Molecular programming modulates hepatic lipid metabolism and adult metabolic risk in the offspring of obese mothers in a sex-specific manner
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Abstract
Male and female offspring of obese mothers are known to differ extensively in their metabolic adaptation and later development of complications. We investigate the sex-dependent responses in obese offspring mice with maternal obesity, focusing on changes in liver glucose and lipid metabolism. Here we show that maternal obesity prior to and during gestation leads to hepatic steatosis and inflammation in male offspring, while female offspring are protected. Females from obese mothers display important changes in hepatic transcriptional activity and triglycerides profile which may prevent the damaging effects of maternal obesity compared to males. These differences are sustained later in life, resulting in a better metabolic balance in female offspring. In conclusion, sex and maternal obesity drive differently transcriptional and posttranscriptional regulation of major metabolic processes in offspring liver, explaining the sexual dimorphism in obesity-associated metabolic risk.
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Reply to the reviewers
We thank the reviewers for their excellent comments. The comments raised by the reviewers have tremendously improved our manuscript and allowed us to provide more clarity of our findings. Please find below the point-by-point answers to the reviewer's comments.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The manuscript from Savva et al. focuses on a long-standing and unresolved challenge of metabolic (and not only) health in mammals: the sexual dimorphism. Authors couple transcriptomics and metabolomics to in-depth molecular phenotyping in offspring of dams fed HFD before conception and throughout pregnancy and lactation to isolate …
Note: This rebuttal was posted by the corresponding author to Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Reply to the reviewers
We thank the reviewers for their excellent comments. The comments raised by the reviewers have tremendously improved our manuscript and allowed us to provide more clarity of our findings. Please find below the point-by-point answers to the reviewer's comments.
Reviewer #2 (Evidence, reproducibility and clarity (Required)):
The manuscript from Savva et al. focuses on a long-standing and unresolved challenge of metabolic (and not only) health in mammals: the sexual dimorphism. Authors couple transcriptomics and metabolomics to in-depth molecular phenotyping in offspring of dams fed HFD before conception and throughout pregnancy and lactation to isolate molecular determinants of sexually dimorphic response to maternal obesity.
**Major comments:**
- While the manuscript present a compelling and exhaustive amount of data, which accurately describes the sexually dimorphic responses to maternal obesity, it lacks mechanistic insights and I personally think this to mainly be a timing issue. For example, I tried hard to find experimental details on the RNA sequencing and could not find much: when is the RNA sequencing performed? If, as I suspect, the sequencing experiments match the metabolomics experiments, I don't think they add much mechanistic insights onto the observed phenomena. They rather contribute to better describe them. Indeed, both metabolomic and transcriptomic profiles might be consequence of the observed phenotypes, rather than be causative (as the authors try to argue). Are these differences already present at birth? what happens to placenta and fetal tissues?
ANSWER: We have clarified the experimental setting and added information about the maternal status. We did not collect the placenta and fetal tissues as the main goal of the study was to investigate the offspring metabolism in a longitudinal study, using the animal as its own control. Therefore, we looked at glucose tolerance, insulin sensitivity and lipid profile in the liver at two different timepoints on the same mouse. At end we sacrificed the mouse and collected tissues for further analysis (lipidomic, RNA sequencing and histology).
Some adult phenotypes - especially metabolic and neurological phenotypes - might also be influenced by different maternal care early postnatal. Are the litters balanced by number and sex ratio? Would cross-fostering maintain the phenotypes?
ANSWER: We agree with the reviewer that phenotype can be influenced by maternal care early postnatal. Each dam delivered litters in different proportions. Dams on CD delivered in average seven to eight littermates (same ratio of female and male offspring) whereas dams on HFD delivered less (five in average). Some of the offspring died at very early age of unknown reasons. The final number of offspring used for this study was 11 females and 12 males born from dams on CD and 11 females and 10 males born from dams on HFD. We did not perform cross-fostering to limit the stress in the animals.
Of the two points above, I would love to see more details on the RNA sequencing, as well as placental and fetal tissues analysed. It would be also interesting to know about any litter balancing measure or at least have more statistics on the litter size and sex ratios.
ANSWER: Each individual was followed over the course of the experimentation for body weight and food intake (six months). However, not all animals were used for every in vivo and ex vivo experiment justified under consideration of the 3Rs, sample throughput capacity and financial constraints (magnetic resonance, lipidomic, qPCR, and tolerance tests). However, all experimentation was performed according to a prior power calculation and published reports (PMID: 30808418, 23446231, 31811898, 25694038 and 31820027). Throughout the study, we opted for randomized experimental design (random selection of the animals for in vivo and ex vivo experiments).
