Lipolysis of bone marrow adipocytes is required to fuel bone and the marrow niche during energy deficits

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.

Keywords: adipose triglyceride lipase; bone; bone marrow adipocyte; caloric restriction; cell biology; hematopoiesis; lipolysis; mouse.

Figure 1.. Generation of a BMAd-specific Cre mouse model (BMAd-Cre).

(A) Efficacy of Osterix-FLPo was evaluated by crossing with FLP-dependent EGFP reporter to yield Osterix-EGFP. (B) Osterix-EGFP male mice at 16 weeks were sacrificed. Fresh tissue confocal microscopy was performed on bisected caudal vertebrae, distal tibia and proximal tibia. Red arrows indicate singly dispersed BMAds. Scale bar; 100 μm. (C) Frozen-sections of proximal tibial slides from Osterix-EGFP were stained with Anti-GFP (green) and DAPI (blue). Scale bar; 50 μm. (D) Schematic of how Osterix-FLPo recombines FLPo-activated Adipoq-Cre (FAC) in BMAd-Cre mice to restrict expression of Cre to BMAds. (E) BMAd-Cre mice were bred with mT/mG reporter mice and the resulting BMAd-mT/mG mice were sacrificed at 16 weeks of age. Cellular fluorescence was evaluated by fresh tissue confocal microscopy. Scale bar; 100 μm. (F) Proximal tibial sections from BMAd-mT/mG mice were stained with antibodies to tdTomato (red) and EGFP (green), and nuclei were counterstained with DAPI (blue). * indicates BMAds. Scale bar; 50 μm.

Figure 1—figure supplement 1.. Genotyping strategy and validation of BMAd-Cre.

(A-C) Schematic of genotyping primer designing for Osterix-FLPo (A) and FLPo activated Adipoq-Cre (FAC; B), and representative genotyping results (C). Mut = Mutation; WT = wildtype; Ori = original band without conversion of reversed Cre cassette; Flipped = FLPo activated Cre (BMAd-Cre). + indicates the presence of insertion; - indicates wild type. (D) The flipped recombination band was found in mRNA of bone, but not WAT depots. Mice expressing Osterix-FLPo with or without FAC were sacrificed and mRNA was isolated. cDNAs from distal tibiae, caudal vertebrae, epididymal and subcutaneous WATs were used for PCR and agarose gel electrophoresis. + indicates the presence of insertion; - indicates wild type. (E) Representative image of genomic PCR sequencing aligned with the endogenous allele. (F) BMAd-Cre mice were bred with mT/mG reporter mice and the resulting BMAd-mT/mG mice were sacrificed at 16 weeks of age. Cellular fluorescence was evaluated by fresh tissue confocal microscopy. Scale bar; 100 μm. (G-I) Efficiency of BMAd-Cre recombinase is age- and allele-dependent. Fresh caudal vertebrae collected from BMAd-mT/mG mice at indicated ages were bisected and used for confocal imaging. Scale bar; 100 μm. (I) Quantification of EGFP+ BMAds.

Figure 1—figure supplement 2.. Insertion of IRES-Cre cassette in the 3'UTR of endogenous Adipoq decreases expression of adiponectin but does not cause a metabolic or bone phenotype.

(A-F) Endogenous adiponectin expression is decreased by IRES-Cre insertion. Mice expressing Osterix-FLPo with or without FAC were sacrificed. Epididymal WAT (eWAT), caudal vertebrae, distal tibiae and serum were collected. + indicates the presence of insertion; - indicates wild type. (A) mRNA expression of adiponectin in eWAT, caudal vertebrae and distal tibiae from BMAd-Cre (OSX⁺FAC⁺) WT, heterozygous, and homozygous mice. (B-C) Lysates from eWAT (B) and caudal vertebrae (C) were used for immunoblot analyses of adiponectin and FABP4, with α-tubulin and ERK2 as loading controls. Quantification was performed using Image J. (D-F) Non-denatured (D) and denatured (E) serum from BMAd-Cre WT, heterozygous, or homozygous mice was used for immunoblot analyses of adiponectin, with albumin as a reference protein. (F) High (HMW), medium (MMW), and low (LMW) molecular weight forms of adiponectin were quantified by Image J. (G-P) Hypoadiponectinemia does not cause detectable phenotypes. Male BMAd-Cre WT, heterozygote, or homozygote mice were sacrificed at 18 weeks of age. Soft tissues and bones were collected. (G-K) Body weight, glucose tolerance, and WAT depot weights were not changed in BMAd-Cre mice. (L-P) Trabecular and cortical bone parameters were determined by μCT. Data are expressed as mean ± SD. * indicates p<0.05 with one-way ANOVA with Tukey’s multiple comparisons test.

