Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits
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Evaluation Summary:
This is an exemplary manuscript describing the creation of a novel mouse model to study bone marrow adipocytes. The authors demonstrate that these cells play a critical role in the pathophysiology of myeloid cell lineage regeneration as well as the maintenance of bone mass and hematopoietic progenitors during times of limited energy (e.g. caloric restriction).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Abstract
To investigate roles for bone marrow adipocyte (BMAd) lipolysis in bone homeostasis, we created a BMAd-specific Cre mouse model in which we knocked out adipose triglyceride lipase (ATGL, Pnpla2 gene). BMAd- Pnpla2 -/- mice have impaired BMAd lipolysis, and increased size and number of BMAds at baseline. Although energy from BMAd lipid stores is largely dispensable when mice are fed ad libitum, BMAd lipolysis is necessary to maintain myelopoiesis and bone mass under caloric restriction. BMAd-specific Pnpla2 deficiency compounds the effects of caloric restriction on loss of trabecular bone in male mice, likely due to impaired osteoblast expression of collagen genes and reduced osteoid synthesis. RNA sequencing analysis of bone marrow adipose tissue reveals that caloric restriction induces dramatic elevations in extracellular matrix organization and skeletal development genes, and energy from BMAd is required for these adaptations. BMAd-derived energy supply is also required for bone regeneration upon injury, and maintenance of bone mass with cold exposure.
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Evaluation Summary:
This is an exemplary manuscript describing the creation of a novel mouse model to study bone marrow adipocytes. The authors demonstrate that these cells play a critical role in the pathophysiology of myeloid cell lineage regeneration as well as the maintenance of bone mass and hematopoietic progenitors during times of limited energy (e.g. caloric restriction).
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
Overall this is a large body of work that in the first part describes the creation of a mouse model that uses Osterix and Adipoq to target the elimination of adipose triglyceride lipase (Pnpla2) to bone marrow adipocytes. The creation and the phenotype of these mice are explored in detail. Then the authors turn to several experiments to flush out the role that these fat stores play in the bone marrow. They convincingly demonstrate through caloric restriction models the role these cells play in the bone marrow such as the role in progenitor survival as well as sustaining osteoblast-mediated bone mass.
The major limitations of the study have been largely noted by the authors who commendably dedicate substantial time to discussion of them - and that is the imperfections in the mouse model that was created for …
Reviewer #1 (Public Review):
Overall this is a large body of work that in the first part describes the creation of a mouse model that uses Osterix and Adipoq to target the elimination of adipose triglyceride lipase (Pnpla2) to bone marrow adipocytes. The creation and the phenotype of these mice are explored in detail. Then the authors turn to several experiments to flush out the role that these fat stores play in the bone marrow. They convincingly demonstrate through caloric restriction models the role these cells play in the bone marrow such as the role in progenitor survival as well as sustaining osteoblast-mediated bone mass.
The major limitations of the study have been largely noted by the authors who commendably dedicate substantial time to discussion of them - and that is the imperfections in the mouse model that was created for the study. Because of these limitations, it is doubtful that in the current iteration the mouse model can be used for more than what has already been accomplished - largely confirming long hypothesized roles of bone marrow Adipocytes.
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Reviewer #2 (Public Review):
This manuscript addresses the role of Pnpla2 gene coding for ATGL protein in bone marrow adipocytes (BMAd) for the maintenance of bone mass and hematopoiesis in basal and under stress conditions. A major strength of this manuscript consists of presenting a new mouse model for tissue-specific genetic modification of BMAd. This model provides a long-sought tool for advancement of research on the role of adipocytes in bone marrow environment.
Using that model, the authors constructed mice with BMAd-specific deletion of Pnpla2 and tested it in several different conditions of increased local energy demands including calorie restriction, de novo hematopoiesis, local bone regeneration, and chronic exposure to cold. Although presented studies have a number of strengths, the conclusions are clouded by experiments …
Reviewer #2 (Public Review):
This manuscript addresses the role of Pnpla2 gene coding for ATGL protein in bone marrow adipocytes (BMAd) for the maintenance of bone mass and hematopoiesis in basal and under stress conditions. A major strength of this manuscript consists of presenting a new mouse model for tissue-specific genetic modification of BMAd. This model provides a long-sought tool for advancement of research on the role of adipocytes in bone marrow environment.
Using that model, the authors constructed mice with BMAd-specific deletion of Pnpla2 and tested it in several different conditions of increased local energy demands including calorie restriction, de novo hematopoiesis, local bone regeneration, and chronic exposure to cold. Although presented studies have a number of strengths, the conclusions are clouded by experiments that are difficult to interpret due to either their design or the way of analysis. Some experimental results are unexpected and counterintuitive in respect to leading hypothesis and require deeper analysis. In general, most experiments consist of phenotyping the model with no mechanistic studies. The hypothesis that BMAd provides NEFA which fuels bone formation, hematopoiesis, bone regeneration, and provides protection from bone loss during cold exposure, is not proven to satisfaction. Contrary, the presented data suggest a possibility of other mechanisms, besides providing energy, as being under ATGL control and having signaling functions. This possibility is neither tested nor discussed. There is an increasing number of reports on proteins with well-defined enzymatic function to have additional signaling activities unrelated to their enzymatic activities. Perhaps ATGL is one of them.