After weaning, up to five littermates were housed per cage. However, some male individuals had to be separated due to aggressive behaviors. When females showed hierarchical behaviors in the cage, we separated them to be sure that each individual had full access to food. Since mice are social animals, no individual was housed alone.
Reviewer #2 (Significance (Required)):
The manuscript from Savva et al. revolves around the unresolved challenge of how sexually dimorphic phenotypes are established. The topic is actual - although already a lot has been published, as acknowledged by the authors as well - and of broad interest to the community of mouse geneticists and physiologists. To understand the molecular underpinnings of sexually dimorphic phenotypes, the authors use in-depth molecular phenotyping in the mouse coupled to metabolomics and transcriptomics. While extremely informative and exhaustive, the actual dataset is - at least for me - purely descriptive, which might reduce its overall impact. I'm a mouse geneticist and a metabolic physiologist and I find the topic of sexual dimorphism extremely interesting.
**Referee Cross-commenting**
I generally agree with the other reviewers' comments. I think the ms is interesting and the dataset compelling although to a certain extent overlapping with previously published studies. There is general agreement on the lack of mechanistic data and the authors should definitely address this point.
ANSWER: In mammals, including humans, biological sex is determined by a pair of sex chromosomes. Genes on the X chromosome (X-linked genes) have distinctive inheritance patterns because they are present in different number between females (XX) and males (XY). Moreover, a plethora of attributes can be determined by sex chromosomes and more importantly by X-linked chromosomes. Therefore, we extracted the sex-linked chromosomes that are affected by the maternal diet or by sex and presented them in figure 5 to give more insight into the molecular underpinning of the sexual dimorphism in metabolism homeostasis.
We have presented the data in figures 4e-4g and commented on page 12 L300-L315 in the result section and in the last paragraph of the discussion section (page 18).
Reviewer #3 (Evidence, reproducibility and clarity (Required)):
In this paper Savva et al. explore how maternal obesity influence hepatic metabolism in a sex-specific fashion. They first assess the contribution of the adipose tissue to the development of insulin resistance and glucose intolerance focusing on inflammation and oxidative phosphorylation pathways. Then they proceed to asses if maternal obesity could remodel the hepatic triglyceride levels and phospholipids using proton magnetic resonance spectroscopy and LC-MS lipidomic, respectively. Finally, they explore hepatic lipid metabolism and genes promoting cancer development.
Despite the methodology is correct and elegant, the study does not explore a possible mechanism of action and some results are contradictory. Indeed, some of the results seems to be driven by the sex of the offspring independently of the maternal feeding.
There are indeed, some limitations for the authors and editors to consider. To this reviewer the manuscript is difficult to read, particularly the results section in which the data are listed without discussing their relevance or their connection to previous research from other groups. Moreover, the discussion could benefit from an extensive rewrite. Indeed, this section lacks of clarity and references that could help elucidate the novel finding of the authors.
ANSWER: We have rewritten the manuscript and we believe that it has been extensively improved in clarity. We have added references and commented on the differences observed, if any. We also have added one complete paragraph on the potential role of sex-biased modulators, especially the sex chromosomes XX or XY. These data are presented in figures 4e-4g and commented on page 12 L300-L315 in the result section and in the last paragraph of the discussion section (page 18).
**Major comments:**
Line 97: How the authors explain similar weight gain in the F-C/HF vs. F-HF/HF? A large body of literature reports that maternal high fat diet influences offspring weight gain, independently of sex, when compared to maternal standard diet (PMID: 23973955; 29872021; 31076636; 3036829).
ANSWER: The literature is still controversial, possibly due to slightly different experimental settings (e.g. the exact composition of the control and high fat diet, exposure time to the different diets). In the current study we used a match control diet of the high fat diet to minimize potential diet-derived signaling molecule effect. We found several publications in line with our findings (PMID: 30405201, 31690792 and 29972240). In addition, in our study, we assessed the changes in body weight over a long time in the offspring, whereas only few studies show detailed measurements over time, which makes it more difficult to compare across studies.
Line 99: Which is the explanation for the reduced body weight in M-HF/HF from birth until 9 week of age? Can the authors show the timeline for food intake?
