Correspondence to editorial on “Downregulation of the MARC1 p.A165 risk allele reduces hepatocyte lipid content by increasing beta-oxidation”

Dear Editor,

We appreciate the editorial attention [1] given to our recent study on the role of MARC1 in metabolic dysfunction-associated steatotic liver disease (MASLD) [2]. However, we find several points in the editorial to be flawed and based on misconceptions of our experimental design, data interpretation, and the broader context of liver physiology.

The editorial suggests that our study fails to establish a direct mechanistic link between MARC1 downregulation and increased β‑oxidation [1]. We respectfully disagree. Our data clearly demonstrates that MARC1 downregulation in primary human hepatocytes (PHHs) homozygous for the p.A165 allele results in higher β‑oxidation activity, as measured by radiolabeled [³H]palmitate oxidation assays [3]. These findings are further supported by transcriptomic and proteomic analyses showing upregulation of genes and pathways involved in fatty acid metabolism and oxidative phosphorylation.

The editorial appears to overlook the robustness of these complementary datasets. By employing RNA-seq and LCMS proteomics, we demonstrated consistent upregulation of key metabolic pathways. Importantly, our findings are supported by human genetics data from 239,075 UK Biobank participants, where carriers of the MARC1 p.T165 variant (a protective allele) exhibited higher plasma 3-hydroxybutyrate levels, a recognized proxy for higher hepatic β‑oxidation (see Fig. 2 of the original paper [2]).

Thus, rather than being speculative, our mechanistic model is grounded in multi-layered evidence that integrates in vitro and population-level data.

The editorial questions whether the observed in vitro effects are translatable to human physiology and criticizes the use of radiolabeled [³H]palmitate for not showing full oxidation to CO₂. We view this critique as conceptually flawed and the proposed experiment unnecessary.

The liver’s physiological role is not primarily to fully oxidize fatty acids for its own energy demands as opposed to muscle, for example. Instead, the liver performs partial β‑oxidation, converting fatty acids into acetyl-CoA and subsequently into exportable ketone bodies (e.g., 3‑hydroxybutyrate) during fasting or carbohydrate restriction. This metabolic partitioning is well established in humans and represents a core function of hepatic energy metabolism [4]. Indeed, detection of elevated 3‑hydroxybutyrate levels in UK Biobank carriers of the protective MARC1 allele provides in vivo evidence of this process.

Moreover, whether fatty acids are fully oxidized to CO₂ in hepatocytes or only partially oxidized to generate ketone bodies does not diminish the fact that β‑oxidation is occurring. The use of ¹⁴C-labeled palmitate to measure CO₂ release would not add mechanistic insight into our study, as our goal was to assess fatty acid catabolism in hepatocyte rather than ATP production per se. Thus, a demonstration of “full oxidation” is redundant and not aligned with liver physiology.

It is incorrect to claim that our findings are based on a single donor. Our primary results were obtained using PHHs from a donor homozygous for the p.A165 allele (see Fig. 1 of the original paper [2]). Then, we validated these findings in four additional hepatocyte-derived cell lines, namely HepG2, HuH7, Hep3B2, and HepaRG (see Supplementary Fig. 1 of the original paper [2]), which originate from four different donors each homozygous for the MARC1 risk allele. Results were then further validated in PHHs from another human donor homozygous for the MARC1 p.A165 risk allele (see Supplementary Fig. 2 of the original paper [2]). Across these diverse cellular backgrounds, MARC1 downregulation consistently reduced intracellular lipid content providing strong support for the generalizability of our observations.

The point regarding the lack of reported clinical characteristics of the donor used is fair given the recent observation that the MARC1 p.A165 allele interacts with BMI in determining liver damage [5]. However, the BMI of the PHHs donors were described in Supplementary Table 1 of the original paper [2]. Additionally, the experiments were conducted in vitro and not ex vivo with several days of culturing in a dish with standard condition and we believe this dilutes the effect of specific features of the donor.

Regarding the interpretation of the results on cells carrying the protective MARC1 allele, we have shown that the amino acid substitution causes an increased proteosomal degradation of the protein with a ~50% reduction in protein levels [6]. However, the results in our current paper suggests that the p.T165 mutant protein is non-functional.

Regarding ROS production due to higher beta-oxidation, we have already discussed this point in the paper. Please refer to the discussion section [2,7,8].

Overall, we believe our study provides compelling evidence that downregulation of the MARC1 p.A165 risk allele enhances β‑oxidation, reduces intracellular lipid accumulation, and attenuates oxidative stress in PHHs.

These findings, in turn, advance the understanding of MASLD genetics and lay the groundwork for novel interventions in this globally prevalent disease.

Abbreviations

MASLD

metabolic dysfunction-associated steatotic liver disease

PHH

primary human hepatocytes