Hepatic synthesis of triacylglycerols containing medium-chain fatty acids is dominated by diacylglycerol acyltransferase 1 and efficiently inhibited by etomoxir

Abstract

Objective Medium-chain fatty acids (MCFAs) play an increasing role in human nutrition. In the liver, one fraction is used for synthesis of MCFA-containing triacylglycerol (MCFA-TG), and the rest is used for oxidative energy production or ketogenesis. We investigated which enzymes catalyse the synthesis of MCFA-TG and how inhibition of MCFA-TG synthesis or fatty acid (FA) oxidation influences the metabolic fate of the MCFAs. Methods FA metabolism was followed by time-resolved tracing of alkyne-labelled FAs in freshly isolated mouse hepatocytes. Quantitative data were obtained by mass spectrometry of several hundred labelled lipid species. Wild-type hepatocytes and cells from diacylglycerol acyltransferase (DGAT)1 −/− mice were treated with inhibitors against DGAT1, DGAT2, or FA β-oxidation. Results Inhibition or deletion of DGAT1 resulted in a reduction of MCFA-TG synthesis by 70%, while long-chain (LC)FA-TG synthesis was reduced by 20%. In contrast, DGAT2 inhibition increased MCFA-TG formation by 50%, while LCFA-TG synthesis was reduced by 5–25%. Inhibition of β-oxidation by the specific inhibitor teglicar strongly increased MCFA-TG synthesis. In contrast, the widely used β-oxidation inhibitor etomoxir blocked MCFA-TG synthesis, phenocopying DGAT1 inhibition. Conclusions DGAT1 is the major enzyme for hepatic MCFA-TG synthesis. Its loss can only partially be compensated by DGAT2. Specific inhibition of β-oxidation leads to a compensatory increase in MCFA-TG synthesis, whereas etomoxir blocks both β-oxidation and MCFA-TG synthesis, indicating a strong off-target effect on DGAT1.

Development of oxaalkyne and alkyne fatty acids as novel tracers to study fatty acid beta-oxidation pathways and intermediates

“…Noteworthy, a coincubation with teglicar did not profoundly influence the pool of secreted labeled lipids but resulted in increased lipid biosynthesis especially of TG confirming earlier findings ( 25 ). These results illustrate a metabolic rerouting of the tracers.…”

“…The isolation of primary hepatocytes by collagenase perfusion followed the literature ( 25 , 30 ) with minor adaptations (permission LANUV NRW, 84–02.04.2015.A381). Eight-week-old male C57BL/6N mice were injected with heparin-sodium (430 U in 100 μl, Ratiopharm) and anaesthetized with a ketamin/xylazin mixture.…”

“…The procedure followed the literature ( 25 ) with minor adaptations. Cellular lipids were extracted by sonication of multi-well plates in MeOH/CHCl 3 5/1 containing internal standards (Cer 18:0;Y;O2/15:1(11Z)[(12,13,14,15)D8]; DG 17:0;Y_15:1(11Z)[(12,13,14,15)D8]; PA 17:0;Y/15:1(11Z)[(12,13,14,15)D8]; PC 17:0;Y/15:1(11Z)[(12,13,14,15)D8]; PE 17:0;Y/15:1(11Z)[(12,13,14,15)D8]; PI 17:0;Y/15:1(11Z)[(12,13,14,15)D8]; PS 17:0;Y/15:1(11Z)[(12,13,14,15)D8]; SE[(26,27D6)] 17:0;Y; TG 16:0_17:0;Y_15:1(11Z)[(12,13,14,15)D8] all ref.…”

“…Clickable lipid tracers were successfully used to monitor protein lipidation, protein-lipid interaction, lipid localization, and metabolism, and this work has been extensively reviewed ( 16 , 17 , 18 , 19 , 20 ). Importantly, alkyne lipids are amenable to various highly sensitive detection procedures including TLC and MS-based lipidomics ( 11 , 21 , 22 , 23 , 24 , 25 , 26 , 27 ). The versatility of the alkyne tag in combination with advanced click reporters, detection technologies, and instrumentation has opened new possibilities in lipid research.…”

“…The versatility of the alkyne tag in combination with advanced click reporters, detection technologies, and instrumentation has opened new possibilities in lipid research. However, the particular developments in alkyne FA tracing have thus far focused on anabolic pathways ( 11 , 22 , 23 , 24 , 25 ). For analysis of catabolism, the potential of alkyne FAs and the new methodologies has not been explored.…”