A bone-specific adipogenesis pathway in fat-free mice defines key origins and adaptations of bone marrow adipocytes with age and disease
- Division of Bone and Mineral Diseases, Department of Medicine, Washington University, United States
- Department of Biomedical Engineering, Washington University, United States
- Department of Orthopaedic Surgery, Washington University, United States
- Division of Endocrinology, Metabolism & Lipid Research, Department of Medicine, Washington University, United States
Figures
Figure 1 with 1 supplement
Adiponectin is expressed by AdipoqCre+/mTmG+ bone marrow stromal cells in vitro.
(A) Bone marrow stromal cells from 16 week old male AdipoqCre+/mTmG+ mice were cultured at low density for 14 days to promote formation of colony-forming units (CFUs). Endogenous fluorescence was …
-
Figure 1—source data 1
Percent total colony count.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig1-data1-v2.xlsx
Figure 1—figure supplement 1
Adiponectin is expressed by BMAT adipocytes in the AdipoqCre+/mTmG+ mouse in vivo.
(A) Representative longitudinal cross-section of the proximal tibial metaphysis and epiphysis at the region of the growth plate (*). Bone marrow adipocytes (BMAds) were identified by expression of …
Figure 2
AdipoqCre+/DTA+ fat free (FF) mice lack white and brown adipose tissues and circulating adiponectin.
(A) Serum adiponectin (ACRP30) of AdipoqCre-/DTA+ control (Con), AdipoqCre+/DTA+ fat free (FF), and Adipoq knockout (KO) mice by western blot. Blood albumin levels by Ponceau S staining (loading …
-
Figure 2—source data 1
Blood glucose, tibia length, and body mass.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig2-data1-v2.xlsx
-
Figure 2—source data 2
Western blot uncropped images.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig2-data2-v2.pdf
Figure 3 with 1 supplement
Trabecular bone is increased in AdipoqCre+/DTA+ fat free (FF) mice.
(A) Representative 3D µCT-based reconstructions of tibiae from 4 month old AdipoqCre+/DTA+ fat free (FF) and AdipoqCre-/DTA+ controls (Con). Scale = 1 mm. (B–F) Quantification of trabecular …
-
Figure 3—source data 1
Trabecular bone parameters.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Cortical parameters are increased in young AdipoqCre+/DTA+ fat free (FF) mice, but tends to decline with age.
(A) Representative ex vivo micro-CT cross sections of 4 month old AdipoqCre+/DTA+ fat free (FF) and AdipoqCre-/DTA+ control (Con) cortical bone at 6 mm, 8 mm, and 12 mm distal to the proximal end of …
-
Figure 3—figure supplement 1—source data 1
Cortical bone parameters.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig3-figsupp1-data1-v2.xlsx
Figure 4
BMAT is present in AdipoqCre+/DTA+ fat free (FF) mice and expands with age.
(A, B) Representative μCT images of osmium-stained tibiae for both male and female AdipoqCre+/DTA+ fat free (FF) and AdipoqCre-/DTA+ control (Con) mice at (A) 4 months and (B) 8 months of age. BMAT …
-
Figure 4—source data 1
BMAT volume.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig4-data1-v2.xlsx
Figure 5 with 1 supplement
Fat free (FF) bone marrow adipocytes express perilipin, but not CD68, and have increased lipid storage relative to controls.
Representative images from both male and female AdipoqCre+/DTA+ fat free (FF) and AdipoqCre-/DTA+ control (Con) mice at 8 months of age. (A) Representative longitudinal hematoxylin and eosin (H&E) …
-
Figure 5—source data 1
BMAd size distribution.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Extra-skeletal adipocytes are depleted in the foot and tail of FF mice.
Representative images from AdipoqCre+/DTA+ fat free (FF) and AdipoqCre-/DTA+ control (Control) mice at 8 months of age. Representative longitudinal hematoxylin and eosin (H&E) stained sections of …
Figure 6
BMAT in AdipoqCre+/DTA+ fat free (FF) mice is not responsive to cold temperature challenge (22°C vs 30°C).