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Reviewer #3 (Public Review):
Bone marrow adipocytes (BMAds) are a major component of the bone marrow and accumulate further in diverse clinical conditions, suggesting that these cells play important roles in both normal physiology and in disease states. BMAds also accumulate during caloric restriction, demonstrating that they are functionally distinct from adipocytes in white or brown adipose tissues. Despite their pathophysiological potential and unique characteristics, the functions of BMAds have remained poorly understood, especially in comparison to adipocytes elsewhere in the body.
In this manuscript, Li et al address this important gap in knowledge by establishing a new mouse model that allows specific transgenic targeting of BMAds. To do so they use an ingenious approach involving the combination of two novel transgenic mouse …
Reviewer #3 (Public Review):
Bone marrow adipocytes (BMAds) are a major component of the bone marrow and accumulate further in diverse clinical conditions, suggesting that these cells play important roles in both normal physiology and in disease states. BMAds also accumulate during caloric restriction, demonstrating that they are functionally distinct from adipocytes in white or brown adipose tissues. Despite their pathophysiological potential and unique characteristics, the functions of BMAds have remained poorly understood, especially in comparison to adipocytes elsewhere in the body.
In this manuscript, Li et al address this important gap in knowledge by establishing a new mouse model that allows specific transgenic targeting of BMAds. To do so they use an ingenious approach involving the combination of two novel transgenic mouse lines. In one line, named Osterix-FLPo mice, a flippase (FLPo) is expressed under the control of the Osterix promoter, which is active in osteoblasts and BMAds but not in white adipocytes. In the second line, named FAC (FLPo-dependent Adipoq-Cre) mice, an inverted Cre sequence, flanked by flippase recombination sites, is inserted into the 3`-untranslated region of the adiponectin gene (Adipoq); importantly, Adipoq is expressed in adipocytes (including BMAds) but not in osteoblasts. Because the Cre sequence is inverted in the FAC mice, in the absence of flippase no functional Cre is expressed. Thus, the authors bred the Osterix-FLPo mice with the FAC mice to generate BMAd-Cre mice, in which flippase is expressed in both osteoblasts and BMAds only. This causes the FAC Cre sequence to be flipped into the correct orientation in BMAds and osteoblasts, but not in other cell types. Because the Adipoq promoter is not active in osteoblasts, Cre expression is therefore specific to BMAds.
After convincingly validating this BMAd-Cre transgenic model, the authors use it to investigate how BMAds influence metabolism, bone remodelling and hematopoiesis. To do so they combine the BMAd-Cre mice with floxed-Pnpla2 mice, thereby deleting Pnpla2 in BMAds but not in other cell types. Pnpla2 encodes adipose triglyceride lipase (ATGL), the first and rate-limiting enzyme in lipolysis, in which triacylglycerols are catabolised to release non-esterified fatty acids and glycerol. The resulting Pnpla2 KO mice, therefore, have BMAds that are unable to undergo stimulated lipolysis. The authors convincingly demonstrate this lipolytic defect and how it results in increased bone marrow adiposity in the KO mice. They then assess the metabolic and skeletal consequences of this lipolytic defect.
On a normal, ad libitum (AL) diet, the BMAd-Pnpla2-KO mice display no overt metabolic or skeletal phenotype compared to BMAd-Pnpla2-WT mice. Therefore, the authors challenged the mice with caloric restriction (CR), reasoning that BMAd lipolysis may be more important under conditions of limited energy availability. During CR the KO mice show a similar metabolic phenotype to the WT mice; however, differences in bone and hematopoietic parameters become apparent. The CR studies are done in males and females and, after observing some sex differences, a further cohort of females is studied following ovariectomy. The molecular basis for these skeletal and hematological phenotypes is then investigated by bulk RNA sequencing of the distal tibial bone marrow samples from the KO and WT males under AL or CR conditions. Finally, the authors move beyond CR to investigate if BMAd lipolysis influences skeletal remodelling following bone injury or during cold exposure.
The authors conclude that BMAd lipolysis is required to support bone homeostasis and myelopoiesis during caloric restriction, at least in male mice. They also show convincingly that deficient BMAd lipolysis compromises post-injury bone regeneration and might exacerbate bone loss during cold exposure. Through comprehensive skeletal phenotyping they conclude that, during CR, BMAd-Pnpla2-KO mice have decreased trabecular bone owing to an impaired ability of osteoblasts to secrete osteoid; this conclusion is further supported by RNA sequencing analyses of distal tibial bone marrow. Most of the authors' other conclusions are also supported by the data presented. Despite some weaknesses, overall this study represents an important advance in our understanding of BMAd function and stands out for establishing a new transgenic model that is likely to be extremely useful for future BMAd research.
