Nathalie Hennuyer

Atherogenic dyslipidemia and chronic inflammation appear strongly associated with residual cardiovascular disease (CVD) risk. Peroxisome proliferator–activated receptor α (PPARα) agonists, which exert both triglyceride-lowering and anti-inflammatory properties, are potential therapeutic candidates to target residual CVD risk. However, CVD outcome studies with these drugs have yielded mixed results for currently unclear reasons. In this study, we compared the impact of hepatocyte anti-inflammatory (transrepression) versus lipid-lowering (transactivation) properties of PPARα agonism on atherosclerosis in Ldlr ^−/− mice. PPARα agonist treatment reduced aortic atherosclerotic plaque surface in Western diet–fed Ldlr ^−/− mice expressing in hepatocytes either PPARα ^WT or a transrepression-selective PPARα mutant (PPARα ^DISS ), despite the lack of atherogenic dyslipidemia improvement with PPARα ^DISS . Mechanistically, ligand-activated hepatocyte-specific transrepressive PPARα activity decreased the pro-inflammatory phenotype of hepatocytes, lowered the recruitment of leukocytes in the liver, reduced plasma IL-1β concentrations, and decreased atherosclerotic plaque macrophage content. These findings identify a distal effect of hepatic PPARα on vascular integrity rather linked to its anti-inflammatory activity than its lipid-lowering actions and highlight an essential role of targeting inflammation in CVD.

Acyl‐coenzyme A synthetase long‐chain family member 4 (ACSL4), a pivotal enzyme in lipid metabolism, has emerged as a therapeutic target for ferroptosis‐related conditions and cancer. However, its reference inhibitor, rosiglitazone, has off‐target activity on peroxisome proliferator‐activated receptor gamma (PPARγ), a key regulator of lipid homeostasis. Here, the discovery of LIBX‐A401, a potent ACSL4 inhibitor derived from rosiglitazone devoid of PPARγ activity, is reported. Its binding to ACSL4 is ATP‐dependent, stabilizing the C‐terminal domain and altering the fatty acid gate region, as shown by Hydrogen‐Deuterium Exchange Mass Spectrometry. Photoaffinity labeling identified A329 within the fatty acid binding site, while molecular dynamics and mutagenesis highlighted Q302 as critical for LIBX‐A401 binding. LIBX‐A401 exhibits anti‐ferroptotic properties in cells, supported by target engagement. These findings establish LIBX‐A401 as a valuable tool to study ACSL4 in ferroptosis and cancer, while its elucidated binding mode paves the way for the rational design of improved inhibitors.

Acyl‐coenzyme A synthetase long‐chain family member 4 (ACSL4), a pivotal enzyme in lipid metabolism, has emerged as a therapeutic target for ferroptosis‐related conditions and cancer. However, its reference inhibitor, rosiglitazone, has off‐target activity on peroxisome proliferator‐activated receptor gamma (PPARγ), a key regulator of lipid homeostasis. Here, the discovery of LIBX‐A401, a potent ACSL4 inhibitor derived from rosiglitazone devoid of PPARγ activity, is reported. Its binding to ACSL4 is ATP‐dependent, stabilizing the C‐terminal domain and altering the fatty acid gate region, as shown by Hydrogen‐Deuterium Exchange Mass Spectrometry. Photoaffinity labeling identified A329 within the fatty acid binding site, while molecular dynamics and mutagenesis highlighted Q302 as critical for LIBX‐A401 binding. LIBX‐A401 exhibits anti‐ferroptotic properties in cells, supported by target engagement. These findings establish LIBX‐A401 as a valuable tool to study ACSL4 in ferroptosis and cancer, while its elucidated binding mode paves the way for the rational design of improved inhibitors.

