Lipidomic analysis of human primary hepatocytes following LXR activation with GW3965 identifies AGXT2L1 as a main target associated to changes in phosphatidylethanolamine

resumo

Liver X receptor (LXR) agonists have the potential to alleviate obesity related diseases, particularly atherosclerosis. However, LXRs are transcriptional regulators that induce de novo lipogenesis and lipid accumulation in hepatocytes which represents a serious adverse effect. In this work, we sought to characterize the LXR agonist GW3965 effects on fatty acid (FA) and phospholipid (PL) remodelling and the correlation with gene expression in order to better understand the underlying effects leading to hepatic pathology upon LXR activation. Human primary hepatocytes treated for 48 h with GW3965 were analysed for changes in lipid metabolism gene expression by qPCR, variations in the FA profile was evaluated by GC-FID and in PL profiles using thin layer chromatography, ESI-MS and MS/MS analysis. Changes in cell membrane biochemical properties were studied using bilayer models generated with CHARMM-GUI. ELOLV6 and SCD1 mRNA increase was consistent with higher C16:1 and C18:1n9 at the expense of C16:0 and C18:0. The reduction of C18:2n6 and increase in C20:2n6 was in agreement with ELOVL5 upregulation. Phosphatydilethanolamine (PE) levels tended to decrease and phosphatidylinositol to increase; although differences did not reach significance, they correlated with changes in AGXT2L1, CDS1 and LPIN1 mRNA levels that were increased. The overall effect of GW3965 on PEs molecular profiles was an increase of long-chain polyunsaturated FA chains and a decrease of C16/C18 saturated and monounsaturated FAs chains. Additionally, PC (32:1) and PC (34:2) were decreased, and PC (36:1) and PC (34:1) were increased. AGXT2L1 is an enzyme with strict substrate specificity for phosphoethanolamine, which is converted into ammonia in GW3965-treated hepatocytes and could explain the PE reduction. In summary, LXR activation by GW3965 targets PE biosynthesis and FA elongation/desaturation, which tends to decrease PE in relation to total PL levels, and remodelling of PC and PE molecular species. We identified the human AGXT2L1 gene as induced by LXR activation by both synthetic and endogenous agonist treatment. The increase in acetaldehyde-induced oxidative stress, and in the lipid species identified have the potential to enhance the inflammatory process and impair membrane function. Future studies should focus on inhibition of AGXT2L1 activity with the aim of reverting the steatosis induced by LXR activation.

palavras-chave

MOLECULAR-DYNAMICS SIMULATIONS; ADDITIVE FORCE-FIELD; LIVER; ACID; RECEPTORS; MEMBRANE; GENE; PHOSPHATIDYLCHOLINE; METHYLATION; PROTEINS

categoria

Biochemistry & Molecular Biology; Endocrinology & Metabolism

autores

Santinha, D; Klopot, A; Marques, I; Ellis, E; Jorns, C; Johansson, H; Melo, T; Antonson, P; Jakobsson, T; Felix, V; Gustafsson, JA; Domingues, MR; Mode, A; Helguero, LA

Grupos

G6 - Materiais Virtuais e Inteligência Artificial

Projectos

Anion transmembrane transport promoted by drug-like molecules: building a library of anion carriers inspired in Ataluren (PTC124) (PTDC/QEQ-SUP/4283/2014)

CICECO - Aveiro Institute of Materials (UID/CTM/50011/2019)

agradecimentos

This work was supported by the Swedish Science Council, the Emerging Technology Fund of Texas under Agreement 300-9-1958, the Robert A Welch Foundation Grant E-0004, Karolinska Institutet funds; The Portuguese Foundation for Science and Technology (FCT) through programmes supported by European Union, QREN, FEDER and COMPETE (PEst-C/QUI/UI0062/2013, PEst-C/QUI/UI0062/2011). Lipidomics work was supported by QOPNA-LAQV (UID/QUI/00062/2019) and Portuguese Mass Spectrometry Network (LISBOA-01-0145-FEDER-402-022125), University of Aveiro, funded by FCT/MCTES through national funds (PIDDAC), and the co-funding by the FEDER, within the PT2020 Partnership Agreement and Compete 2020. The molecular modelling was supported by projects PTDC/QEQ-SUP/4283/2014 and CICECO - Aveiro Institute of Materials (FCT UID/CTM/50011/2019), financed by National Funds through the FCT/MEC and, when applicable, co-financed by FEDER through COMPETE, under the PT2020 Partnership Agreement. I. M. is grateful for a post-doc grant (BPD/UI98/6065/2018) under the Integrated Programme of SR&TD pAGE - Protein aggregation Across the Lifespan (reference CENTRO-01-0145-FEDER-000003), co-funded by the Centro 2020 programme, Portugal 2020, European Union, through the European Regional Development Fund. LAH research is supported by FCT project UID/BIM/04501/2019 granted to Institute for Biomedicine.