Figure 2.. Ablation of adipose triglyceride lipase (ATGL; gene name Pnpla2) increases size and number of BMAd.

(A-J) Male mice of the indicated genotypes at 24 weeks of age were euthanized for investigation of white adipose tissue (WAT) and bone. (A) RNA was extracted from WAT and caudal vertebrae, and converted to cDNA. PCR products for wildtype (WT; 1257 bp) Pnpla2 and exon 2–7 knockout (Mut; 553 bp) bands were visualized. (B) Decalcified proximal tibiae were sectioned and used for immunofluorescent staining for ATGL (green) expression. Slides were counterstained with DAPI (blue) for nuclei. Scale bar; 50 μm. (C) Immunoblot analyses of ATGL, α-tubulin, and ERK2 in lysates from subcutaneous WAT and caudal vertebral. (D-F) Decalcified tibiae were stained with osmium tetroxide and visualized by μCT (D). BMAT in proximal (E) and distal (F) tibia was quantified. Data are expressed as mean ± SD. * indicates p<0.05 with a two-sample t-test. (G–H) Decalcified tibiae were paraffin-sectioned and stained with Hematoxylin & Eosin. Representative pictures were taken from proximal (G) and distal (H) tibia. Scale bar; 200 μm. (I-J) BMAd sizes from proximal (I) and distal (J) tibiae were quantified with MetaMorph software. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA with Sidak’s multiple comparisons test. (K–M) Distal tibial BMAT was flushed from female BMAd-Pnpla2^-/- and their wildtype littermates at 24 weeks of age. For each n, distal tibial explants from two mice were combined per well and cultured in 2% BSA-HBSS solution (K). Subgroups from each genotype were treated with forskolin (FSK, 5 μM) or vehicle (Veh, DMSO). Glycerol (L) and non-esterified fatty acid (NEFA; M) in culture media at indicated time points were measured by colorimetric assay (n=3–4 per treatment). * indicates Pnpla2^+/+ (FSK) different from Pnpla2^+/+ (Veh) and from Pnpla2^-/- (FSK) with p<0.05 with three-way ANOVA followed by Sidak’s multiple comparisons test.

Figure 3.. BMAd lipolysis is required to maintain bone homeostasis in male mice under CR conditions, but not when mice are fed ad libitum.

(A-C) Male BMAd-Pnpla2^-/- and their BMAd-Pnpla2^+/+ littermates with ad libitum feeding were euthanized at 24 weeks of age. Two independent age- and sex- matched cohorts were plotted together. Tibiae from ad libitum mice were analyzed by μCT for indicated trabecular (Tb.) bone variables. BV/TV: bone volume fraction; Conn. Dens: connective density; BMD: bone mineral density; N: number; Th: thickness; Sp: separation. Scale bars indicate 500 μm. (D-M) Male mice at 18 weeks of age underwent 30% CR for 6 weeks. Two independent age- and sex- matched cohorts were plotted together for μCT parameters (D-F), one of those two cohorts was used for ELISA, and static or dynamic histomorphometry (G-M). (D-F) Tibiae from CR mice were analyzed by μCT for indicated trabecular bone variables. Scale bar; 500 μm. (G-H) Static histomorphometry analyses were performed to calculate osteoblast number (Ob. N), osteoclast number (OC. N) and osteoclast surface (Oc. S) per bone surface (BS). (I-J) Concentrations of circulating P1NP and TRACP5b in CR mice were measured. (K) Osteoid quantification was performed on undecalcified plastic sections with Goldner’s Trichrome staining. (L-M) Dynamic histomorphometry was performed on calcein-labelled trabecular bone from proximal tibia. sLS: single-labelled surface; MS: mineralized surface; Ir.L.Wi: inter-label width; MAR: mineral apposition rate. Data are expressed as mean ± SD. * indicates p<0.05 with a two-sample t-test. In addition, multiple unpaired t tests had been performed crossing all parameters, p values were adjusted for multiple comparisons using Two-stage step-up (Benjamini, Krieger, and Yekutieli) with FDR method. Adjusted p values are shown in Figure 3—source data 1.

