The arachidonic acid preferred long-chain acyl-CoA synthetase 4 (ACSL4) is a key enzyme for fatty acid metabolism in various metabolic tissues. In this study, we utilized hamsters fed a normal chow diet, a high-fat diet or a high cholesterol and high fat diet (HCHFD) as animal models to explore novel transcriptional regulatory mechanisms for ACSL4 expression under hyperlipidemic conditions. Through cloning hamster ACSL4 homolog and tissue profiling ACSL4 mRNA and protein expressions we observed a selective upregulation of ACSL4 in testis and liver of HCHFD fed animals. Examination of transcriptional activators of the ACSL family revealed an increased hepatic expression of PPARδ but not PPARα in HCHFD fed hamsters. To explore a role of PPARδ in dietary cholesterol-mediated upregulation of ACSL4, we administered a PPARδ specific agonist L165041 to normolipidemic and dyslipidemic hamsters. We observed significant increases of hepatic ACSL4 mRNA and protein levels in all L165041-treated hamsters as compared to control animals. The induction of ACSL4 expression by L165041 in liver tissue in vivo was recapitulated in human primary hepatocytes and hepatocytes isolated from hamster and mouse. Moreover, employing the approach of adenovirus-mediated gene knockdown, we showed that depletion of PPARδ in hamster hepatocytes specifically reduced ACSL4 expression. Finally, utilizing HepG2 as a model system, we demonstrate that PPARδ activation leads to increased ACSL4 promoter activity, mRNA and protein expression, and consequently higher arachidonoyl-CoA synthetase activity. Taken together, we have discovered a novel PPARδ-mediated regulatory mechanism for ACSL4 expression in liver tissue and cultured hepatic cells.

BACKGROUND AND AIMS:Globally, NAFLD is one of the most common liver disorders, with an estimated prevalence rate of more than 30% in men and 15% in women and an even higher prevalence in people with type 2 diabetes mellitus. Optimal pharmacologic therapeutic approaches for NAFLD are an urgent necessity. APPROACH AND RESULTS:In this study, we showed that compared with healthy controls, hepatic ACSL4 levels in patients with NAFLD were found to be elevated. Suppression of ACSL4 expression promoted mitochondrial respiration, thereby enhancing the capacity of hepatocytes to mediate β-oxidation of fatty acids and to minimize lipid accumulation by up-regulating peroxisome proliferator-activated receptor coactivator-1 alpha. Moreover, we found that abemaciclib is a potent and selective ACSL4 inhibitor, and low dose of abemaciclib significantly ameliorated most of the NAFLD symptoms in multiple NAFLD mice models. CONCLUSIONS:Therefore, inhibition of ACSL4 is a potential alternative therapeutic approach for NAFLD.

BACKGROUND:Clear cell renal cell carcinoma (ccRCC), the predominant subtype of kidney cancer, possesses characteristic alterations to multiple metabolic pathways, including the accumulation of cytosolic lipid droplets. However, the pathways that drive lipid droplet accumulation in ccRCC cells and their importance to cancer biology remain poorly understood. METHODS:We sought to identify the carbon sources necessary for lipid droplet accumulation using Oil red O staining and isotope-tracing lipidomics. The role of the acyl-CoA synthetase (ACSL) family members, an important group of lipid metabolic enzymes, was investigated using siRNA and drug mediated inhibition. CTB and XTT assays were performed to determine the effect of ACSL3 knockdown and lipid starvation on ccRCC cell viability and shRNA was used to study the effect of ACSL3 in an orthotopic mouse model. The relationship between ferroptosis susceptibility of ccRCC and ACSL3 controlled lipid metabolism was examined using CTB and FACS-based assays. The importance of 5-LOX in ferroptosis susceptibility in ccRCC was shown with XTT survival assays, and the expression level and predictive value of 5-LOX in TCGA ccRCC data was assessed. RESULTS:We found that ccRCC cells obtain the necessary substrates for lipid droplet accumulation by metabolizing exogenous serum derived lipids and not through de novo lipogenesis. We show that this metabolism of exogenous fatty acids into lipid droplets requires the enzyme acyl-CoA synthetase 3 (ACSL3) and not other ACSL family proteins. Importantly, genetic or pharmacologic suppression of ACSL3 is cytotoxic to ccRCC cells in vitro and causes a reduction of tumor weight in an orthotopic mouse model. Conversely, ACSL3 inhibition decreases the susceptibility of ccRCC cells to ferroptosis, a non-apoptotic form of cell death involving lipid peroxidation. The sensitivity of ccRCC to ferroptosis is also highly dependent on the composition of exogenous fatty acids and on 5-lipoxygenase (5-LOX), a leukotriene producing enzyme which produces lipid peroxides that have been implicated in other cancers but not in ccRCC. CONCLUSIONS:ACSL3 regulates the accumulation of lipid droplets in ccRCC and is essential for tumor growth. In addition, ACSL3 also modulates ferroptosis sensitivity in a manner dependent on the composition of exogenous fatty acids. Both functions of ACSL3 could be exploited for ccRCC therapy.

