Dear Editor,
We sincerely commend the authors (Ciociola et al.) for their rigorous investigation into the protective mechanisms of the
MARC1 p.A165T variant in metabolic dysfunctionassociated steatotic liver disease (MASLD) pathogenesis [
1]. The elegant integration of primary human hepatocyte studies, multi-omics analyses, and validation in the UK Biobank cohort provides compelling evidence that
MARC1 downregulation enhances β-oxidation while reducing ferroptosis and ROS production. This work significantly advances our understanding of genetic modifiers in hepatic steatosis and offers promising therapeutic directions. Given the clinical urgency of developing effective MASLD therapies, we did make some comments on the article before, but we assure that none of our comments have any malicious or hostile intentions [
2]. Then, we will reply to correspondence on “Downregulation of the MARC1 p.A165 risk allele reduces hepatocyte lipid content by increasing beta-oxidation”. We wish to engage in constructive dialogue regarding several methodological considerations that could further strengthen the translational impact of these findings.
Donor heterogeneity and metabolic context: The reliance on PHHs from single donors per genotype (
MARC1 p.A165 vs. p.T165) warrants careful consideration. MASLD exhibits well-documented phenotypic heterogeneity driven by met-abolic variables including insulin resistance, adiposity, and dyslipidemia [
3]. The protective effect of p.T165 may be modulated by such factors—for instance, Liao et al. [
4] established a NASH mouse model through a methionine choline deficiency (MCD) diet to investigate the effects of Formononetin (FMNT) on fatty acid beta oxidation and NASH. The research results indicated that FMNT intervention affected insulin sensitivity, activated the SIRT1/PGC-1α/PPARα pathway, and promoted fatty acid beta oxidation, regulated liver lipid metabolism, and ultimately improves hepatic steatosis in NASH mice. We suggest future studies might include PHHs from 3-5 donors stratified by metabolic phenotype (e.g., BMI, HOMA-IR) to determine whether the observed lipid-lowering effects are conserved across metabolic subgroups. Such stratification would clarify clinical applicability, particularly for lean MASLD populations who exhibit distinct pathobiology.
Mechanistic paradox in protective allele: The absence of phenotypic changes following
MARC1 knockdown in p.T165 hepatocytes—despite 50% lower baseline protein expression—presents a fascinating mechanistic conundrum, perhaps we may encounter such situations during the actual experimental process. We hypothesize potential compensatory adaptations, such as induction of alternative lipid-catabolic pathways (e.g., autophagy-mediated lipolysis or PPARα-CPT1A axis activation) [
5,
6] that maintain lipid homeostasis when MARC1 is further suppressed. Alternatively, a threshold effect may exist whereby residual MARC1 activity in p.T165 cells is sufficient for metabolic function. Transcriptomic comparison of p.T165 PHHs pre/postknockdown could illuminate such adaptations, potentially revealing novel regulatory networks relevant to MASLD treatment.
Physiological relevance of cell models: While HepG2 cells provide valuable preliminary data, their metabolic reprogramming toward glycolysis and suppressed β-oxidation may limit physiological relevance to non-cancerous hepatocytes. The observed lipid reduction in
MARC1-KO HepG2 cells might reflect cancer-specific survival adaptations rather than bona fide MASLD mechanisms. We suggest that if there are further research works or ideas in the future, supplementary validation can be attempted in metabolic regulation models, such as: (1)
MARC1-edited iPSC-derived hepatocytes from MASLD patients, (2) 3D spheroid cultures preserving hepatic polarization, or (3) primary hepatocytes from
Marc1 KO mice fed steatogenic diets. Cross-referencing results with human adipocyte data [
7] would further clarify tissue-specific functions.
β-oxidation assay refinement: Fatty acid beta-oxidation is the main way the body breaks down fatty acids and plays a key role in energy production [
8]. Fatty acid beta-oxidation is a complex metabolic process: Fatty acids are first activated into acyl CoA in the cytoplasm, and acyl CoA is transported into mitochondria through carnitine, Finally, acyl CoA is dehydrogenated, hydrated, dehydrogenated and thiolysed in mitochondria to generate acetyl CoA (
Fig. 1). When hunger, high-fat and low sugar diet or diabetes occur, when the utilization of sugar in the body is impaired, fatty acid oxidation is enhanced. Fatty acids in the liver undergo β-oxidation to produce acetyl CoA, and two molecules of acetyl CoA can condense to form acetoacetic acid. Acetoacetic acid can be decarboxylated to produce acetone, and can also be reduced to produce β-hydroxybutyric acid. Acetoacetic acid, β-hydroxybutyric acid, and acetone are collectively referred to as ketone bodies. The liver cannot utilize ketone bodies and must be transported through the bloodstream to extrahepatic tissues, especially muscles and kidneys, where it is converted into acetyl CoA and oxidized for utilization. The ³H palmitate assay elegantly demonstrates increased acid-soluble metabolites (ASMs), but cannot distinguish between complete oxidation (yielding CO
2) and incomplete oxidation generating ketone bodies or acylcarnitines. Given that mitochondrial efficiency determines metabolic outcomes—incomplete oxidation may increase ROS despite lipid reduction— some researchers [
9] prefered to use
14C palmitate→
14CO
2 tracers to quantify complete respiratory flux. Additionally, mass spectrometry-based acylcarnitine profiling (e.g., C16:0-carnitine accumulation) could identify potential β-oxidation bottlenecks masked in current analyses [
10].
