Photo-downregulation of SIRT4 mitigates aging in mice by enhancing H3K9ac via fatty acid metabolism

Reviewer #1 (Public review):

Summary:

Deng and colleagues pursue the possibility that red light exposure can provide some benefits and anti-senescence effects in aged mouse models. In addition, they show how red light influences metabolism in cultured keratinocytes. The authors provide a long dissection of the potential paths involved in the changes promoted by red light exposure, identifying CytC oxidase, SIRT4, PPARa and MCD as key players.

Strengths:

The authors did a thorough exploration of the multiple potential avenues by which red light exposure influences metabolism. The in vitro and in vivo evidence nicely complement each other.

Weaknesses:

This is a challenging hypothesis that would require some additional experimental controls. The pathway dissection, while extensive, is sometimes approached in unconvincing ways, and the results are not always evident to judge or interpret. Technically, the western blots and transcriptomic analyses require notable improvements.

Reviewer #2 (Public review):

Summary:

This work identifies a previously unknown way that red light can slow ageing. The authors show that red light lowers the level of a protein called SIRT4 in skin cells. Reducing SIRT4 boosts fatty acid use and increases a type of histone modification that keeps genes active. These changes help cells clear away signs of ageing, reduce inflammation, and restore normal metabolism. The findings open the possibility of developing new treatments that target SIRT4 to reverse age‑related decline.

Strengths:

The evidence is solid because the authors use several complementary methods. They test red light in both cultured cells and naturally aged mice, and they confirm the key role of SIRT4 by silencing its gene. Measurements of metabolism, protein changes, and ageing markers all point in the same direction. However, the exact way red light lowers SIRT4 levels is not fully explained, which leaves a minor gap. Overall, the conclusions are well supported and convincing.

Weaknesses:

The paper does not evolve to use the mechanistic discoveries of the manuscript to help our community to identify the mechanism of photobiomodulation, which is not known so far.

I would like to draw attention to a recently published paper by Herrera et al. (FEBS Letters 2025, doi:10.1002/1873-3468.70195), which shows that red light (660 nm) stimulates mitochondrial fatty acid oxidation in keratinocytes via AMPK‑dependent phosphorylation of ACC, without altering expression of electron transport chain complexes. I believe this paper is highly complementary to the current study.

Herrera et al. demonstrate that red light increases basal, ATP‑linked, and maximal oxygen consumption rates in keratinocytes specifically through enhanced fatty acid oxidation (inhibited by etomoxir). This independently validates the central finding of the current manuscript, i.e., red light boosts lipid metabolism, strengthening the robustness of this concept.

While the current manuscript focuses on the SIRT4‑MCD axis, Herrera et al. identify AMPK phosphorylation and ACC inhibition as key effectors. The authors can integrate and expand their discussion, since SIRT4 downregulation may converge on AMPK activation, or they may represent parallel, reinforcing mechanisms. This would enrich the mechanistic model and open new hypotheses.

The mechanism of photobiomodulation: Herrera et al. explicitly challenge the prevailing paradigm that red light acts solely via cytochrome c oxidase (by showing long‑lasting effects, unchanged OXPHOS protein levels, and no difference in permeabilised cells). The current finding (red light acts through SIRT4 downregulation, i.e., not direct enzymatic activation) aligns perfectly with Herrera´s critique.

Long‑term metabolic effects - Herrera et al. show that a single red light exposure elevates oxygen consumption for up to 2 days. The current study focuses on changes at 12‑24 h. Their data extend the time window and suggest that the metabolic reprogramming you describe may persist longer than currently discussed, which is clinically relevant.

Discussing Herrera et al.'s results would not only acknowledge independent, corroborating evidence but would also allow the authors to position their SIRT4‑centric mechanism within a broader, emerging understanding of red‑light photobiomodulation.

Author response:

Reviewer #1 (Public review):

Weaknesses:

This is a challenging hypothesis that would require some additional experimental controls. The pathway dissection, while extensive, is sometimes approached in unconvincing ways, and the results are not always evident to judge or interpret. Technically, the western blots and transcriptomic analyses require notable improvements.

We would like to thank the reviewer for the careful and patient examination of the issues identified in our manuscript. The poor quality of some of the Western blot bands in Figure 4 may have been caused by inappropriate electrophoresis conditions during the Western blot experiments. In the revised manuscript, we will optimize the electrophoresis conditions to obtain higher-quality protein bands and update the quantitative data. Regarding the quantification format, we believe that heatmaps provide a more intuitive representation of trends in protein expression across different treatment groups. This approach more accurately reflects the results of our biological replicates than simply analyzing the significance of differences in the grayscale values of protein bands. For the analysis of transcriptomic data, we will conduct a more detailed analysis of signal pathway enrichment and the identified differentially expressed genes to ensure that predicted genes are excluded from our current results and redundant data presentation is removed.

