Review Reports

Round 1

Reviewer 1 Report

Comments and Suggestions for Authors

In this study, the authors generated a Dnmt3b knockout specifically in adipocyte progenitor cells to investigate its role in adipose progenitor biology and its downstream impact on energy metabolism in adipose tissue. The study shows that 16 weeks of high-fat diet (HFD) feeding improves body weight regulation, glucose homeostasis, and overall energy metabolism in female—but not male—mice lacking Dnmt3b. The observed sexual dimorphism in the metabolic response is particularly intriguing. However, the underlying mechanisms driving this sex-specific effect were not explored in this study. Overall, the study is well designed, but the following comments would help clarify and strengthen the manuscript:

1. What are the expression levels of Dnmt1, Dnmt3b, and Dnmt3a in male and female mice across all three fat depots? Authors show Dnmt3b mRNA in female iWAT and iBAT (Supplemental Fig. 1); please include female gWAT and all corresponding male depots to complete the figure.
2. Any difference in ERα and ERβ expression in male vs female in any fat depots ?
3. Does circulating leptin /VO2/RER changes in Male mice? If authors already have the data, they should consider showing it.

4. Food intake data shown just for 3 days (Supplemental Fig. 2)- does the authors measured food intake at the end of the study ? Male mice has any differences in food intake ?

5. Authors has collected complete data shown in Supplemental Fig. 3 - it should be a main figure to increase data visibility. 

 

Author Response

Reviewer 1

 

1. What are the expression levels of Dnmt1, Dnmt3b, and Dnmt3a in male and female mice across all three fat depots? Authors show Dnmt3b mRNA in female iWAT and iBAT (Supplemental Fig. 1); please include female gWAT and all corresponding male depots to complete the figure.

We thank the reviewer for this suggestion. In response, we conducted additional experiments by measuring the expression of all three Dnmts across three fat depots in both male and female mice and presented the data in Supplemental Figure 1.

 

2. Any difference in ERα and ERβ expression in male vs female in any fat depots?

We thank the reviewer for this thoughtful suggestion. In response, we performed additional experiments by measuring the expression of Esr1 (ERα), the ER isoform we previously reported to be regulated by DNA methylation in adipocytes (Wu et al., JCI Insight 2025), across adipose depots in both male and female mice. These new data are now presented in Figure 7.

 

We found that Esr1 expression is increased in all fat depots including iBAT, iWAT and gWAT of female PD3bKO mice, whereas in male PD3bKO mice, Esr1 expression is increased only in iBAT and shows a trend toward increase in iWAT. Thus, Esr1 expression is elevated in both sexes, but only female PD3bKO mice display a reduction in adiposity and improved insulin sensitivity. Although the precise mechanism remains unclear, it is plausible that estrogen, acting as the ligand for ERα/ESR1, amplifies the impact of increased Esr1 expression in females. The presence of circulating estrogen in females may synergistically engage upregulated ERα in adipocytes, resulting in a protective metabolic effect compared with males. We added these discussions to the manuscript.

 

3. Does circulating leptin /VO2/RER changes in Male mice? If authors already have the data, they should consider showing it.

We appreciate the reviewer’s suggestion. In the present study, we did not perform indirect calorimetry (VO₂/RER) in male PD3bKO mice. Because male knockout mice did not show significant differences in body weight or adiposity compared with their controls, we prioritized energy metabolism phenotyping in females, where the protective effects on body weight and thermogenic programming were robust. Running a full set of metabolic cage experiments requires dedicated cohorts and substantial resources, and we do not currently have an appropriate male cohort available for such studies. We agree that VO₂/RER and leptin data in males would be informative, and we now acknowledge this as a limitation in the Discussion, noting that future work will specifically address sex-dependent differences in energy expenditure and endocrine profiles in PD3bKO mice.

 

4. Food intake data shown just for 3 days (Supplemental Fig. 2)- does the authors measured food intake at the end of the study? Male mice have any differences in food intake?