ANSWER: Excess gestational weight gain is associated with health risk for both the infant and the mother. A large number of epidemiological studies have demonstrated a direct relationship between birth weight and BMI attained in later life. Lower birth weight seems to be associated with later risk for central obesity, which also confers increased cardiovascular risk. It has been previously demonstrated that offspring born from obese mother have lower body weight at birth than control diet/lean mother’s littermates (PMID: 15116085 and 24936914). Here, offspring after weaning are all put on the HFD until six months of age (before sacrifice).
We do not have timeline of food intake but we measured average food intake twice a week during three weeks at about 4-month of age, we presented the data in Fig.S1a and in the result section page 5 (Line 104). Interestingly, food intake tended to be induced and reduced by MO in female and male, respectively.
Line 101: How the authors explain the increase in final weight of the male compared to the female if no differences in total fat, VAT or SAT was observed between the offspring?
ANSWER: We have presented the graphs in relative amount body fat (% TF), and TF is corrected for the total body weight. Figs.1d-1f. When fat is reported to the body weight males have lower relative fat content than females. However, males are taller (bigger) and have bigger bone and muscle mass than females. Therefore, males weight more than females despite lower relative amount of fat.
Line 116: The authors state: "The ratio between the total SAT and the Abd SAT revealed that MO redistributed SAT outside of the abdominal region in females but not in males" but Fig.1g displays no significant differences between F-C/HF and F-HF/HF. Please explain.
ANSWER: Sex differences are observed (at END, MO tended to increase the ratio in F and decrease in M) to a lower level than F. Lower SAT on VAT ratio in obese males than in females is well recognized, however here we observed that MO tends to redistribute fat differently between sexes, and fat distribution has been strongly correlated to metabolic risks.
Line 128-130: The authors state: "At MID, glucose tolerance was highly diet- and sex-dependent, and males but not females showed impaired glucose tolerance by MO." However, in Fig.1h no significant differences in glucose peak or glucose AUC were observed between M-C/HF and M-HF/F. Please explain. This is correct, however fasting glucose (T0) was higher and T60 and T120 as well. In addition, Ins levels during the OGTT was increased at T0, T30 and T120.
ANSWER: We have clarified the sentence and agree with the reviewer that the males are not affected by MO but are already insulin resistant when born from lean mothers. We added the quantitative insulin-sensitivity check index (QUICKI) in Fig.1n and demonstrate that indeed males are less insulin sensitive than females, but MO does not impact the insulin sensitivity in both sexes.
Line 130: OGTT only provides information on insulin secretion and action but does not directly yield a measure of insulin sensitivity. Please rephrase.
ANSWER: We have measured the insulin level during the OGTT, this information, combined with the glucose disappearance curve gives important information on the ability of pancreatic beta cell to release insulin in response to a glucose load and on the ability of insulin to store the glucose into the cells i.e. insulin sensitivity.
We have added the quantitative insulin-sensitivity check index (QUICKI) in Fig.1n and confirmed that males are less insulin sensitive than females with no effect of MO. Line 125
Authors should rephrase the conclusion of the paragraph since MO does not seems to influence fat distribution or insulin resistance. Looking at figure 1 it seems that the only differences observed are driven by the sex of the offspring independently of maternal feeding.
ANSWER: We have changed the conclusion and focused on the sex differences.
Do the dams were insulin resistant? Indeed, hyperinsulinemia and insulin resistance are key programming factor of offspring metabolic syndrome.
ANSWER: We agree that it would have been good to have the insulin levels of the mothers before mating. Unfortunately, we took only the body weight and the glucose level after 2 h fasting. We saw no differences between CD and HFD fed mothers. We included these results in the Figure 1b. Of note, we have performed long term HFD in young female mice for other purposes and noticed that females are resistant to hyperinsulinemia when fed a HFD in a long term, so we assumed that feeding a HFD for 6 weeks before mating would not affect insulin levels.
Line 162: "There were no significant differences between the sexes. However, it is interesting to note that several pathways were regulated differently between sexes between the C/HF and HF/HF groups." Can the authors rephrase the concept, it is unclear.
ANSWER: We have revised the sentences and gave some examples in P6, Lines 132-139.
Line 170: The authors state: "insulin secretion pathway is reduced in males only". How this results are in line with the data reported in Fig.1i were both M-C/HF and M-HF/HF display increased insulin secretion?
ANSWER: We agree with the reviewer that this is controversial. However, males from obese mothers showed slightly increased insulin levels during the OGTT and slightly reduced QUICKI as compared to males from lean mothers. Moreover, here we measured the total insulin level and not the C-peptide level that is more representative of the “active insulin”. One can be that males have higher insulin level but lower active insulin.
