Paternal multigenerational exposure to an obesogenic diet drives epigenetic predisposition to metabolic diseases in mice
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Abstract
Obesity is a growing societal scourge. Recent studies have uncovered that paternal excessive weight induced by an unbalanced diet affects the metabolic health of offspring. These reports mainly employed single-generation male exposure. However, the consequences of multigenerational unbalanced diet feeding on the metabolic health of progeny remain largely unknown. Here, we show that maintaining paternal Western diet feeding for five consecutive generations in mice induces an enhancement in fat mass and related metabolic diseases over generations. Strikingly, chow-diet-fed progenies from these multigenerational Western-diet-fed males develop a ‘healthy’ overweight phenotype characterized by normal glucose metabolism and without fatty liver that persists for four subsequent generations. Mechanistically, sperm RNA microinjection experiments into zygotes suggest that sperm RNAs are sufficient for establishment but not for long-term maintenance of epigenetic inheritance of metabolic pathologies. Progressive and permanent metabolic deregulation induced by successive paternal Western-diet-fed generations may contribute to the worldwide epidemic of metabolic diseases.
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###Reviewer #3:
In this interesting study, the authors explored the effect of five consecutive generations of high-fat high-sugar diet (WD) in mice and their offspring's metabolic performance under a normal chow diet. It is very interesting to find that the chow-diet-fed progenies from these multigenerational western-diet-fed males develop a "healthy" overweight phenotype (which means without problem of glucose metabolism and fatty liver abnormalities) that persist 4 subsequent generations. In parallel, the authors also performed zygotic sperm RNA injection using sperm RNAs from the WD-fed males (both from first generation and five generations of feeding) and showed that the sperm RNA indeed induce offspring metabolic phenotypes in F1 mice and some phenotypes persist to F2-F3, but none persist to F4, which is different from the mating …
###Reviewer #3:
In this interesting study, the authors explored the effect of five consecutive generations of high-fat high-sugar diet (WD) in mice and their offspring's metabolic performance under a normal chow diet. It is very interesting to find that the chow-diet-fed progenies from these multigenerational western-diet-fed males develop a "healthy" overweight phenotype (which means without problem of glucose metabolism and fatty liver abnormalities) that persist 4 subsequent generations. In parallel, the authors also performed zygotic sperm RNA injection using sperm RNAs from the WD-fed males (both from first generation and five generations of feeding) and showed that the sperm RNA indeed induce offspring metabolic phenotypes in F1 mice and some phenotypes persist to F2-F3, but none persist to F4, which is different from the mating induced phenotype (last 4 generations). The study is overall well-performed and the comprehensive examinations (especially on phenotypes) represent an advance to the mammalian epigenetic inheritance field. I have a few concerns and suggestions for further improvement.
In the abstract, I strongly recommend the authors to clarify what is a "healthy" overweight phenotype, which in the current paper means normal glucose metabolism and without fatty liver. This will make the information in the abstract more informative and precise. In fact, this is the major novel discovery in the phenotypic exploration, not only for social-medical implications, but also from the perspective of evolution. It looks like the five-generational western-diet-fed males have evolved to develop a protective mechanism in glucose and liver fat metabolism that can be inherited by the offspring. The underlying mechanism is intriguing and worth exploring in the future using this model. More extensive discussions on the social-medical and evolutionary aspects could be included.
Regarding the phenotype induced by sperm RNA injection, the description should be more precise as the current description is not all consistent with the data presented. In Figure.4, some parameter changes persist to F2-F3, this already suggests transgenerational inheritance rather than merely intergenerational transmission. The more precise description should be that sperm RNAs can unequivocally induce intergenerational phenotype, but may induce some transgenerational features - although the effect is weaker than the effect induced by whole sperm. In fact, in a previous study using a mental-stress induced model, sperm RNA injection can also induce phenotype in both F1 and F2 generations (Nat Neurosci. 2014 May;17(5):667-9.).