We thank the reviewer for this comment. The 3-day food intake data shown were collected automatically by the TSE metabolic cage system during the indirect calorimetry experiments in female mice and were not repeated at the terminal time point. We did not measure food intake in male mice, as they were not placed in metabolic cages and we did not perform separate manual food intake measurements in this cohort.

 

To provide additional insight into energy balance in males, we have now taken advantage of existing stored tissue samples to measure thermogenic gene expression in iBAT and iWAT from male PD3bKO and control mice. These new data have been added to Figure 5F and 5G in the revised manuscript and show that thermogenic gene expression in male iBAT and iWAT is not significantly altered, which is consistent with the absence of changes in body weight and adiposity in male PD3bKO mice. We acknowledge the lack of food intake measurements in males as a limitation and note in the Discussion that future studies will include comprehensive assessment of food intake and energy expenditure in both sexes.

 

5. Authors has collected complete data shown in Supplemental Fig. 3 - it should be a main figure to increase data visibility.

We thank the reviewer for this helpful suggestion. In response, the original Supplemental Figure 3 has been moved into the main text and is now presented as Figure 5.

Reviewer 1

 

1. What are the expression levels of Dnmt1, Dnmt3b, and Dnmt3a in male and female mice across all three fat depots? Authors show Dnmt3b mRNA in female iWAT and iBAT (Supplemental Fig. 1); please include female gWAT and all corresponding male depots to complete the figure.

We thank the reviewer for this suggestion. In response, we conducted additional experiments by measuring the expression of all three Dnmts across three fat depots in both male and female mice and presented the data in Supplemental Figure 1.

 

2. Any difference in ERα and ERβ expression in male vs female in any fat depots?

We thank the reviewer for this thoughtful suggestion. In response, we performed additional experiments by measuring the expression of Esr1 (ERα), the ER isoform we previously reported to be regulated by DNA methylation in adipocytes (Wu et al., JCI Insight 2025), across adipose depots in both male and female mice. These new data are now presented in Figure 7.

 

We found that Esr1 expression is increased in all fat depots including iBAT, iWAT and gWAT of female PD3bKO mice, whereas in male PD3bKO mice, Esr1 expression is increased only in iBAT and shows a trend toward increase in iWAT. Thus, Esr1 expression is elevated in both sexes, but only female PD3bKO mice display a reduction in adiposity and improved insulin sensitivity. Although the precise mechanism remains unclear, it is plausible that estrogen, acting as the ligand for ERα/ESR1, amplifies the impact of increased Esr1 expression in females. The presence of circulating estrogen in females may synergistically engage upregulated ERα in adipocytes, resulting in a protective metabolic effect compared with males. We added these discussions to the manuscript.

 

3. Does circulating leptin /VO2/RER changes in Male mice? If authors already have the data, they should consider showing it.

We appreciate the reviewer’s suggestion. In the present study, we did not perform indirect calorimetry (VO₂/RER) in male PD3bKO mice. Because male knockout mice did not show significant differences in body weight or adiposity compared with their controls, we prioritized energy metabolism phenotyping in females, where the protective effects on body weight and thermogenic programming were robust. Running a full set of metabolic cage experiments requires dedicated cohorts and substantial resources, and we do not currently have an appropriate male cohort available for such studies. We agree that VO₂/RER and leptin data in males would be informative, and we now acknowledge this as a limitation in the Discussion, noting that future work will specifically address sex-dependent differences in energy expenditure and endocrine profiles in PD3bKO mice.

 

4. Food intake data shown just for 3 days (Supplemental Fig. 2)- does the authors measured food intake at the end of the study? Male mice have any differences in food intake?

We thank the reviewer for this comment. The 3-day food intake data shown were collected automatically by the TSE metabolic cage system during the indirect calorimetry experiments in female mice and were not repeated at the terminal time point. We did not measure food intake in male mice, as they were not placed in metabolic cages and we did not perform separate manual food intake measurements in this cohort.