Male AdipoqCre+/DTA+ FF mice and controls were maintained in thermoneutral housing (30°C) or moved to room temperature (22°C) at 3–5 weeks of age. Bones were analyzed after 3–4 months, at 15–17 …
-
Figure 6—source data 1
Cold challenge source data.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig6-data1-v2.xlsx
Figure 7
BMAT in AdipoqCre+/DTA+ fat free (FF) mice is not regulated by β3-adrenergic stimulation.
Male AdipoqCre+/DTA+ FF and AdipoqCre-/DTA+ controls (Con) were treated with CL316,243, a β3-adrenergic receptor (β3-AR) agonist, using a new chronic treatment regimen. Eight daily subcutaneous …
-
Figure 7—source data 1
Serum glycerol, average BMAd size, and BMAe gene expression.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig7-data1-v2.xlsx
Figure 8 with 3 supplements
Subcutaneous fat transplant prevents BMAT expansion in AdipoqCre+/DTA+ fat free (FF) mice.
Male and female AdipoqCre+/DTA+ FF and AdipoqCre-/DTA+ controls (Con) underwent sham surgery or were transplanted subcutaneously with WT inguinal white adipose tissue (iWAT) at 3–5 weeks of age. …
-
Figure 8—source data 1
Fat transplant source data.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig8-data1-v2.xlsx
Figure 8—figure supplement 1
Engrafted fat transplant fragments resemble subcutaneous white adipose tissue at the terminal end point.
Male and female AdipoqCre+/DTA+ fat free and AdipoqCre-/DTA+ controls underwent sham surgery or were transplanted subcutaneously with control adipose tissue at 3–5 weeks of age. After surgery, mice …
Figure 8—figure supplement 2
Trabecular and cortical bone phenotypes remain unchanged after subcutaneous fat transplant.
Male and female AdipoqCre+/DTA+ fat free (FF) and AdipoqCre-/DTA+ control (Con) mice underwent sham surgery or were transplanted subcutaneously with WT adipose tissue at 3–5 weeks of age. After …
-
Figure 8—figure supplement 2—source data 1
Fat transplant bone parameters.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig8-figsupp2-data1-v2.xlsx
Figure 8—figure supplement 3
Adipose tissue masses remain unchanged after subcutaneous fat transplant.
Male and female AdipoqCre+/DTA+ fat free (FF) and AdipoqCre-/DTA+ control (Con) mice underwent sham surgery or were transplanted subcutaneously with WT adipose tissue at 3–5 weeks of age. After …
-
Figure 8—figure supplement 3—source data 1
Fat transplant adipose tissue masses.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig8-figsupp3-data1-v2.xlsx
Figure 9
Fat-free (FF) bone marrow adipocytes are mostly adiponectin negative and have decreased cytokine expression relative to controls.
(A) Representative immunostains of bone marrow adipocytes in femur and tibia from both male and female AdipoqCre+/DTA+/mTmG+ triple mutant mice (FFmTmG) at 4 months of age in vivo. Assessed relative …
-
Figure 9—source data 1
Percent total BMAd count, CFU per million cells, and BMAe gene expression.
- https://cdn.elifesciences.org/articles/66275/elife-66275-fig9-data1-v2.xlsx
Figure 10
Summary model.