The sperm small RNA analysis part (Fig. S4) is relatively weak. The datasets generated are in fact quite valuable as they include the sperm from the control diet, first-generation WD and the Fifth-generation WD. This is an opportunity to explore the difference especially between the first-generation WD and Fifth-generation WD as no one has done this before. The current data analyses are crude and did not show these differences in an informative way. It is needed to at least provide the overall length distribution of each datasets with the annotation of different types of small RNAs. The authors have shown some difference regarding miRNAs and tRNA-derived small RNAs (tsRNAs) in Fig.S4, it would be interesting to also look at the rRNA-derived small RNAs (rsRNAs) because rsRNAs are also extensively discovered in both mouse and human sperm and these sperm rsRNAs are sensitive to dietary changes (Nat Cell Biol. 2018 May;20(5):535-540; PLoS Biol. 2019 Dec 26;17(12):e3000559.), closely associated with mammalian epigenetic inheritance and thus represent a component of the recently proposed sperm RNA code in epigenetic inheritance (Nat Rev Endocrinol. 2019 Aug;15(8):489-498). The reanalysis of the datasets could be done by SPORTS1.0 (Genomics Proteomics Bioinformatics. 2018 Apr;16(2):144-151.), which provide the annotation and analyses of miRNAs, tsRNA, rsRNAs and piRNAs that have been used in the above mentioned publications (Nat Cell Biol. 2018 May;20(5):535-540; PLoS Biol. 2019 Dec 26;17(12):e3000559)
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###Reviewer #2:
Raad et al. examined the effects of multigenerational paternal exposure to an obesogenic diet on epigenetic and metabolic alterations at somatic and germ cell levels. The experimental work addresses an important question. The findings are intriguing that sperm mRNA and natural crosses have different effects on offspring metabolic states. The major tissue of interest explored was WAT. Fat cell size, no and gene expression were reported. The intriguing thing about these data is that the sperm RNA microinjection did not fully recapitulate the effect across multiple generations - there is little explanation of potential mechanisms.
There is no detailed coverage of the gene changes, small RNAs, piRNAs etc observed and the pathways implicated. This would be a welcome addition.
As this is such a complex design, more overall …
###Reviewer #2:
Raad et al. examined the effects of multigenerational paternal exposure to an obesogenic diet on epigenetic and metabolic alterations at somatic and germ cell levels. The experimental work addresses an important question. The findings are intriguing that sperm mRNA and natural crosses have different effects on offspring metabolic states. The major tissue of interest explored was WAT. Fat cell size, no and gene expression were reported. The intriguing thing about these data is that the sperm RNA microinjection did not fully recapitulate the effect across multiple generations - there is little explanation of potential mechanisms.
There is no detailed coverage of the gene changes, small RNAs, piRNAs etc observed and the pathways implicated. This would be a welcome addition.
As this is such a complex design, more overall schematics would be helpful.
Number of mice per group ranges widely, and it is unclear how many matings this represents. Fig 3 legend states 4 WD1 and 9 WD5 males from different littermates were mated with CD females - again, unclear - do you mean from different litters? Numbers shown in panel A do not seem to concur with those in panels B, C
Figure 1 shows outcomes for WD 1,2,3,4,5 and largely focuses on gWAT. Gene expression changes are only briefly summarised. Only 1 CD generation is represented.
It is unclear why mice were studied at the various ages- eg Across data sets, ages shown range from 10 weeks, 12 weeks, 16 weeks, 18 weeks. Note there are inconsistencies regarding figure formats and some details are missing, which makes it hard to understand what the authors found. Fig S3 and S5- no n values given. Labels in S4 D, E hard to follow.
In several of the figures, it is not clear what the significance (*) is being compared to - is it always CD? Eg Figure 3, Figure 4
It appears that variability increases from WD1 to WD5- with larger ranges evident- is this why n increases across generations? And is this a consistent observation across paternal studies of this kind?
The effect of paternal WD on BW, GTT and adiposity is relatively larger in mice than rats- have the authors considered species differences?
One page 10 the authors state that the diet used is not associated with hepatic steatosis - but I would have thought there was good evidence of this occurring in mice, over the timeframe described here.
The intriguing thing about these data is that the sperm RNA microinjection did not fully recapitulate the effect across multiple generations - there is little explanation of potential mechanisms.
It is surprising that there is no detailed coverage of the gene changes observed and the metabolic pathways implicated. The story is undersold.
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###Reviewer #1:
While this study is focusing on an interesting hypothesis and attempting to address the molecular mechanisms at play, there are numerous flaws in the study design and the statistical test that prevail from drawing conclusions.
