis of these data, ETO-carnitine was successfully differentiated from isobaric structures (e.g., 3-hydroxy-C18:0 carnitine and 3-hydroxy- C18:1 carnitine). Mechanistically, generation of ETOcarnitine from mitochondria required exogenous Mg2+, ATP or ADP, CoASH, and L-carnitine, indicating that thioesterification by long-chain acyl-CoA synthetase to form ETO-CoA precedes its conversion to ETO-carnitine by CPT1. CPT1-dependent generation of ETO-carnitine was substantiated by an orthogonal approach using ST1326 (a CPT1 inhibitor), which effectively inhibits mitochondrial ETOcarnitine production. Surprisingly, purified ETOcarnitine potently inhibited calcium-independent PLA2γ and PLA2β as well as mitochondrial respiration independent of CPT1. Robust production and release of ETO-carnitine from HepG2 cells incubated in the presence of ETO was also demonstrated. Collectively, this study identifies the chemical mechanism for the biosynthesis of a novel pharmacometabolite of ETO, ETO-carnitine, that is generated by CPT1 in mitochondria and likely impacts multiple downstream (non-CPT1 related) enzymes and processes in multiple subcellular compartments.
AB - Mitochondrial fatty acid oxidation serves as an essential process for cellular survival, differentiation, proliferation, and energy metabolism. Numerous studies have utilized etomoxir (ETO) for the irreversible inhibition of carnitine palmitoylcarnitine transferase 1 (CPT1), which catalyzes the rate-limiting step for mitochondrial long-chain fatty acid β-oxidation to examine the bioenergetic roles of mitochondrial fatty acid metabolism in many tissues in multiple diverse disease states. Herein, we demonstrate that intact mitochondria robustly metabolize ETO to etomoxir-carnitine (ETO-carnitine) prior to nearly complete ETO-mediated inhibition of CPT1. The novel pharmaco-metabolite, ETOcarnitine, was conclusively identified by accurate mass, fragmentation patterns, and isotopic fine structure. On the basis of these data, ETO-carnitine was successfully differentiated from isobaric structures (e.g., 3-hydroxy-C18:0 carnitine and 3-hydroxy- C18:1 carnitine). Mechanistically, generation of ETOcarnitine from mitochondria required exogenous Mg2+, ATP or ADP, CoASH, and L-carnitine, indicating that thioesterification by long-chain acyl-CoA synthetase to form ETO-CoA precedes its conversion to ETO-carnitine by CPT1. CPT1-dependent generation of ETO-carnitine was substantiated by an orthogonal approach using ST1326 (a CPT1 inhibitor), which effectively inhibits mitochondrial ETOcarnitine production. Surprisingly, purified ETOcarnitine potently inhibited calcium-independent PLA2γ and PLA2β as well as mitochondrial respiration independent of CPT1. Robust production and release of ETO-carnitine from HepG2 cells incubated in the presence of ETO was also demonstrated. Collectively, this study identifies the chemical mechanism for the biosynthesis of a novel pharmacometabolite of ETO, ETO-carnitine, that is generated by CPT1 in mitochondria and likely impacts multiple downstream (non-CPT1 related) enzymes and processes in multiple subcellular compartments.
KW - carnitine palmitoyltransferase (CPT)
KW - etomoxir
KW - etomoxir-carnitine
KW - off-target effects
KW - pharmacometabolite
UR - http://www.scopus.com/inward/record.url?scp=85205403343&partnerID=8YFLogxK
U2 - 10.1016/j.jlr.2024.100611
DO - 10.1016/j.jlr.2024.100611
M3 - Article
C2 - 39094773
AN - SCOPUS:85205403343
SN - 0022-2275
VL - 65
JO - Journal of lipid research
JF - Journal of lipid research
IS - 9
M1 - 100611
ER -