Adiponectin is highly expressed by mature bone marrow adipocytes (BMAds) and by a subset of bone marrow stromal progenitor cells. Adiponectin-expressing progenitors overlap with Cxcl12-abundant …
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Strain, strain background (Mus musculus) | C57BL/6 J: Adipoq-Cre | Jackson Laboratories | 028020RRID:IMSR_JAX:028020 | |
| Strain, strain background (Mus musculus) | C57BL/6 J: Rosa26< lsl-mTmG/+> | Jackson Laboratories | 007676RRID:IMSR_JAX:007676 | |
| Strain, strain background (Mus musculus) | C57BL/6 J: Rosa26< lsl-DTA/DTA> | Jackson Laboratories | 009669RRID:IMSR_JAX:009669 | |
| Strain, strain background (Mus musculus) | C57BL/6 J: Adipoq-/- | Jackson Laboratories | 008195RRID:IMSR_JAX:008195 | |
| Chemical compound, drug | 10 % neutral buffered formalin | Fisher Scientific | 23–245684 | |
| Chemical compound, drug | EDTA | Sigma-Aldrich | E5134 | |
| Chemical compound, drug | Hydrogen Peroxide | Sigma-Aldrich | 216,763 | 30 wt. % in H2O |
| Commercial assay or kit | IMMPRESS HRP Anti-Rabbit IgG kit | Vector Laboratories | MP-7401 RRID:AB_2336529 | |
| Chemical compound, drug | DAPI | Sigma-Aldrich | D9542 | 1 mg/mL |
| Chemical compound, drug | Hematoxylin | Ricca Chemical | 3536–16 | |
| Other | OCT mounting media | Fisher HealthCare | 23-730-571 | |
| Commercial assay or kit | MesenCult Expansion Kit (Mouse) | STEMCELL Technologies | 05513 | |
| Chemical compound, drug | Triton X-100 | Sigma-Aldrich | 9002-93-1 | |
| Other | Fluoromount-G | Thermo Fisher Scientific | 00-4958-02 | |
| Chemical compound, drug | Osmium tetroxide | Electron Microscopy Sciences | 19,170 | 4 % Aqueous Solution |
| Chemical compound, drug | Potassium dichromate | Sigma-Aldrich | 24–4520 | 1/60 M |
| Commercial assay or kit | Serum Triglyceride Determination Kit | Sigma-Aldrich | TR0100 | |
| Chemical compound, drug | CL316,243 | Sigma-Aldrich | C5976 | ≥ 98 % (HPLC) |
| Other | Donkey serum | Sigma-Aldrich | D9663 | |
| Other | 4 x NuPage LDS Buffer | Thermo Fisher Scientific | NP0007 | |
| Chemical compound, drug | Ponceau S | Fisher Scientific | BP103-10 | |
| Other | DNase and protease free bovine serum albumin | Fisher Scientific | BP9706 | |
| Other | HBSS Buffer | Gibco | 13150016 | |
| Commercial assay or kit | NucleoSpin RNA XS Kit | Takara Biosciences | 740,902 | |
| Commercial assay or kit | SuperScript IV VILO Master Mix with ezDNase Enzyme | Thermo Fisher Scientific | 11766050 | |
| Commercial assay or kit | qPCRBIO SyGreen Mix Lo-ROX | PCR Biosystems | PB20.11–51 | |
| Antibody | Antibodies for immunostaining and western blot | Detailed in Supplementary file 1 | ||
| Software, algorithm | GraphPad Prism | GraphPad | v8.4.3RRID:SCR_002798 | |
| Software, algorithm | SCANCO Medical microCT systems | Scanco Medical AG | RRID:SCR_017119 | |
| Software, algorithm | NDP.view2 | Hamamatsu Photonics | U12388-01 | |
| Software, algorithm | Fiji | ImageJ | RRID:SCR_002285 | |
| Software, algorithm | Microsoft Excel | Microsoft | RRID:SCR_016137 | |
| Other | Scanco µCT 40 | Scanco Medical AG | Imaging system | |
| Other | 2.0-HT NanoZoomer System | Hamamatsu Photonics | Imaging system | |
| Other | Spinning Disk Confocal Microscope | Nikon | Imaging system | |
| Other | LI-COR Odyssey Imager | LI-COR | Imaging system | |
| Other | Glucometer | Contour Next | ||
| Other | Digital Caliper | iKKEGOL | 5486 | |
| Other | PicoLab Rodent Diet 20 | LabDiet | 5053 | |
| Other | Microplate Spectrophotometer | BioTek | Epoch | |
| Other | QuantStudio 3 Real-Time PCR System | Thermo Fisher Scientific A28136 | A28136 |
Additional files
-
Supplementary file 1
Antibodies used for western blot and immunostaining.
- https://cdn.elifesciences.org/articles/66275/elife-66275-supp1-v2.docx
-
Supplementary file 2
qPCR Primers.
- https://cdn.elifesciences.org/articles/66275/elife-66275-supp2-v2.docx
-
Transparent reporting form
- https://cdn.elifesciences.org/articles/66275/elife-66275-transrepform1-v2.docx
