IDDF2023-ABS-0084 Ambient fine particulate matter (PM2.5) exposure triggered liver metabolic reprogramming via cysteine oxidation on malate dehydrogenase 2 (MDH2) and carnitine palmitoyltransferase ii (CPT2) in mice

Background:
Air pollution, particularly PM2.5, remains the top environmental death cause worldwide. Epidemiological and experimental evidence demonstrated an association between PM2.5 exposure and fatty liver diseases featured by reactive oxidative species (ROS) mediated metabolic reprogramming, while exact targets remained vague. By profiling PM2.5-oxidized cysteine residues, we aim to identify oxidation events that contributed to liver metabolic reprogramming in an ambient PM2.5 exposed mouse model.

Methods:
Adult male mice (6 per group) were exposed to filtered clean air or ambient air with high PM2.5 in Taiyuan, Shanxi during the coal-burning season from November 2018 to May 2019 (IDDF2023-ABS-0084 Figure 1). Redox proteomics analysis was conducted for isolated liver mitochondria to obtain relative oxidation abundance and occupancy of each identified cysteine residue (IDDF2023-ABS-0084 Figure 2A). Molecular dynamics (MD) simulation was conducted by AutoDock Vina.

Results:
A total of 2805 unique Cysteine-containing peptides from 704 proteins were identified and quantified. Multiple cysteine sites on mitochondria enzymes regulating energy metabolism were found significantly oxidized (IDDF2023-ABS-0084 Figure 2B-D). In silica screening by MD revealed key enzymes with reduced substrate/cofactor affinities including GPD2, CPT2, MDH2, GOT2, and ACADL (IDDF2023-ABS-0084 Figure 3), which modulate mitochondria shuttles or beta-oxidation. Targeted metabolomics and activity assay using liver tissues from exposed mice confirmed that reduced MDH activity caused malfunction of the malate shuttle, the primary mitochondria shuttle used in the liver for NADH/NAD transportation and rebalancing (IDDF2023-ABS-0084 Figure 4A). Consequently, ROS stress increased via NAD/NADH ratio and glycolysis was repressed (IDDF2023-ABS-0084 Figure 4B-C). Reduced CPT2 activity was confirmed by accumulated beta-oxidation fatty acids C14-C18 (IDDF2023-ABS-0084 Figure 4C). A metabolic reprogramming trend lipogenesis was therefore identified and confirmed by elevated TG abundance (IDDF2023-ABS-0084 Figure 4C). The PM2.5-induced metabolic reprogramming was further confirmed in PM2.5-exposed human liver HepG2 cells using the seahorse fuel flex test (IDDF2023-ABS-0084 Figure 5A-B).

Conclusions:
Redox proteomics and MD simulation revealed PM2.5 modulated mice liver MDH2 and CPT2 via cysteine oxidation. In vivo and in vitro functional validation confirmed that malate shuttle and beta-oxidation were repressed upon PM2.5 exposure, leading to metabolic reprogramming that favours lipogenesis. Our study, therefore, provides a potential mechanism for PM2.5 mediated liver metabolic reprogramming (IDDF2023-ABS-0084 Figure 5C).