Lines 174-175: All the genes reported in Fig. 1o, except for LPIN1, do not seems to be altered by MO. Please rephrase.
ANSWER: Lpin1 and Pdk1 are reduced by MO in females. We have rephrased P6, Lines 142-147
Line 184: The authors state that the signaling pathways was assessed both at transcriptional and post-transcriptional levels. Where are depicted the data of the post-transcriptional levels?
ANSWER: We have corrected this sentence and rephrased it.
Similarly to glucose metabolism and fat depot results, also in the case of the liver steatosis the increased number of lipid droplets seems to be linked to the sex of the animal rather than the maternal diet. Since the authors also investigated inflammatory pathways it could be of interest to assess CD68 infiltration by immunohistochemistry and Picrosirius red staining for the assessment of fibrosis.
ANSWER: We agree with the reviewer that immune cells infiltration quantification would have been excellent. However, due to the lock down and the moving of our lab we could not performed the IHC.
Liver histology in Fig.5a (M-C/HFD) is completely different from the one depicted in Fig.4a in terms of steatosis. Can the authors please explain this difference and report the magnification used in Fig.4a. Please also report the scale in Fig.5a.
ANSWER: We have corrected this mistake and we apologize for the confusion. The Fig.4a described a M-moHF offspring liver. The pictures have been changed and magnification has been added.
Changes in placental function are thought to be a key link between the maternal and intrauterine milieu and long-term health of the offspring (PMID:24107818). Alterations in placental function and structure in response to obesity and their underlying molecular mechanisms have been explored both in humans and in animal models (PMID:24484739; 22303323; 28291256). Others have shown that maternal hyperinsulinemia is strongly associated with offspring insulin resistance and excess placental lipid deposition and hypoxia (PMID: 28291256). Excessive lipid deposition leads to a lipotoxic placental environment that is associated with increased markers of inflammation and oxidative stress (PMID: 24333048). Can the authors could provide some data?
ANSWER: We agree with the reviewer that maternal and intrauterine milieu are crucial to determine long-term health status of the offspring. However, in the current study we wanted to explore the sex differences in offspring fed a HFD and born from either lean or obese mothers in the long term.
We did not focus the current project on the maternal status, but on the offspring and the sexual dimorphism in metabolic risks later in life.
**Minor:**
Fig. 2m Acox1 is not reduced by MO in female.
ANSWER: We have corrected the sentence.
Supp. TableS1 do not report Pdk1, Lpin1, Nox4 and Prlr.
ANSWER: The reason why these genes do not appear in the TableS1 is because the table S1 shows all the significantly regulated genes extracted from the KEGG pathways. The genes mentioned above Pdk1, Lpin1, Nox4 and Prlr do not appear in the KEEG pathway.
Supp. TableS2 do not report PCSK9 and PNPLA3
ANSWER: Same as above, the Table S2 is based on KEGG pathway analysis and the genes Pcsk9 and Pnpla3 do not appear in the KEGG pathway.
Reviewer #3 (Significance (Required)):
The prevalence of obesity during pregnancy continues to increase at alarming rates. This is concerning as in addition to immediate impacts on maternal wellbeing, obesity during pregnancy has detrimental effects on the long-term health of the offspring. This paper is connected to an extended research field aiming at prevent the detrimental effect of maternal obesity on the offspring.
An important limitation in the ability to design intervention strategies to prevent the detrimental effects of maternal obesity on offspring health is that it is currently unclear which of the many potential variables associated with obesity is the key programming factor mediating the effects on the offspring.
**Reviewer field of expertise:**
Molecular Biology, Type 2 Diabetes, Obesity and NAFLD.
**Referee Cross-commenting**
I agree with the other reviewers' comments, particularly on the lack of mechanistic data.
ANSWER: In mammals, including humans, biological sex is determined by a pair of sex chromosomes. Genes on the X chromosome (X-linked genes) have distinctive inheritance patterns because they are present in different number between females (XX) and males (XY). Moreover, a plethora of attributes can be determined by sex chromosomes and more importantly by X-linked chromosomes. Therefore, we extracted the sex-linked chromosomes that are affected by the maternal diet or by sex and presented them in figure 5 to give more insight into the molecular underpinning of the sexual dimorphism in metabolism homeostasis. We have presented the data page 12 L300-L315in the result section and in the last paragraph of the discussion section (page 18).