 

To provide additional insight into energy balance in males, we have now taken advantage of existing stored tissue samples to measure thermogenic gene expression in iBAT and iWAT from male PD3bKO and control mice. These new data have been added to Figure 5F and 5G in the revised manuscript and show that thermogenic gene expression in male iBAT and iWAT is not significantly altered, which is consistent with the absence of changes in body weight and adiposity in male PD3bKO mice. We acknowledge the lack of food intake measurements in males as a limitation and note in the Discussion that future studies will include comprehensive assessment of food intake and energy expenditure in both sexes.

 

5. Authors has collected complete data shown in Supplemental Fig. 3 - it should be a main figure to increase data visibility.

We thank the reviewer for this helpful suggestion. In response, the original Supplemental Figure 3 has been moved into the main text and is now presented as Figure 5.

Reviewer 1

 

1. What are the expression levels of Dnmt1, Dnmt3b, and Dnmt3a in male and female mice across all three fat depots? Authors show Dnmt3b mRNA in female iWAT and iBAT (Supplemental Fig. 1); please include female gWAT and all corresponding male depots to complete the figure.

We thank the reviewer for this suggestion. In response, we conducted additional experiments by measuring the expression of all three Dnmts across three fat depots in both male and female mice and presented the data in Supplemental Figure 1.

 

2. Any difference in ERα and ERβ expression in male vs female in any fat depots?

We thank the reviewer for this thoughtful suggestion. In response, we performed additional experiments by measuring the expression of Esr1 (ERα), the ER isoform we previously reported to be regulated by DNA methylation in adipocytes (Wu et al., JCI Insight 2025), across adipose depots in both male and female mice. These new data are now presented in Figure 7.

 

We found that Esr1 expression is increased in all fat depots including iBAT, iWAT and gWAT of female PD3bKO mice, whereas in male PD3bKO mice, Esr1 expression is increased only in iBAT and shows a trend toward increase in iWAT. Thus, Esr1 expression is elevated in both sexes, but only female PD3bKO mice display a reduction in adiposity and improved insulin sensitivity. Although the precise mechanism remains unclear, it is plausible that estrogen, acting as the ligand for ERα/ESR1, amplifies the impact of increased Esr1 expression in females. The presence of circulating estrogen in females may synergistically engage upregulated ERα in adipocytes, resulting in a protective metabolic effect compared with males. We added these discussions to the manuscript.

 

3. Does circulating leptin /VO2/RER changes in Male mice? If authors already have the data, they should consider showing it.

We appreciate the reviewer’s suggestion. In the present study, we did not perform indirect calorimetry (VO₂/RER) in male PD3bKO mice. Because male knockout mice did not show significant differences in body weight or adiposity compared with their controls, we prioritized energy metabolism phenotyping in females, where the protective effects on body weight and thermogenic programming were robust. Running a full set of metabolic cage experiments requires dedicated cohorts and substantial resources, and we do not currently have an appropriate male cohort available for such studies. We agree that VO₂/RER and leptin data in males would be informative, and we now acknowledge this as a limitation in the Discussion, noting that future work will specifically address sex-dependent differences in energy expenditure and endocrine profiles in PD3bKO mice.

 

4. Food intake data shown just for 3 days (Supplemental Fig. 2)- does the authors measured food intake at the end of the study? Male mice have any differences in food intake?

We thank the reviewer for this comment. The 3-day food intake data shown were collected automatically by the TSE metabolic cage system during the indirect calorimetry experiments in female mice and were not repeated at the terminal time point. We did not measure food intake in male mice, as they were not placed in metabolic cages and we did not perform separate manual food intake measurements in this cohort.