ROS reduction mechanism/ferroptosis and therapeutic translation: The observed decrease in ROS despite elevated β-oxidation is particularly intriguing, as increased elec-tron flux through ETC complexes typically heightens radical generation [
11]. We speculate three non-exclusive mechanisms worthy of exploration: (1) Enhanced mitochondrial uncoupling (e.g., UCP2 upregulation), (2) Induction of antioxidant enzymes (SOD2, GPX4) via NRF2 activation, or (3) Improved ETC complex assembly reducing electron leakage. Reanalysis of proteomic data for antioxidant pathways or functional assays measuring mitochondrial membrane potential could resolve this paradox and strengthen the proposed antioxidant phenotype.
Ferroptosis is a distinct form of regulated cell death characterized by substantial iron accumulation and significant lipid peroxidation. This process plays a critical role in diverse disease pathologies associated with cellular demise. Given the liver’s central role in iron and lipid metabolism alongside its vulnerability to oxidative damage, ferroptosis may contribute to various stages of MASLD progression through multiple pathways and distinct mechanisms [
12]. The upregulation of ferroptosis suppressors (e.g., GPX4, SLC7A11) after
MARC1 knockdown aligns with recent evidence implicating ferroptosis in MASLD progression. To assess functional significance, ferroptosis challenge assays using erastin or RSL3 in knockdown PHHs could quantify cytoprotection. Given the authors’ therapeutic focus, we are further concerned about whether
MARC1 inhibition shows efficacy in advanced MASLD models with established fibrosis, where ferroptosis drivers like ACSL4 are upregulated. Such data would inform clinical development stages for MARC1-targeted agents.
In a word, these considerations are offered in the spirit of collaborative scientific refinement. China has the highest number of hepatitis patients or carriers, and also has the highest number of MASLD. The number of liver disease patients in China has exceeded 400 million, including 60 million patients with alcoholic liver disease and 200 million patients with MASLD. The prevalence rate of MASLD among people over 40 years old in China will be as high as 40.3%, and MASLD may become the main pathogenic factor of hepatocellular carcinoma, we have gained more experience by studying the guidelines for MASLD from other countries [
13,
14]. The foundational work by Ciociola et al. has unequivocally established
MARC1 modulation as a promising therapeutic strategy. Addressing these nuances will not only resolve current paradoxes, but also accelerate clinical translational therapy by identifying patient subgroups most likely to benefit from MARC1-targeted therapies. We eagerly anticipate future studies from this group that will further illuminate the therapeutic landscape for MASLD.
Finally, I would like to express my gratitude once again to the authors (Ciociola et al.) for sharing their highly valuable research articles on MASLD.
FOOTNOTES
-
Authors’ contribution
XJ and WG, ZW collected the literature, XJ wrote the manuscript. XJ, ZW, WG and LJD finally approved the publication. All authors read and approved the final manuscript.
-
Acknowledgements
Supported by Sichuan Natural Science Foundation (2024NSFSC1933, 2024NSFSC1896), Affiliated Hospital of North Sichuan Medical College, High-level Talent Research Grant/Doctoral Research Initiation Grant (2024GC017); Research and development program of Affiliated Hospital of North Sichuan Medical College (2023ZD005, 2023PTZK017, 2023LC001, 2023LC007); Central Guidance for Local Science and Technology Projects (2025ZYD0168); Key Scientific Research Project of Affiliated Hospital of North Sichuan Medical College (2025ZD003).
-
Conflicts of Interest
The authors declare no conflict of interest.
Figure 1.The process of fatty acid beta oxidation Fatty acids need to be activated in the cytosol firstly, under the catalysis of acyl CoA synthase, it is activated into acyl-CoA in the presence of ATP, CoA SH and Mg2+, and acyl-CoA can enter the mitochondrial matrix through carnitine transport. At the same time, it needs the assistance of lipoyl transferases I and II. The entry of acyl-CoA into mitochondria is the rate limiting step of fatty acid oxidation. Finally, the oxidation process of acyl-CoA: dehydrogenation, hydration, redehydrogenation and thiolysis. The acetyl CoA generated by β-oxidation of fatty acids enters the tricarboxylic acid cycle and is fully oxidized to water and CO2. At the same time, it can also be transformed into other metabolic intermediates (such as ketone bodies).
Abbreviations
metabolic dysfunction-associated steatotic liver disease
methionine choline deficiency
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