Figure 3—figure supplement 1.. Blocking BMAd-lipolysis does not influence global metabolism when mice are fed ad libitum or calorically restricted.

(A-I) BMAd-Pnpla2^-/- male mice and littermate controls (Pnpla2^+/+) were fed ad libitum until 24 weeks of age. Body weight (A), glucose tolerance test (B), and weights of subcutaneous WAT (sWAT), epididymal WAT (eWAT), and liver (C-E) were recorded. Decalcified tibiae were used for osmium tetroxide-staining and quantified by μCT analyses to measure the BMAT volume from proximal to distal ends, as indicated by boxed regions (F-G). Concentrations of glycerol and NEFA in serum (H) and bone marrow supernatant (I) were measured with colorimetric assay kits. Glycerol and NEFA contents in bone marrow supernatant were normalized to protein concentrations. (J-U) BMAd-Pnpla2^-/- male mice and littermate controls (Pnpla2^+/+) at 18 weeks of age and underwent a 30% CR for 6 weeks. Body weight changes (J) and glucose tolerance (K) were recorded. sWAT (L), eWAT (M), and liver (N) weights were measured during dissection. BMAT volume at different locations were quantified in osmium tetroxide-stained bones following μCT scanning (O-P). Representative images of proximal tibial rBMAT are shown (Q). Quantitative analyses of BMAd sizes were performed using MetaMorph software (R-S). Concentrations of glycerol and NEFA in serum (T) and bone marrow supernatant (U) were measured using colorimetric assays. Glycerol and NEFA contents in bone marrow supernatant were normalized to protein concentrations. (V) Immunoblot analyses of circulating adiponectin under non-reducing and non-heat-denaturing conditions (top panel), and denaturing conditions (middle panel), with albumin as a loading control (low panel). HMW: high molecular weight forms; MMW: medium molecular weight forms; LMW: low molecular weight. Data are expressed as mean ± SD. * indicates p<0.05 with a two-sample t-test.

Figure 3—figure supplement 2.. Cortical bone variables in BMAd-Pnpla2^-/- mice and other possible mechanisms for bone loss in BMAd-Pnpla2^-/- CR mice.

(A-D) Mouse tibiae from 24 weeks old ad libitum (A-B) or CR (C-D) mice were collected. Cortical bone area (CT. BA/TA) and thickness (Ct. Th) were measured by μCT. Scale bar; 500 μm. (E-J) BMAd-Pnpla2^-/- male mice and littermate controls (Pnpla2^+/+) were fed ad libitum until 24 weeks of age. (E-F) Proximal tibial static histomorphometry was performed to calculate osteoblast number (Ob. N), osteoclast number (OC. N) and osteoclast surface (Oc. S) per bone surface (BS). (G-J) Circulating P1NP, RANKL, CTX-1 and TRACP5b were measured with commercially available ELISA kits. (K-N) BMAd-Pnpla2^-/- male mice and littermate controls (Pnpla2^+/+) at 18 weeks of age underwent a 30% CR for 6 weeks. (K-L) Circulating RANKL and CTX-1 were measured with commercially available ELISA kits. (M-N) Histomorphometry analysis for osteoid surface and osteoid maturation time (Omt). Data are expressed as mean ± SD. * indicates p<0.05 with a two-sample t-test.

Figure 3—figure supplement 3.. BMAd lipolysis is not required in female mice to maintain bone homeostasis under CR conditions.