 

To provide additional insight into energy balance in males, we have now taken advantage of existing stored tissue samples to measure thermogenic gene expression in iBAT and iWAT from male PD3bKO and control mice. These new data have been added to Figure 5F and 5G in the revised manuscript and show that thermogenic gene expression in male iBAT and iWAT is not significantly altered, which is consistent with the absence of changes in body weight and adiposity in male PD3bKO mice. We acknowledge the lack of food intake measurements in males as a limitation and note in the Discussion that future studies will include comprehensive assessment of food intake and energy expenditure in both sexes.

 

5. Authors has collected complete data shown in Supplemental Fig. 3 - it should be a main figure to increase data visibility.

We thank the reviewer for this helpful suggestion. In response, the original Supplemental Figure 3 has been moved into the main text and is now presented as Figure 5.

Reviewer 1

 

1. What are the expression levels of Dnmt1, Dnmt3b, and Dnmt3a in male and female mice across all three fat depots? Authors show Dnmt3b mRNA in female iWAT and iBAT (Supplemental Fig. 1); please include female gWAT and all corresponding male depots to complete the figure.

We thank the reviewer for this suggestion. In response, we conducted additional experiments by measuring the expression of all three Dnmts across three fat depots in both male and female mice and presented the data in Supplemental Figure 1.

 

2. Any difference in ERα and ERβ expression in male vs female in any fat depots?

We thank the reviewer for this thoughtful suggestion. In response, we performed additional experiments by measuring the expression of Esr1 (ERα), the ER isoform we previously reported to be regulated by DNA methylation in adipocytes (Wu et al., JCI Insight 2025), across adipose depots in both male and female mice. These new data are now presented in Figure 7.

 

We found that Esr1 expression is increased in all fat depots including iBAT, iWAT and gWAT of female PD3bKO mice, whereas in male PD3bKO mice, Esr1 expression is increased only in iBAT and shows a trend toward increase in iWAT. Thus, Esr1 expression is elevated in both sexes, but only female PD3bKO mice display a reduction in adiposity and improved insulin sensitivity. Although the precise mechanism remains unclear, it is plausible that estrogen, acting as the ligand for ERα/ESR1, amplifies the impact of increased Esr1 expression in females. The presence of circulating estrogen in females may synergistically engage upregulated ERα in adipocytes, resulting in a protective metabolic effect compared with males. We added these discussions to the manuscript.

 

3. Does circulating leptin /VO2/RER changes in Male mice? If authors already have the data, they should consider showing it.

We appreciate the reviewer’s suggestion. In the present study, we did not perform indirect calorimetry (VO₂/RER) in male PD3bKO mice. Because male knockout mice did not show significant differences in body weight or adiposity compared with their controls, we prioritized energy metabolism phenotyping in females, where the protective effects on body weight and thermogenic programming were robust. Running a full set of metabolic cage experiments requires dedicated cohorts and substantial resources, and we do not currently have an appropriate male cohort available for such studies. We agree that VO₂/RER and leptin data in males would be informative, and we now acknowledge this as a limitation in the Discussion, noting that future work will specifically address sex-dependent differences in energy expenditure and endocrine profiles in PD3bKO mice.

 

4. Food intake data shown just for 3 days (Supplemental Fig. 2)- does the authors measured food intake at the end of the study? Male mice have any differences in food intake?

We thank the reviewer for this comment. The 3-day food intake data shown were collected automatically by the TSE metabolic cage system during the indirect calorimetry experiments in female mice and were not repeated at the terminal time point. We did not measure food intake in male mice, as they were not placed in metabolic cages and we did not perform separate manual food intake measurements in this cohort.

 

To provide additional insight into energy balance in males, we have now taken advantage of existing stored tissue samples to measure thermogenic gene expression in iBAT and iWAT from male PD3bKO and control mice. These new data have been added to Figure 5F and 5G in the revised manuscript and show that thermogenic gene expression in male iBAT and iWAT is not significantly altered, which is consistent with the absence of changes in body weight and adiposity in male PD3bKO mice. We acknowledge the lack of food intake measurements in males as a limitation and note in the Discussion that future studies will include comprehensive assessment of food intake and energy expenditure in both sexes.