Regarding additional experimental controls, such as incorporating experimental data under blue light treatment conditions as a control for red light. While exploring the optimal red light irradiation dose at the cellular level, we simultaneously conducted experiments on the effects of blue light irradiation at the same dose on keratinocyte activity. The results indicated that as the blue light irradiation dose increased (0–160 J/cm²), the keratinocyte activity exhibited a dose-dependent decline. This indicates that blue light is phototoxic to keratinocytes. The relevant experimental results have already been published in our previous study (Communications Biology 2024, doi: 10.1038/s42003-024-06973-1). Taken together with the data from our study, this demonstrates that the anti-aging effects of red light reported in the current manuscript are indeed driven by red light.

Reviewer #2 (Public review):

Weaknesses:

The paper does not evolve to use the mechanistic discoveries of the manuscript to help our community to identify the mechanism of photobiomodulation, which is not known so far.

I would like to draw attention to a recently published paper by Herrera et al. (FEBS Letters 2025, doi:10.1002/1873-3468.70195), which shows that red light (660 nm) stimulates mitochondrial fatty acid oxidation in keratinocytes via AMPK‑dependent phosphorylation of ACC, without altering expression of electron transport chain complexes. I believe this paper is highly complementary to the current study.

Herrera et al. demonstrate that red light increases basal, ATP-linked, and maximal oxygen consumption rates in keratinocytes specifically through enhanced fatty acid oxidation (inhibited by etomoxir). This independently validates the central finding of the current manuscript, i.e., red light boosts lipid metabolism, strengthening the robustness of this concept.

While the current manuscript focuses on the SIRT4-MCD axis, Herrera et al. identify AMPK phosphorylation and ACC inhibition as key effectors. The authors can integrate and expand their discussion, since SIRT4 downregulation may converge on AMPK activation, or they may represent parallel, reinforcing mechanisms. This would enrich the mechanistic model and open new hypotheses.

The mechanism of photobiomodulation: Herrera et al. explicitly challenge the prevailing paradigm that red light acts solely via cytochrome c oxidase (by showing long-lasting effects, unchanged OXPHOS protein levels, and no difference in permeabilised cells). The current finding (red light acts through SIRT4 downregulation, i.e., not direct enzymatic activation) aligns perfectly with Herrera´s critique.

Long-term metabolic effects-Herrera et al. show that a single red light exposure elevates oxygen consumption for up to 2 days. The current study focuses on changes at 12-24 h. Their data extend the time window and suggest that the metabolic reprogramming you describe may persist longer than currently discussed, which is clinically relevant.

Discussing Herrera et al.'s results would not only acknowledge independent, corroborating evidence but would also allow the authors to position their SIRT4-centric mechanism within a broader, emerging understanding of red-light photobiomodulation.

We would like to thank the reviewer for providing us with constructive suggestions for discussion. Our results showed that under red light conditions, both glycolipid and lipid metabolism were activated in keratinocytes, and cellular metabolic flux increased. The activation of lipid metabolism directly led to an increase in metabolism-associated H3K9ac and drove the upregulation of anti-aging-related genes; we believe this is key to the anti-aging effects of red light. Mechanistic analysis combining proteomics and acetylation proteomics revealed that red light significantly downregulated SIRT4 expression and increased the acetylation of MCD, a protein regulated by SIRT4 that governs cellular fatty acid oxidation rates. Through validation using cell-level knockdown and inhibitors, we confirmed that SIRT4 inhibition exerts anti-aging effects in vitro and that inhibiting MCD function under red light conditions suppresses H3K9ac. These results establish the role of the SIRT4-MCD signalling axis in mediating the anti-aging effects of red light.

The study by Herrera et al. included a substantial body of validation data confirming the role of red light in promoting fatty acid oxidation, providing robust empirical support for our research. Furthermore, Herrera et al. revealed that red light-induced fatty acid oxidation depends on AMPK and ACC phosphorylation. This mechanism of red-light photobiomodulation may refute the notion that its bio-regulatory effects rely solely on the action of mitochondrial cytochrome c oxidase. Furthermore, together with our study revealing that red light exerts anti-aging photobiomodulatory effects via the SIRT4-MCD signalling axis, these findings independently confirm that red light regulates cellular fatty acid oxidation, thereby demonstrating the pivotal role of activated fatty acid oxidation in the bio-regulatory effects of red light. In the revised manuscript, we will include a discussion on the potential link between the red light-driven downregulation of SIRT4 and the phosphorylation of AMPK/ACC. This will be of positive value in elucidating how SIRT4 exerts its anti-aging effects by regulating lipid metabolism, as well as in explaining the possible mechanisms by which red light downregulates SIRT4.