BMAd-Pnpla2^-/- female mice and their wildtype controls (Pnpla2^+/+) at 18 weeks of age were fed ad libitum (AL) or underwent a 30% CR for another 6 weeks. (A-B) Final body weight and random glucose levels were measured (A). sWAT, parametrial WAT (pmWAT), liver and spleen weights were recorded during dissection (B). (C) Representative images from proximal tibiae were collected from decalcified and paraffin-sectioned bones. Scale bar; 200 μm. Quantification of BMAT surface area was finished by Image J. (D-E) Trabecular and cortical bone variables were determined by μCT analysis. (F) Complete blood counts (CBC) were performed to measure white and red blood cells in circulation. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA analysis followed by Šídák’s multiple comparisons test. Significant effects of genotype or diet as well as trends are shown.

Figure 3—figure supplement 4.. BMAd-lipolysis impairment in estrogen-deficient female mice does not affect CR-induced bone changes.

Female mice at 16 weeks of age underwent ovariectomy and recovered for 2 weeks, which were followed by 30% CR for 12 weeks.+ indicates OVX or CR, - indicates sham or ad libitum, respectively. Changes in body weight (A) and glucose tolerance test (B) were recorded. Femoral and tibial lengths were measured (C). Weights of sWAT, pmWAT, liver, spleen, and uterus were measured during dissection (D). Representative images of proximal tibial BMAT were shown (E). Trabecular bone parameters were determined by μCT (F). Tb.: trabecular bone; BV/TV: bone volume fraction; BMD: bone mineral density; N: number; Th: thickness; Sp: separation. Green dots show bone variable measurements from a sex- and age- matched independent cohort of sham ad libitum (OVX -; CR -) mice. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA analysis followed by Šídák’s multiple comparisons test. Significant effects of genotype or diet as well as trends are shown. Sham ad libitum group is not included for statistical analyses.

Figure 4.. BMAd-Pnpla2 deficiency impairs myelopoiesis.

(A-H) BMAd-Pnpla2^-/- mice and littermate controls (Pnpla2^+/+) were caloric restricted (CR; +) for 20 weeks or remained on an ad libitum diet (-), and then received whole-body irradiation (6 Gy). Mice were euthanized 9 days post-irradiation. Femurs were collected for flow cytometry to measure the regeneration of hematopoietic cells. Bone marrow mononuclear cells (BMNCs), monocytes, B and T lymphocytes and neutrophils were quantified. (I-L) CFU assays. Femora and tibial bone marrow cells were isolated from BMAd-Pnpla2^-/- mice and littermate controls (Pnpla2^+/+), which had been fed ad libitum (-) or a caloric restricted (CR; +) diet for 20 weeks. After counting, 1 × 10⁴ cells were plated for CFU assays. Colonies were counted by an independent expert in a blinded manner 7 days after plating. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA analyses followed by Šídák’s multiple comparisons test. Significant effects of genotype, diet, or their interactions are shown, as are trends.

Figure 4—figure supplement 1.. Flow cytometry strategies for hematopoietic cells and sublethal irradiation-induced hematopoietic regeneration in BMAd-Pnpla2^-/- mice.

(A-B) Hematopoietic cell gating strategies in hematopoietic stem/progenitor cells (HSPC, A) and mature leukocytes (B). (C-G) BMAd-Pnpla2^-/- mice and littermate controls (Pnpla2^+/+) underwent 30% caloric restriction (CR; +) for 20 weeks or remained on ad libitum (AL; -) diet, and then received a whole-body irradiation (6 Gy). Tail vein blood (~50 µl) was collected every 2–3 days to monitor hematopoietic cell recovery. Mice were euthanized at day 9 post-irradiation. (C-E) Data from complete blood cell counts shows dynamic changes of white and red blood cells before and after irradiation. Three-way ANOVA analyses with Sidak’s multiple comparisons test were performed. (F-G) Hematopoietic cells from femurs were stained with cell markers to identify hematopoietic stem and progenitor cells. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANONA analysis followed by Šídák’s multiple comparisons test. Significant effects of genotype, diet, or their interactions are shown.

Figure 5.. BMAd-Pnpla2 deficiency causes extensive alterations to the bone marrow transcriptome only when coupled with CR.