 

5. Authors has collected complete data shown in Supplemental Fig. 3 - it should be a main figure to increase data visibility.

We thank the reviewer for this helpful suggestion. In response, the original Supplemental Figure 3 has been moved into the main text and is now presented as Figure 5.

Reviewer 1

 

1. What are the expression levels of Dnmt1, Dnmt3b, and Dnmt3a in male and female mice across all three fat depots? Authors show Dnmt3b mRNA in female iWAT and iBAT (Supplemental Fig. 1); please include female gWAT and all corresponding male depots to complete the figure.

We thank the reviewer for this suggestion. In response, we conducted additional experiments by measuring the expression of all three Dnmts across three fat depots in both male and female mice and presented the data in Supplemental Figure 1.

 

2. Any difference in ERα and ERβ expression in male vs female in any fat depots?

We thank the reviewer for this thoughtful suggestion. In response, we performed additional experiments by measuring the expression of Esr1 (ERα), the ER isoform we previously reported to be regulated by DNA methylation in adipocytes (Wu et al., JCI Insight 2025), across adipose depots in both male and female mice. These new data are now presented in Figure 6.

 

We found that Esr1 expression is increased in all fat depots including iBAT, iWAT and gWAT of female PD3bKO mice, whereas in male PD3bKO mice, Esr1 expression is increased only in iBAT and shows a trend toward increase in iWAT. Thus, Esr1 expression is elevated in both sexes, but only female PD3bKO mice display a reduction in adiposity and improved insulin sensitivity. Although the precise mechanism remains unclear, it is plausible that estrogen, acting as the ligand for ERα/ESR1, amplifies the impact of increased Esr1 expression in females. The presence of circulating estrogen in females may synergistically engage upregulated ERα in adipocytes, resulting in a protective metabolic effect compared with males. We added these discussions to the manuscript.

 

3. Does circulating leptin /VO2/RER changes in Male mice? If authors already have the data, they should consider showing it.

We appreciate the reviewer’s suggestion. In the present study, we did not perform indirect calorimetry (VO₂/RER) in male PD3bKO mice. Because male knockout mice did not show significant differences in body weight or adiposity compared with their controls, we prioritized energy metabolism phenotyping in females, where the protective effects on body weight and thermogenic programming were robust. Running a full set of metabolic cage experiments requires dedicated cohorts and substantial resources, and we do not currently have an appropriate male cohort available for such studies. We agree that VO₂/RER and leptin data in males would be informative, and we now acknowledge this as a limitation in the Discussion, noting that future work will specifically address sex-dependent differences in energy expenditure and endocrine profiles in PD3bKO mice.

 

4. Food intake data shown just for 3 days (Supplemental Fig. 2)- does the authors measured food intake at the end of the study? Male mice have any differences in food intake?

We thank the reviewer for this comment. The 3-day food intake data shown were collected automatically by the TSE metabolic cage system during the indirect calorimetry experiments in female mice and were not repeated at the terminal time point. We did not measure food intake in male mice, as they were not placed in metabolic cages and we did not perform separate manual food intake measurements in this cohort.

 

To provide additional insight into energy balance in males, we have now taken advantage of existing stored tissue samples to measure thermogenic gene expression in iBAT and iWAT from male PD3bKO and control mice. These new data have been added to Figure 5F and 5G in the revised manuscript and show that thermogenic gene expression in male iBAT and iWAT is not significantly altered, which is consistent with the absence of changes in body weight and adiposity in male PD3bKO mice. We acknowledge the lack of food intake measurements in males as a limitation and note in the Discussion that future studies will include comprehensive assessment of food intake and energy expenditure in both sexes.

