The ferroptosis mediator ACSL4 fails to prevent disease progression in mouse models of MASLD

Abstract

Background: Metabolic dysfunction-associated steatotic liver disease (MASLD) is an increasingly prevalent condition and a major risk factor for chronic liver damage, potentially leading to steatohepatitis and HCC. It is already known that patients with MASLD show increased systemic and hepatic iron concentrations as well as perturbed lipid metabolism, suggesting the involvement of ferroptosis in the development and progression of MASLD. Consequently, inhibition of ferroptosis represents a potential therapeutic option for patients with MASLD.

Methods: We investigated whether liver parenchymal cell-specific deletion (LPC-KO) of the pro-ferroptotic gene acyl-CoA synthetase long-chain family member 4 (ACSL4LPC-KO) reduces MASLD onset and progression in mice. ACSL4LPC-KO and wild-type littermates were fed a choline-deficient high-fat diet (CD-HFD) or a Western diet for 20 weeks (CD-HFD and Western diet) or 40 weeks (CD-HFD only) to monitor MASLD progression and metabolic syndrome development.

Results: In contrast to the recently published studies by Duan et al, our results show no significant differences between ACSL4LPC-KO and wild-type mice with regard to the development of MASLD or the progression of metabolic syndrome. Furthermore, no differences were observed in metabolic parameters (ie, weight gain, glucose tolerance test, hepatic steatosis) or MASLD-associated inflammatory response.

Conclusions: Our analyses, therefore, suggest that loss of ACSL4 has no effect on the progression of MASLD induced by CD-HFD or the Western diet. The discrepancy between our and previously published results could be due to differences in the diets or the influence of a distinct microbiome, so the results obtained with hepatocyte-specific ACSL4LPC-KO should be taken with caution.

Keywords: MAFLD; MASH; NASH; cell death; metabolic syndrome.

FIGURE 1

ACSL4^LPC-KO has no impact on weight gain and glucose tolerance under CD-HFD. (A) ACSL4 protein levels in the livers of ACSL4^LPC-KO and loxP-flanked control mice fed normal chow were assessed by Western blot. (B) ACSL4^LPC-KO resulted in distinct alterations in the lipid profile of whole liver tissue in LPCh and LPE species, as measured through LC/MS. (C, D) ACSL4^LPC-KO showed similar weight gain compared to WT under CD-HFD over the indicated time period, both in terms of relative weight gain (C) and absolute weight (D). (E) Feed intake was lower in CD-HFD than in SD, but did not differ between ACSL4^LPC-KO and WT. (F, G) CD-HFD led to impaired glucose tolerance compared to SD measured by glucose tolerance tests. In 20-week-old (F) and 40-week-old mice (G) fed with indicated diets, no difference between ACSL4^LPC-KO and WT was observed. Mann-Whitney U test, * for p ≤ 0.05. Abbreviations: ACSL4^LPC-KO, liver parenchymal cell–specific deletion of pro-ferroptotic gene acyl-CoA synthetase long-chain family member 4; CD-HFD, choline-deficient high-fat diet; LC-MS, liquid chromatography-mass spectrometry; LPCh, lysophosphatidylcholine; LPE, lysophosphatidylethanolamine; SD, standard diet; WT, wild-type.

FIGURE 2

ACSL4^LPC-KO does not protect against liver injury or the development of MASLD, despite providing protection against lipid peroxidation. (A) ACSL4^LPC-KO mice and WT mice both develop steatosis hepatitis after 20 and 40 weeks of feeding. (b) No difference between ACSL4^LPC-KO and WT in the development of steatohepatitis was observed in the NAS score on histology in 20-week-old and 40-week-old mice. (C) As indicated by 4-HNE staining, pronounced lipid peroxidation was detected in WT mice compared to ACSL4^LPC-KO mice when fed a CD-HFD for 40 weeks and a WD for 20 weeks. The liver of tamoxifen-induced hepatocyte-specific GPX4 KO (Alb-creER^T2;Gpx4^fl/fl) mice was used as a positive control 5 days after tamoxifen injection (GPX4^Δhep). (D, E) After 20 weeks (D) and 40 weeks (E), the CD-HFD led to an increase in serum values showing liver damage (AST, ALT, GLDH, and LDH) and aberrant parameters of lipid metabolism (cholesterol) as indicated. No difference could be detected between ACSL4^LPC-KO and WT mice. Mann-Whitney U test, ** for p ≤0.01. Abbreviations: ACSL4^LPC-KO, liver parenchymal cell–specific deletion of pro-ferroptotic gene acyl-CoA synthetase long-chain family member 4; CD-HFD, choline-deficient high-fat diet; MASLD, metabolic dysfunction–associated steatotic liver disease; WD, Western diet; WT, wild-type.

FIGURE 3

qPCR-based and immunohistochemical examinations showed no differences between ACSL4^LPC-KO and WT in terms of tissue inflammation and fibrogenesis. (A–C) Immunohistochemical staining did not show any significant differences of CD3⁺ T cells (A), B220⁺ B cells (B), and F4/80⁺ cells (C) between the liver of ACSL4^LPC-KO and WT mice fed with a CD-HFD for 40 weeks. (D) qPCR-based studies indicated no differences in inflammatory parameters (cytokines, distinct receptors, and alarmins) between ACSL4^LPC-KO and WT mice fed with the CD-HFD for 40 weeks. (E) Analysis of distinct fibrogenesis and fat metabolism–associated genes in whole liver tissue after 40 weeks of CD-HFD showed no difference between WT and ACSL4^LPC-KO. Abbreviations: ACSL4^LPC-KO, liver parenchymal cell–specific deletion of pro-ferroptotic gene acyl-CoA synthetase long-chain family member 4; CD-HFD, choline-deficient high-fat diet; WT, wild-type.

FIGURE 4

ACSL4^LPC-KO does not influence weight gain under the Western diet. (A, B) ACSL4^LPC-KO showed similar weight gain compared to WT under CD-HFD over the indicated time period, both in terms of relative weight gain (A) and absolute weight (B). Abbreviations: ACSL4^LPC-KO, liver parenchymal cell–specific deletion of pro-ferroptotic gene acyl-CoA synthetase long-chain family member 4; CD-HFD, choline-deficient high-fat diet; WT, wild-type.