Strengths:
1. The authors have taken an ingenious approach for specific transgenic targeting of BMAds, establishing the first BMAd-specific model. This should be an extremely useful tool for the burgeoning field of BMAd research. The authors convincingly demonstrate the specificity of this BMAd-Cre model, including the use of lineage tracing and PCR to show that Cre is expressed in BMAds but not in other cell types. One exception is Cre expression in a small subset of stromal/dendritic cells within the bone marrow, but this is only a minor limitation that should not compromise the utility of the model for further exploring BMAd function. However, there are two more-substantial limitations to the model: unexpected effects on circulating adiponectin, and relatively low Cre expression in younger mice (see below under 'Weaknesses').
2. The effects of CR are studied in males and females, highlighting sex differences in the phenotypes.
3. For the CR studies, the skeletal phenotyping and RNAseq analyses have generally been done to a very high standard. The authors conclude that, under CR, BMAds provide energy to maintain osteoblasts' secretion of collagen matrix for osteoid synthesis. The RNAseq data identify molecular changes consistent with these conclusions. However, there is one minor limitation to how the RNAseq data are analysed (see 'Weaknesses').
Weaknesses:
1. One of the biggest limitations concerns the BMAd-Cre model: compared to non-Cre controls, the BMAd-Cre mice have decreased adiponectin protein expression in white adipose tissue and bone marrow adipose tissue and a ~70% decrease in circulating adiponectin concentrations. The authors speculate that this may result from the insertion of the inverted Cre sequence within the 3`-UTR impairing translation of adiponectin protein. One previous mouse CR study found that adiponectin knockout limits BMAd accumulation and bone loss during CR; thus, decreased circulating adiponectin in the BMAd-Cre mice might confound the interpretation of how the BMAd-Pnpla2-KO is impacting bone homeostasis during CR. Despite this possibility, the authors do not measure circulating adiponectin in the BMAd-Pnpla2-WT or BMAd-Pnpla2-KO mice on AL or CR diets. This should be measured to determine if CR is still capable of increasing circulating adiponectin in the BMAd-Pnpla2-WT and BMAd-Pnpla2-KO mice. It would also be informative to compare their circulating adiponectin concentrations to those of non-Cre Pnpla2-fl/fl mice, both on AL and CR diets, as an additional control.
Another limitation, highlighted by the authors, is that, because of the architecture of the FAC transgene, the BMAd-Cre mice have relatively low expression of Cre, resulting in lower rates of recombination in younger mice. This may limit the use of the model to mice older than 16 weeks of age.
2. The second general limitation of the study is that the effects of the KO (i.e. BMAd-Pnpla2-WT vs BMAd-Pnpla2-KO) are generally assessed within each diet (AL or CR), thereby missing the possibility of detecting genotype-diet interactions. Moreover, for the male CR studies data for metabolic and bone parameters are presented completely separately for the AL mice and for the CR mice, which prevents analysis of CR effects. For some studies the four groups of mice (i.e. with diet and genotype as the two independent variables) are shown on the same graphs, yet the data are analysed only using 1-way ANOVA. As a result, it is not possible to detect genotype-diet interactions. It would be hugely informative for all the data to be presented with the four groups alongside each other, and to analyse these using 2-way ANOVA. This would confirm if the CR intervention has had expected metabolic and skeletal effects and may reveal new effects of the BMAd-Pnpla2-KO that currently are going undetected.
3. The bone regeneration studies are done in male mice only, while the cold-exposure studies are in females only. Given the sex differences in how BMAd-Pnpla2-KO impacts CR, it would be informative to know if similar sex differences occur in these other contexts.
4. The cold exposure studies present only a limited analysis of the bone phenotype that reveals only minor effects. Therefore, the conclusion from these studies (that BMAd lipolysis is needed to maintain bone mass during cold exposure) is less convincing than the conclusions from the CR or bone regeneration studies.
5. In the RNAseq analysis, the authors first compare the effects of CR in the BMAd-Pnpla2-WT mice. To determine how BMAd-Pnpla2-KO influences the CR response, they then compare transcript expression between the CR BMAd-Pnpla2-KO mice and the AL BMAd-Pnpla2-WT mice. This comparison is invalid because it is being made across two independent variables (diet and genotype) and therefore it may miss some of the true effects of the KO on the CR response. However, it appears that transcript expression is similar between the KO and WT mice on an AL diet, and therefore correcting this analysis (so that CR KO mice are compared to AL KO mice) may not substantially alter the authors' conclusions.
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