 

5. Authors has collected complete data shown in Supplemental Fig. 3 - it should be a main figure to increase data visibility.

We thank the reviewer for this helpful suggestion. In response, the original Supplemental Figure 3 has been moved into the main text and is now presented as Figure 5.

Reviewer 2 Report

Comments and Suggestions for Authors

(1) The study does not provide direct evidence to link Dnmt3b to epigenetic regulation of thermogenic genes. Without DNA methylation of key gene, the claim that Dnmt3b modulates thermogenesis remains speculative.

(2) PDGFRα-Cre may target non-adipocyte tissues, potentially confounding the similar observed phenotypes. 

(3)  While the paper highlights stark sex differences in metabolic outcomes, it does not involve in any mechanistic investigation or deep discussion.

(4) there are D3bKO and PD3bKO, creating confusion.

Author Response

Reviewer 2

(1) The study does not provide direct evidence to link Dnmt3b to epigenetic regulation of thermogenic genes. Without DNA methylation of key gene, the claim that Dnmt3b modulates thermogenesis remains speculative.

We appreciate this important point and agree that we have not directly measured DNA methylation at specific thermogenic gene loci in this study. Our current data demonstrate that Dnmt3b deletion in adipocyte progenitor cells leads to: (a) robust upregulation of thermogenic genes in iBAT (Ucp1, Pgc1α, Dio2, Elovl3), (b) increased oxygen consumption, and (c) improved whole-body metabolic outcomes in female PD3bKO mice. Together, these findings establish a strong functional link between loss of Dnmt3b in adipocyte progenitors and enhanced thermogenic capacity/energy expenditure, even though the exact CpG or non-CpG methylation events have not yet been mapped.

 

Our interpretation that Dnmt3b contributes to epigenetic regulation of thermogenesis is based on extensive literatures and our own previous work: (a) DNMT3B has been shown to regulate non-CpG methylation of the PGC-1α promoter and thereby control mitochondrial biogenesis and oxidative metabolism in skeletal muscle. (Barres et al., Cell Metab 2009; ref. 28 in the manuscript.). (b) Ucp1 enhancer methylation is known to influence Ucp1 expression in adipose tissues (Shore et al., Diabetologia 2010; ref. 27). (c) Our previous studies demonstrated critical roles of DNA methyltransferases (DNMT1, DNMT3A, and DNMT3B) in adipocyte development and thermogenic regulation in brown fat, including models in which Dnmt3b deficiency in mature brown adipocytes ameliorates obesity in females (Li et al., Life 2021; ref. 34).

 

In this revised manuscript, we have toned down our wording to more clearly distinguish between (a) the direct experimental evidence provided here (i.e., Dnmt3b deletion leading to increased thermogenic gene expression and energy expenditure) and (b) the inferred mechanistic link that this likely reflects epigenetic regulation by Dnmt3b. Specifically, in the Discussion we now state that our data “support a role for Dnmt3b in regulating thermogenic programming, potentially via epigenetic mechanisms” rather than implying that we have directly demonstrated locus-specific methylation changes. We have also added explicit language recognizing the absence of direct DNA methylation measurements as a limitation and highlighting the need for future work (e.g., locus-specific bisulfite sequencing or methylome analysis in progenitors and mature brown adipocytes) to define the precise epigenetic mechanisms.

 

(2) PDGFRα-Cre may target non-adipocyte tissues, potentially confounding the similar observed phenotypes.

We thank the reviewer for raising this important point regarding Cre-driver specificity. We agree that PDGFRα-Cre is not exclusively restricted to adipocyte progenitors and can be expressed in other mesenchymal lineages, including fibroblasts and stromal cells, and potentially in certain non-adipose tissues depending on developmental context. We chose PDGFRα-Cre because it is widely used and well-validated for targeting adipocyte progenitor cells in vivo and has been extensively characterized in lineage-tracing and functional studies (Berry & Rodeheffer, Nat Cell Biol 2013; Sun et al., Cell Stem Cell 2020; Krueger et al., Stem Cell Reports 2014; refs. 16-19).