In line 72, the authors state that "the average body weight of the WD-fed male mice increased gradually with multigenerational WD feeding", however, the results of the test indicating gradual increase is not reported. As described in the legend of Figure 1, the test performed tested differences in body weight between the control group and each individual generation, not the generations to each other. Visually, it rather seems that in fact, body weight was not gradually increased for instance, comparison of WD1 and WD3, or WD2 and WD5, does not support the "gradual increase" in body weight that the …
###Reviewer #1:
While this study is focusing on an interesting hypothesis and attempting to address the molecular mechanisms at play, there are numerous flaws in the study design and the statistical test that prevail from drawing conclusions.
In line 72, the authors state that "the average body weight of the WD-fed male mice increased gradually with multigenerational WD feeding", however, the results of the test indicating gradual increase is not reported. As described in the legend of Figure 1, the test performed tested differences in body weight between the control group and each individual generation, not the generations to each other. Visually, it rather seems that in fact, body weight was not gradually increased for instance, comparison of WD1 and WD3, or WD2 and WD5, does not support the "gradual increase" in body weight that the authors are claiming.
There is a lack of clarity in the methods in regards to numbers of animals used in each generation, the number of founders, and what constitutes the control group. In the legend of Figure 1, it is stated that 5 males were used from WD2 and on. However, the method section states "(...) 4 to 6 independent males of WD1 group". The reviewer assumes that the authors know how many animals were used in the WD1 group, and that the authors meant 4 to 6 animals per WD generation. However, if the details indicated in the legend of Figure 1 are accurate (5 fathers per group from WD2), how is it possible that 4 to 6 animals were used? The reviewer suggests to clarify this in the text, as well as in a more detailed experimental setup diagram stating the number of fathers in each generation, the number of offspring studied in each litter, and the total number of offspring studied for each generation.
In Supplemental Figure 1I, the CD1 group appears to be composed of 7 individuals and the CD2 group of 10 individuals. This is not consistent with the numbers reported in Figure 1A (10 in CD1 and 13 in WD3) and Figure 1B (22 visible dots). It is thus difficult for the reviewer to trust that body weights were truly compared between all animals in CD1 and CD5. Regardless, the reviewer is intrigued by the choice of the authors to only study control animals from the first generation (CD1), and the fifth generation (CD5) offspring, as they describe in the methods that, for the control group, they followed the same procedure as the WD group, which should have led to the generation of control animals in all F1, F2, F3, F4 and F5 generations. The authors should clarify on this, and if they indeed generated these animals, they should use body weight data in each generation of controls and compare them to their respective generation WD group (i.e. CD1 to WD1, CD2 to WD2 etc..). By having different sample size in the various groups, the authors are biasing results of the statistical test being made, as greater sample size is likely to compare statistically different than a group with lower sample size (as with CD(22 observations) and WD2(12observations) in Figure 1B, but also with the RNA-seq results). In the same line, there were more animals studied in WD4 and WD5 compared to WD1-3 which is likely biasing statistical analysis. Again, if the study design described in the methods section is accurately reported, it implies that an average of 3 offspring per fathers were used in WD1-3, and 8-10 (a full litter) for the WD4-5.
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##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript. David E James (The University of Sydney) served as the Reviewing Editor.
###Summary:
In this manuscript, Raad and colleagues exposed male mice to a western diet before conception for 5 consecutive generations and measured body weight, adiposity and various metabolic markers in the offspring. Sequencing of small RNA in sperm from founders identified several differentially expressed tRF and miRNA species. Microinjection of RNAs recapitulated some, but not all effects on body weight and metabolism. The authors report an …
##Preprint Review
This preprint was reviewed using eLife’s Preprint Review service, which provides public peer reviews of manuscripts posted on bioRxiv for the benefit of the authors, readers, potential readers, and others interested in our assessment of the work. This review applies only to version 1 of the manuscript. David E James (The University of Sydney) served as the Reviewing Editor.
###Summary:
In this manuscript, Raad and colleagues exposed male mice to a western diet before conception for 5 consecutive generations and measured body weight, adiposity and various metabolic markers in the offspring. Sequencing of small RNA in sperm from founders identified several differentially expressed tRF and miRNA species. Microinjection of RNAs recapitulated some, but not all effects on body weight and metabolism. The authors report an aggravation of adiposity along generations and a phenotype that persists for 4 consequent generations. Such persistence of phenotype was not observed in animals originating from microinjection of total RNAs, suggesting other epigenetic mechanisms are at play in the persistence of phenotype. Overall the studies were considered to be of interest by the referees but one major overarching problem identified by them concerned the study design and the statistical analyses that limited interpretation of the study. These issues need to be seriously addressed by the authors. These and other points are listed below.
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