Ferroptosis is a new form of regulated cell death characterized by excessive iron accumulation and uncontrollable lipid peroxidation. The role of ferroptosis in metabolic dysfunction-associated fatty liver disease (MAFLD) is not fully elucidated. In this study we compared the therapeutic effects of ferroptosis inhibitor liproxstatin-1 (LPT1) and iron chelator deferiprone (DFP) in MAFLD mouse models. This model was established in mice by feeding a high-fat diet with 30% fructose in water (HFHF) for 16 weeks. The mice then received LPT1 (10 mg·kg·d, ip) or DFP (100 mg·kg·d, ig) for another 2 weeks. We showed that both LPT1 and DFP treatment blocked the ferroptosis markers ACSL4 and ALOX15 in MAFLD mice. Furthermore, LPT1 treatment significantly reduced the liver levels of triglycerides and cholesterol, lipid peroxidation markers 4-hydroxynonenal (4-HNE) and malondialdehyde (MDA), and ameliorated the expression of lipid synthesis/oxidation genes (Pparα, Scd1, Fasn, Hmgcr and Cpt1a), insulin resistance, mitochondrial ROS content and liver fibrosis. Importantly, LPT1 treatment potently inhibited hepatic apoptosis (Bax/Bcl-xL ratio and TUNEL cell number), pyroptosis (cleavages of Caspase-1 and GSDMD) and necroptosis (phosphorylation of MLKL). Moreover, LPT1 treatment markedly inhibited cleavages of PANoptosis-related caspase-8 and caspase-6 in MAFLD mouse liver. In an in vitro MAFLD model, treatment with LPT1 (100 nM) prevented cultured hepatocyte against cell death induced by pro-PANoptosis molecules (TNF-α, LPS and nigericin) upon lipid stress. On the contrary, DFP treatment only mildly attenuated hepatic inflammation but failed to alleviate lipid deposition, insulin resistance, apoptosis, pyroptosis and necroptosis in MAFLD mice. We conclude that ferroptosis inhibitor LPT1 protects against steatosis and steatohepatitis in MAFLD mice, which may involve regulation of PANoptosis, a coordinated cell death pathway that involves apoptosis, pyroptosis and necroptosis. These results suggest a potential link between ferroptosis and PANoptosis.

Lipid metabolic reprogramming plays a pivotal role in hepatocellular carcinoma (HCC) development, but the underlying mechanisms are incompletely characterized. Long chain acyl CoA synthetase 4 (ACSL4), a member of acyl-CoA synthetases (ACS) family, has been identified as a novel marker of alpha-fetoprotein-high subtype HCC and as an oncogene. Here, we identified a new function of ACSL4 in HCC lipid metabolism. ACSL4 can modulate de novo lipogenesis by accumulating intracellular triglycerides, cholesterols, and lipid droplets in HCC. Mechanistically, ACSL4 upregulates the master lipogenesis regulator sterol regulatory element binding protein 1 (SREBP1) and its downstream lipogenic enzymes in HCC cells via c-Myc. Moreover, SREBP1 is crucial for ACSL4-mediated regulation of lipogenesis as well as HCC cell proliferation and metastasis, as SREBP1 overexpression rescues lipogenic deficiency and decreased oncogenic capabilities associated with ACSL4 suppression in vitro and in vivo. Clinically, our data showed that the expression of ACSL4 was positively correlated with that of SREBP1 in HCC patients, and the combinational biomarkers showed strong predictive value for HCC. Together, our findings uncover a new mechanism by which ACSL4 modulates aberrant lipid metabolism and promotes the progression of HCC.