Male control and BMAd-Pnpla2^-/- mice at 24 weeks of age were either fed AL or underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice was pooled as one sample for RNAseq analyses (n of 3 or 4 per treatment). (A) Differential genes with our criteria (padjj <0.05 and |Log2 fold change|>1) between BMAd-Pnpla2^+/+ CR and BMAd-Pnpla2^+/+ad libitum (AL) were grouped into 4 clusters. (B) Genes different between BMAd-Pnpla2^+/+ CR and BMAd-Pnpla2^+/+ AL were ordered from maximum to minimum log2 fold change (red dots), and compared to corresponding data from BMAd-Pnpla2^-/- CR versus BMAd-Pnpla2^-/- AL (blue dots). Venn diagram shows the differential genes between BMAd-Pnpla2^+/+ CR versus BMAd-Pnpla2^+/+ AL BMAT; and BMAd-Pnpla2^-/- CR versus BMAd-Pnpla2^-/- AL BMAT. (C) Pathway analyses of genes significantly changed by CR in BMAd-Pnpla2^+/+ mice, but not in CR mice lacking Pnpla2 (indicated by * area in panel B). Pathways further analyzed by heatmap indicated with blue arrows. (D-F) Expression Z-scores of genes related to adipogenesis (D), skeletal system development (E) and extracellular matrix organization (F) were shown as heatmap. Effects of genotype and diet, and their interactions were analyzed by three-way ANOVA.

Figure 5—figure supplement 1.. CR causes profound changes in BMAT transcriptome.

Twenty-four weeks old male mice underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice were pooled as one sample for RNA sample preparation. High-quality RNA samples were submitted for RNAseq analyses. (A) Principal component analysis (PCA) plot shows the distinct transcriptional characters in CR groups with (purple dots) or without (aqua dots) Pnpla2 in BMAds. (B) Volcano plots show the differential genes with padj <0.05 & |Log2 fold change|>1 in comparisons between BMAd-Pnpla2^+/+ CR versus ad libitum (AL) (left), BMAd-Pnpla2^-/- versus BMAd-Pnpla2^+/+ at AL (middle) and BMAd-Pnpla2^-/- versus BMAd-Pnpla2^+/+ at CR (right). (C-F) Differential genes from comparison between BMAd-Pnpla2^+/+ CR versus AL were grouped into 4 clusters according to the alteration patterns. Pathway analyses were performed on each cluster except cluster 2, which gene set was not enriched in any pathways.

Figure 5—figure supplement 2.. BMAd-Pnpla2 deficiency causes extensive alterations to the bone marrow transcriptome only when coupled with CR.

Twenty-four weeks old male mice underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice were pooled as one sample for RNA sample preparation. High-quality RNA samples were submitted for RNAseq analyses. (A) Pathway analysis of gene set that respond to CR independent of Pnpla2 deficiency in BMAds, indicated by * area. (B) qPCRs were performed to confirm the changes of adipogenesis genes in BMAT. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA analysis followed by Šídák’s multiple comparisons test. (C-D) Heat maps for genes related to fatty acid biosynthesis (C) and myeloid leukocyte differentiation (D). (E) Collagen genes that upregulated by CR in BMAd-Pnpla2^+/+ mice.

Figure 5—figure supplement 3.. BMAd-Pnpla2 deficiency alters gene expression in response to CR.

Male mice at 24 weeks of age underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice were pooled as one sample for RNA sample preparation. High-quality RNA samples were submitted for RNAseq analyses. (A) Differential genes with our criteria (padj <0.05 and |Log2 fold change|>1) between BMAd-Pnpla2^-/- CR and BMAd-Pnpla2^+/+CR were grouped into three clusters. (B-C) Pathway analyses were performed on cluster 2 (B). Heat maps for genes related to hematopoietic progenitor cell differentiation and positive regulation of osteoblast differentiation (C). (D-E) Pathway analyses were performed on cluster 3 (D). Heat maps for genes related to regulation of lipid catabolic process and leukocyte migration (E).

Figure 6.. Energy from BMAd is required for trabecular bone regeneration and protects against bone loss caused by chronic cold exposure.