 

In our model, we confirmed efficient Dnmt3b deletion in both WAT and BAT, as evidenced by significant reduction of Dnmt3b mRNA in these depots (Supplemental Figure 1), which is consistent with deletion of Dnmt3b in adipocyte progenitor cells. The major metabolic phenotypes we report here include reduced adiposity, enhanced brown fat thermogenic gene expression, increased oxygen consumption, lower RER, and altered insulin sensitivity, which are all classic readouts of adipose-derived alterations in energy balance.

 

At the same time, we fully recognize that the PDGFRα-Cre line may also be active in other progenitor populations and possibly in certain central nervous system. Indeed, in the Discussion we already speculated that the reduced food intake in female PD3bKO mice could involve either adipose-derived factors or potential Dnmt3b deletion in neuronal progenitors, and we now expand this section to explicitly acknowledge that: (a) PDGFRα-Cre is not perfectly adipose-restricted, and extra-adipose deletion may contribute to some aspects of the phenotype. (b) Our interpretation is that the primary driver of the observed metabolic changes is adipose tissue, based on the strong and consistent adipose phenotypes and thermogenic gene changes. (c) Future studies using additional Cre drivers (e.g., more restricted adipocyte-lineage Cre lines or intersectional genetic strategies) will be needed to disentangle purely adipose-intrinsic effects from potential contributions of other PDGFRα-expressing lineages.

 

We believe that documenting the phenotype in this widely used PDGFRα-Cre driver still provides valuable insight into how Dnmt3b in adipose progenitors and related mesenchymal lineages influences energy metabolism and obesity risk.

 

(3)  While the paper highlights stark sex differences in metabolic outcomes, it does not involve in any mechanistic investigation or deep discussion.

We appreciate the reviewer’s emphasis on the importance of the sex-dependent phenotype. The observation that female PD3bKO mice are protected against diet-induced obesity and insulin resistance, whereas male PD3bKO mice tend to exhibit worsened insulin sensitivity without significant weight loss, is indeed an intriguing aspect of our study.

 

We acknowledge that we have not performed mechanistic experiments specifically designed to dissect the basis of this sexual dimorphism (e.g., ovariectomy, estrogen receptor blockade, or sex-specific methylome profiling). Such studies would require a substantial expansion of experimental scope and are therefore beyond the scope of the present work. However, we fully share the reviewer’s view that the sex differences deserve deeper conceptual treatment. Accordingly, we have now expanded the Discussion to provide a more detailed framework, including: (a) A more thorough summary of established sex differences in adiposity, fat distribution, lipolysis, and lipid handling in humans and rodents (Gallagher et al., Am J Clin Nutr 2000; Palmer & Clegg, Mol Cell Endocrinol 2015; Schmidt et al., J Appl Physiol 2014; refs. 35-37). (b) A more explicit discussion of the role of estrogen and estrogen receptors in shaping metabolic phenotypes, including how estrogen signaling can protect females from HFD-induced metabolic dysfunction (Monteiro et al., Mediators Inflamm 2014; ref. 38). (c) Discussion of our own recent work showing that epigenetic programming of estrogen receptor in adipocytes contributes to obesity-induced inflammation and metabolic disease (Wu et al., JCI Insight 2025; ref. 21), and how similar mechanisms could interact with Dnmt3b-dependent methylation pathways to create sex-specific outcomes. Future work will specifically test the hypothesis that estrogen and estrogen receptor signaling intersect with Dnmt3b-mediated epigenetic pathways in adipose tissue and/or the central nervous system to produce the observed female-specific protection and male-specific vulnerability.

 

(4) “there are D3bKO and PD3bKO, creating confusion.”

We now consistently use “PD3bKO” across the entire manuscript.