Reviewer #4 (Evidence, reproducibility and clarity (Required)):
Maternal obesity is a common condition in western society. There is abundant literature showing the deleterious metabolic consequences of MO in the offspring. In this manuscript, Savva et al. characterized the transcriptomic and lipidomic profiles of the liver in male and female progeny of female mice that were fed a high-fat diet during and before pregnancy. After weaning, mice were also fed a high-fat diet. They found that both transcription and lipid composition were different in males and females, and they show that females are protected to metabolic and liver disease, whereas males develop insulin resistance, liver steatosis, and are prone to develop liver cancer. The first part of the study where the authors characterize the metabolism of the progeny, including weight, fat mass in the distinct depots, glucose and insulin tolerance, is not novel. Several publications have previously reported these findings (Programming effects of maternal and gestational obesity on offspring metabolism and metabolic inflammation. Sci Rep. 2019 Nov 5;9(1):16027). It was also previously reported in the cited publication, increased liver weight, steatosis and TG content, similar to the results of the present study. Some novelty of the manuscript is the in-depth analysis of the lipid content of the liver in the models used, as well as the transcriptional profile. Despite the substantial amount of data that the authors generated to prove differences between the male and female offspring, there is not, however, any cross analysis that could link both omics data. Overall, as discussed below the results do not support some conclusions. In particular this reviewer has the following concerns and suggestions.
- The metabolic status of the obese mothers has a direct impact on the offspring. It was previously reported that differences in glucose tolerance on the mother has a strong impact on the sex dimorphism in the metabolism of the progeny (please see the review: Sex and gender differences in developmental programming of metabolism. Molecular Metabolism 15 (2018) 8-19. What are the levels of glucose tolerance on the mothers used in the study? The weight of the mothers could also be shown.
ANSWER: We added the body weight and the Dam glucose level after 2 h fasting. We observed an increased BW in obese mother and no differences in glucose level compared to control diet fed mothers. We included these results in the Figure 1b.
The fact that mice were fed a high-fat diet after weaning could lead to confounding effects. Indeed, the lack of a group of mice fed a normal diet after weaning makes difficult to establish which is the relative contribution to the phenotype of the diet of the progeny, compared to the diet of the mother.
ANSWER: We published a paper in 2021 (PMID: 33398027) where we show that offspring born from obese mothers have sex specific hepatic modulation and provide molecular evidence of sex dependent in utero metabolic adaptation in the offspring born from obese mothers. In this study offspring were fed the CD after weaning and we demonstrated some reversal effect of CD feeding in male offspring.
Changes in food intake could also explain some differences.
ANSWER: We measured average food intake twice a week during three weeks at about 4-month of age, we presented the data in Fig.S1a and in the result section page 5 (Line 104). Males weighed significantly more than females regardless of the maternal diet (Fig.1c), with higher food intake (Fig.S1a). Interestingly, food intake tended to be induced and reduced by MO in female and male, respectively.
Lipidomics data show major differences between males e and females. However, the impact of these differences in the distinct metabolic phenotypes is not addressed.
ANSWER: We have reformulated the major lipidomic data to integrate them into the sex dependent metabolic phenotypes we observed.
The estrogen family and its two respective receptors, ERα and ERβ, have been widely suggested to be protective against obesity, diabetes, and cardiovascular disease. Does the transcriptional profile consistent with changes in the activity of estrogen receptor signaling?
ANSWER: We have at the expression level of Era (we did not find Erb) in the liver of offspring and presented the data in Figure S1b. Era was higher expressed in females than males, independently of the maternal diet. MO had no effect on the expression level of Era. Interestingly, androgen receptor (Ar) was higher expressed in the liver of F-moC than M-moC, these differences vanished in moHF-offspring because MO tended to reduce and induce the expression level of Ar in female and male, respectively.
Epigenetic modifications likely underlie the differences between males and females. Histone modifications or DNA methylation analysis could further improve the study.
ANSWER: We agree with the reviewer that there is likely a sex dependent epigenetic modification. How maternal in utero environment can differently affect female and male offspring born from the same mother remain to be elucidated.
In the last figure the authors claim that MO prevents HCC, but no data about HCC is shown, only gene expression analysis.
ANSWER: We agree with the reviewer that showing HCC markers would have strength the conclusion on the possible role of MO and sex in HCC development. However, we did not have the possibility to run western blot or IHC on our livers. Nevertheless, H&E staining showed bigger cell proliferation spots in females than in males and a reduction of the size of these spots in females born from obese mothers as compared to those born from lean mothers, in line with the pathways analysis and gene expression. Further studies focusing on HCC development in obesity would be needed to unravel the mechanism behind.