Lipid metabolism affects many cellular processes essential for homeostasis, and its disruption is linked to various diseases. A key enzyme in these processes, acyl-coenzyme A synthetase long-chain family member 4 (ACSL4), is a promising target for treating conditions involving ferroptosis and certain cancers. Rosiglitazone (ROSI) is a known ACSL4 inhibitor but its potent activity on peroxisome proliferator-activated receptor gamma (PPARγ), a nuclear receptor strongly involved in lipid metabolism constitutes an important limitation. This study focuses on developing novel ACSL4 inhibitors derived from ROSI, which lack PPARγ activity. Binding of the most potent compound of this series (9) relies on the prior binding of ATP. Hydrogen-Deuterium Exchange Mass Spectrometry (HDx-MS) demonstrated that ATP binding stabilizes the ACSL4 C-terminus, an effect enhanced by compound 9, which also alters important peptide sequences, including the fatty acid gate-domain. Photoaffinity Labeling (PAL) with a diazirine-based probe identified residue A329 in the fatty acid pocket. Molecular dynamics simulations and site-directed mutagenesis highlighted Q302 as critical for compound 9 binding. Thus, compound 9 (LIBX-A401) is a promising tool for studying ACSL4 in ferroptosis-related diseases and cancer, and the elucidation of its binding mode paves the way to the rational design of optimized inhibitors

Dual agonists activating the peroxisome proliferator-activated receptors alpha and gamma (PPARɑ/ɣ) have beneficial effects on glucose and lipid metabolism in patients with type 2 diabetes, but their development was discontinued due to potential adverse effects. Here we report the design and preclinical evaluation of a molecule that covalently links the PPARɑ/ɣ dual-agonist tesaglitazar to a GLP-1 receptor agonist (GLP-1RA) to allow for GLP-1R-dependent cellular delivery of tesaglitazar. GLP-1RA/tesaglitazar does not differ from the pharmacokinetically matched GLP-1RA in GLP-1R signalling, but shows GLP-1R-dependent PPARɣ-retinoic acid receptor heterodimerization and enhanced improvements of body weight, food intake and glucose metabolism relative to the GLP-1RA or tesaglitazar alone in obese male mice. The conjugate fails to affect body weight and glucose metabolism in GLP-1R knockout mice and shows preserved effects in obese mice at subthreshold doses for the GLP-1RA and tesaglitazar. Liquid chromatography–mass spectrometry-based proteomics identified PPAR regulated proteins in the hypothalamus that are acutely upregulated by GLP-1RA/tesaglitazar. Our data show that GLP-1RA/tesaglitazar improves glucose control with superior efficacy to the GLP-1RA or tesaglitazar alone and suggest that this conjugate might hold therapeutic value to acutely treat hyperglycaemia and insulin resistance.

Background: NAFLD affects nearly 25% of the global population. Cardiovascular disease (CVD) is the most common cause of death among patients with NAFLD, in line with highly prevalent dyslipidemia in this population. Increased plasma triglyceride (TG)‐rich lipoprotein (TRL) concentrations, an important risk factor for CVD, are closely linked with hepatic TG content. Therefore, it is of great interest to identify regulatory mechanisms of hepatic TRL production and remnant uptake in the setting of hepatic steatosis. Approach and Results: To identify liver‐regulated pathways linking intrahepatic and plasma TG metabolism, we performed transcriptomic analysis of liver biopsies from two independent cohorts of obese patients. Hepatic encoding apolipoprotein F (APOF) expression showed the fourth‐strongest negatively correlation with hepatic steatosis and the strongest negative correlation with plasma TG levels. The effects of adenoviral‐mediated human ApoF (hApoF) overexpression on plasma and hepatic TG were assessed in C57BL6/J mice. Surprisingly, hApoF overexpression increased both hepatic very low density lipoprotein (VLDL)‐TG secretion and hepatic lipoprotein remnant clearance, associated a ~25% reduction in plasma TG levels. Conversely, reducing endogenous ApoF expression reduced VLDL secretion in vivo, and reduced hepatocyte VLDL uptake by ~15% in vitro. Transcriptomic analysis of APOF‐overexpressing mouse livers revealed a gene signature related to enhanced ApoB‐lipoprotein clearance, including increased expression of Ldlr and Lrp1, among others. Conclusion: These data reveal a previously undescribed role for ApoF in the control of plasma and hepatic lipoprotein metabolism by favoring VLDL‐TG secretion and hepatic lipoprotein remnant particle clearance.