(A-D) BMAd-Pnpla2^+/+ and BMAd-Pnpla2^-/- male mice at 24 weeks of age fed with chow diet (-) or underwent 30% CR for six weeks (+). A 0.7 mm proximal tibial defect was created 1–2 mm distal to the growth plate. Tibiae were collected 9 days after surgery. MicroCT was performed to analyze trabecular (endocortical) and cortical bone regeneration. (A) Representative analyzing images of bone defect. Example of μCT showing trabecular (endocortical) region of interest (ROI; green) and cortical ROI (yellow). Newly generated woven bone in trabecular (endocortical; red) and cortical compartment (purple) is indicated. (B) Quantification of regenerated bone in the endocortical compartment - bone volume fraction (BV/TV), mineral density (BMD) and mineral content (BMC). Data are expressed as mean ± SD. (C) Safranin O/ Fast Green (SO/FG) staining of new cortical compartment formation in defect sites. Scale bar; 200 μm. (D) Quantification of regenerated cortical woven bone volume fraction (BV/TV), mineral density (BMD) and mineral content (BMC). (E-I) Female BMAd-Pnpla2^+/+ and BMAd-Pnpla2^-/- mice at 20 weeks of age were singly housed at 4°C for three weeks without enrichments. Tibiae were collected for sectioning and μCT analyses. (E) Proximal tibiae were decalcified and paraffin-sectioned for H&E staining. Representative images for proximal tibia are shown. Scale bar, 100 μm. (F-G) Quantification of BMAds from H&E-stained slides using MetaMorph software. Comparison of BMAd size at room temperature (RT) versus cold exposure (CE) in mice of indicated genotypes. (H) Trabecular bone volume fraction (BV/TV) and mineral density (BMD) were quantified by μCT. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA analyses followed by Šídák’s multiple comparisons test. Significant effects of genotype, diet, or their interactions are shown, as are trends.

Figure 6—figure supplement 1.. Energy from BMAd protects against changes in trabecular microstructures caused by chronic cold exposure.

(A-D) Female BMAd-Pnpla2^+/+ and BMAd-Pnpla2^-/- mice at 20 weeks of age were singly housed at 4°C for 3 weeks. Tibiae were decalcified for paraffin sectioning. (A) H&E-stained slides were used to quantify osteoblast number (Ob. N) and bone surface (BS). (B-C) TRAP-stained slides were used for osteoclast number (Oc. N) and surface (Oc. S) quantification, which were normalized to BS. (D) Serum P1NP concentrations were measured. (E-J) Male BMAd-Pnpla2^+/+ and BMAd-Pnpla2^-/- mice at 17–20 weeks of age were singly housed at 4°C for 3 weeks. Tibiae were collected for μCT analyses. Trabecular bone volume fraction (BV/TV), mineral density (BMD), connective density (Conn. Dens), trabecular thickness (Th), number (N) and separation (Sp) were quantified by μCT. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA analyses followed by Šídák’s multiple comparisons test. Significant effects of genotype, temperature, or their interactions are shown.

Author response image 1.. BMAd-Pnpla2 deficiency causes alterations in secretory protein gene expression.

A. Differential genes regulated by CR in BMAd-Pnpla2^+/+ mice were mapped with two independent secretome datasets: MetaZSecKB and VerSaDa. B. Overlapping genes between two secretome datasets were used for pathway analyses. . Z-Scores of genes enriched in hematopoietic stem cell proliferation pathway.

Author response image 2.. BMAd lipolysis is required to maintain bone homeostasis in male mice under conditions of CR, but not when mice are fed ad libitum.

Author response image 3.. Phenotypes of Osterix-FLPo wild type, heterozygous and homozygous mice.

Osterix-FLPo mice were euthanized at 24 weeks of age. GTT was performed at 22 weeks of age (A). Body weights, tissue weights and bone lengths were measured during dissection (B-I). Tibiae were collected for mCT analyses; trabecular (J-O) and cortical bone (P-Q) parameters were measured.

Author response image 4.. Quantification of rBMAT in tibial diaphysis region.

Author response image 5.. Comparison of gene set with different control groups.

Author response image 6.. Z-scores of Dpp4 expression in BMAT in response to CR and Pnpla2 deficiency.