Reviewer #4 (Significance (Required)):
The first part of the study where the authors characterize the metabolism of the progeny, including weight, fat mass in the distinct depots, glucose and insulin tolerance, is not novel. Several publications have previously reported these findings (Programming effects of maternal and gestational obesity on offspring metabolism and metabolic inflammation. Sci Rep. 2019 Nov 5;9(1):16027). It was also previously reported in the cited publication, increased liver weight, steatosis and TG content, similar to the results of the present study.
**Referee Cross-commmenting**
I agree with the comments. I also think that the MS is difficult to read. No conexion between the OMICS data.
ANSWER: The current version of the manuscript has been extensively revised and we believe that it has improved in clarity and in novelty.
-
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Referee #3
Evidence, reproducibility and clarity
Maternal obesity is a common condition in western society. There is abundant literature showing the deleterious metabolic consequences of MO in the offspring. In this manuscript, Savva et al. characterized the transcriptomic and lipidomic profiles of the liver in male and female progeny of female mice that were fed a high-fat diet during and before pregnancy. After weaning, mice were also fed a high-fat diet. They found that both transcription and lipid composition were different in males and females, and they show that females are protected to metabolic and liver disease, whereas males develop insulin resistance, liver steatosis, …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #3
Evidence, reproducibility and clarity
Maternal obesity is a common condition in western society. There is abundant literature showing the deleterious metabolic consequences of MO in the offspring. In this manuscript, Savva et al. characterized the transcriptomic and lipidomic profiles of the liver in male and female progeny of female mice that were fed a high-fat diet during and before pregnancy. After weaning, mice were also fed a high-fat diet. They found that both transcription and lipid composition were different in males and females, and they show that females are protected to metabolic and liver disease, whereas males develop insulin resistance, liver steatosis, and are prone to develop liver cancer. The first part of the study where the authors characterize the metabolism of the progeny, including weight, fat mass in the distinct depots, glucose and insulin tolerance, is not novel. Several publications have previously reported these findings (Programming effects of maternal and gestational obesity on offspring metabolism and metabolic inflammation. Sci Rep. 2019 Nov 5;9(1):16027). It was also previously reported in the cited publication, increased liver weight, steatosis and TG content, similar to the results of the present study. Some novelty of the manuscript is the in-depth analysis of the lipid content of the liver in the models used, as well as the transcriptional profile. Despite the substantial amount of data that the authors generated to prove differences between the male and female offspring, there is not, however, any cross analysis that could link both omics data. Overall, as discussed below the results do not support some conclusions. In particular this reviewer has the following concerns and suggestions.
- The metabolic status of the obese mothers has a direct impact on the offspring. It was previously reported that differences in glucose tolerance on the mother has a strong impact on the sex dimorphism in the metabolism of the progeny (please see the review: Sex and gender differences in developmental programming of metabolism. Molecular Metabolism 15 (2018) 8-19. What are the levels of glucose tolerance on the mothers used in the study? The weight of the mothers could also be shown.
- The fact that mice were fed a high-fat diet after weaning could lead to confounding effects. Indeed, the lack of a group of mice fed a normal diet after weaning makes difficult to establish which is the relative contribution to the phenotype of the diet of the progeny, compared to the diet of the mother.
- Changes in food intake could also explain some differences.
- Lipidomics data show major differences between males e and females. However, the impact of these differences in the distinct metabolic phenotypes is not addressed.
- The estrogen family and its two respective receptors, ERα and ERβ, have been widely suggested to be protective against obesity, diabetes, and cardiovascular disease. Does the transcriptional profile consistent with changes in the activity of estrogen receptor signaling?
- Epigenetic modifications likely underlie the differences between males and females. Histone modifications or DNA methylation analysis could further improve the study.
- In the last figure the authors claim that MO prevents HCC, but no data about HCC is shown, only gene expression analysis.
Significance
The first part of the study where the authors characterize the metabolism of the progeny, including weight, fat mass in the distinct depots, glucose and insulin tolerance, is not novel. Several publications have previously reported these findings (Programming effects of maternal and gestational obesity on offspring metabolism and metabolic inflammation. Sci Rep. 2019 Nov 5;9(1):16027). It was also previously reported in the cited publication, increased liver weight, steatosis and TG content, similar to the results of the present study.
Referee Cross-commmenting
I agree with the comments. I also think that the MS is difficult to read. No conexion between the OMICS data.
-
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In this paper Savvaet al. explore how maternal obesity influence hepatic metabolism in a sex-specific fashion. They first assess the contribution of the adipose tissue to the development of insulin resistance and glucose intolerance focusing on inflammation and oxidative phosphorylation pathways. Then they proceed to asses if maternal obesity could remodel the hepatic triglyceride levels and phospholipids using proton magnetic resonance spectroscopy and LC-MS lipidomic, respectively. Finally, they explore hepatic lipid metabolism and genes promoting cancer development.
Despite the methodology is correct and elegant, the study does …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #2
Evidence, reproducibility and clarity
In this paper Savvaet al. explore how maternal obesity influence hepatic metabolism in a sex-specific fashion. They first assess the contribution of the adipose tissue to the development of insulin resistance and glucose intolerance focusing on inflammation and oxidative phosphorylation pathways. Then they proceed to asses if maternal obesity could remodel the hepatic triglyceride levels and phospholipids using proton magnetic resonance spectroscopy and LC-MS lipidomic, respectively. Finally, they explore hepatic lipid metabolism and genes promoting cancer development.
Despite the methodology is correct and elegant, the study does not explore a possible mechanism of action and some results are contradictory. Indeed, some of the results seems to be driven by the sex of the offspring independently of the maternal feeding.
There are indeed, some limitations for the authors and editors to consider. To this reviewer the manuscript is difficult to read, particularly the results section in which the data are listed without discussing their relevance or their connection to previous research from other groups. Moreover, the discussion could benefit from an extensive rewrite. Indeed, this section lacks of clarity and references that could help elucidate the novel finding of the authors.
Major comments:
Line 97: How the authors explain similar weight gain in the F-C/HF vs. F-HF/HF? A large body of literature reports that maternal high fat diet influences offspring weight gain, independently of sex, when compared to maternal standard diet (PMID: 23973955; 29872021; 31076636; 3036829).
Line 99: Which is the explanation for the reduced body weight in M-HF/HF from birth until 9 week of age? Can the authors show the timeline for food intake?
Line 101: How the authors explain the increase in final weight of the male compared to the female if no differences in total fat, VAT or SAT was observed between the offspring?
Line 116: The authors state: "The ratio between the total SAT and the Abd SAT revealed that MO redistributed SAT outside of the abdominal region in females but not in males" but Fig.1g displays no significant differences between F-C/HF and F-HF/HF. Please explain.
Line 128-130: The authors state: "At MID, glucose tolerance was highly diet- and sex-dependent, and males but not females showed impaired glucose tolerance by MO." However, in Fig.1h no significant differences in glucose peak or glucose AUC were observed between M-C/HF and M-HF/F. Please explain.
Line 130: OGTT only provides information on insulin secretion and action but does not directly yield a measure of insulin sensitivity. Please rephrase.
Authors should rephrase the conclusion of the paragraph since MO does not seems to influence fat distribution or insulin resistance. Looking at figure 1 it seems that the only differences observed are driven by the sex of the offspring independently of maternal feeding. Do the dams were insulin resistant? Indeed, hyperinsulinemia and insulin resistance are key programming factor of offspring metabolic syndrome.
Line 162: "There were no significant differences between the sexes. However, it is interesting to note that several pathways were regulated differently between sexes between the C/HF and HF/HF groups." Can the authors rephrase the concept, it is unclear.
Line 170: The authors state: "insulin secretion pathway is reduced in males only". How this results are in line with the data reported in Fig.1i were both M-C/HF and M-HF/HF display increased insulin secretion?
Lines 174-175: All the genes reported in Fig. 1o, except for LPIN1, do not seems to be altered by MO. Please rephrase.
Line 184: The authors state that the signaling pathways was assessed both at transcriptional and post-transcriptional levels. Where are depicted the data of the post-transcriptional levels?
Similarly to glucose metabolism and fat depot results, also in the case of the liver steatosis the increased number of lipid droplets seems to be linked to the sex of the animal rather than the maternal diet. Since the authors also investigated inflammatory pathways it could be of interest to assess CD68 infiltration by immunohistochemistry and Picrosirius red staining for the assessment of fibrosis.
Liver histology in Fig.5a (M-C/HFD) is completely different from the one depicted in Fig.4a in terms of steatosis. Can the authors please explain this difference and report the magnification used in Fig.4a. Please also report the scale in Fig.5a.
Changes in placental function are thought to be a key link between the maternal and intrauterine milieu and long-term health of the offspring (PMID:24107818). Alterations in placental function and structure in response to obesity and their underlying molecular mechanisms have been explored both in humans and in animal models (PMID:24484739; 22303323; 28291256). Others have shown that maternal hyperinsulinemia is strongly associated with offspring insulin resistance and excess placental lipid deposition and hypoxia (PMID: 28291256). Excessive lipid deposition leads to a lipotoxic placental environment that is associated with increased markers of inflammation and oxidative stress (PMID: 24333048). Can the authors could provide some data?
Minor:
Fig. 2m Acox1 is not reduced by MO in female. Supp. TableS1 do not report Pdk1, Lpin1, Nox4 and Prlr. Supp. TableS2 do not report PCSK9 and PNPLA3
Significance
The prevalence of obesity during pregnancy continues to increase at alarming rates. This is concerning as in addition to immediate impacts on maternal wellbeing, obesity during pregnancy has detrimental effects on the long-term health of the offspring. This paper is connected to an extended research field aiming at prevent the detrimental effect of maternal obesity on the offspring.
An important limitation in the ability to design intervention strategies to prevent the detrimental effects of maternal obesity on offspring health is that it is currently unclear which of the many potential variables associated with obesity is the key programming factor mediating the effects on the offspring.
Reviewer field of expertise:
Molecular Biology, Type 2 Diabetes, Obesity and NAFLD.
Referee Cross-commenting
I agree with the other reviewers' comments, particularly on the lack of mechanistic data.
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Referee #1
Evidence, reproducibility and clarity
The manuscript from Savva et al. focuses on a long-standing and unresolved challenge of metabolic (and not only) health in mammals: the sexual dimorphism. Authors couple transcriptomics and metabolomics to in-depth molecular phenotyping in offspring of dams fed HFD before conception and throughout pregnancy and lactation to isolate molecular determinants of sexually dimorphic response to maternal obesity.
Major comments:
- While the manuscript present a compelling and exhaustive amount of data, which accurately describes the sexually dimorphic responses to maternal obesity, it lacks mechanistic insights and I personally think this to …
Note: This preprint has been reviewed by subject experts for Review Commons. Content has not been altered except for formatting.
Learn more at Review Commons
Referee #1
Evidence, reproducibility and clarity
The manuscript from Savva et al. focuses on a long-standing and unresolved challenge of metabolic (and not only) health in mammals: the sexual dimorphism. Authors couple transcriptomics and metabolomics to in-depth molecular phenotyping in offspring of dams fed HFD before conception and throughout pregnancy and lactation to isolate molecular determinants of sexually dimorphic response to maternal obesity.
Major comments:
- While the manuscript present a compelling and exhaustive amount of data, which accurately describes the sexually dimorphic responses to maternal obesity, it lacks mechanistic insights and I personally think this to mainly be a timing issue. For example, I tried hard to find experimental details on the RNA sequencing and could not find much: when is the RNA sequencing performed? If, as I suspect, the sequencing experiments match the metabolomics experiments, I don't think they add much mechanistic insights onto the observed phenomena. They rather contribute to better describe them. Indeed, both metabolomic and transcriptomic profiles might be consequence of the observed phenotypes, rather than be causative (as the authors try to argue). Are these differences already present at birth? what happens to placenta and fetal tissues?
- Some adult phenotypes - especially metabolic and neurological phenotypes - might also be influenced by different maternal care early postnatal. Are the litters balanced by number and sex ratio? Would cross-fostering maintain the phenotypes?
Of the two points above, I would love to see more details on the RNA sequencing, as well as placental and fetal tissues analysed. It would be also interesting to know about any litter balancing measure or at least have more statistics on the litter size and sex ratios.
Significance
The manuscript from Savva et al. revolves around the unresolved challenge of how sexually dimorphic phenotypes are established. The topic is actual - although already a lot has been published, as acknowledged by the authors as well - and of broad interest to the community of mouse geneticists and physiologists. To understand the molecular underpinnings of sexually dimorphic phenotypes, the authors use in-depth molecular phenotyping in the mouse coupled to metabolomics and transcriptomics. While extremely informative and exhaustive, the actual dataset is - at least for me - purely descriptive, which might reduce its overall impact. I'm a mouse geneticist and a metabolic physiologist and I find the topic of sexual dimorphism extremely interesting.
Referee Cross-commenting
I generally agree with the other reviewers' comments. I think the ms is interesting and the dataset compelling although to a certain extent overlapping with previously published studies. There is general agreement on the lack of mechanistic data and the authors should definitely address this point.
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