(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 …
(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 …
(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 …
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-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 …
Ablation of adipose triglyceride lipase (ATGL; gene name Pnpla2) increases size and number of BMAd.
(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 …
BMAd lipolysis is required to maintain bone homeostasis in male mice under CR conditions, but not when mice are fed ad libitum.
(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), …
Blocking BMAd-lipolysis does not influence global metabolism when mice are fed ad libitum or calorically restricted.
(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…
Cortical bone variables in BMAd-Pnpla2-/- mice and other possible mechanisms for bone loss in BMAd-Pnpla2-/- CR mice.
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 …
BMAd lipolysis is not required in female mice to maintain bone homeostasis under CR conditions.
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. …
BMAd-lipolysis impairment in estrogen-deficient female mice does not affect CR-induced bone changes.
(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). …
BMAd-Pnpla2 deficiency impairs myelopoiesis.
(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% …
Flow cytometry strategies for hematopoietic cells and sublethal irradiation-induced hematopoietic regeneration in BMAd-Pnpla2-/- mice.
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 …
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 …
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 …
BMAd-Pnpla2 deficiency causes extensive alterations to the bone marrow transcriptome only when coupled with 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 …
(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 …
Energy from BMAd is required for trabecular bone regeneration and protects against bone loss 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 …
Energy from BMAd protects against changes in trabecular microstructures caused by chronic cold exposure.
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 …
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 …
Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
---|---|---|---|---|
Strain, strain background (Mus musculus, SJLxC57BL/6J) | Osterix-FLPo | Generated in University of Michigan Transgenic Animal Model Core | Donated to JAX: Stock No. 037208 | Male and female |
Strain, strain background (Mus musculus, SJLxC57BL/6J) | FLPo-dependent Adipoq-Cre (FAC) | Generated in University of Michigan Transgenic Animal Model Core | N/A | Male and female |
Strain, strain background (Mus musculus, SJLxC57BL/6J) | Frt-floxed EGFP | Generated in University of Michigan Transgenic Animal Model Core | N/A | Male and female |
Strain, strain background (Mus musculus, C57BL/6J) | mT/mG | Jackson Laboratory | Stock No. 007676 | Male and female |
Strain, strain background (Mus musculus, C57BL/6J) | Pnpla2flox/flox | Jackson Laboratory | Stock No. 024278 | Male and female |
Antibody | Anti-Adiponectin (rabbit monoclonal) | Sigma-Aldrich | A6354 | WB (1:1000) |
Antibody | Anti-FABP4/A-FABP Antibody (goat polyclonal) | R&D Systems | AF1443 | WB (1:1000) |
Antibody | Anti-alpha Tubulin Antibody (rat monoclonal) | Thermo Fisher Scientific | MA180017 | WB (1:1000) |
Antibody | Anti-ERK 2 Antibody (mouse monoclonal) | Santa Cruz Biotechnology | sc-1647 | WB (1:1000) |
Antibody | Anti-Albumin antibody (rabbit monoclonal) | Abcam | Ab207327 | WB (1:1000) |
Antibody | Anti-ATGL Antibody (rabbit polyclonal) | Cell Signaling Technology | 2138S | WB (1:1000) |
Antibody | Anti-ATGL Antibody (rabbit monoclonal) | Abcam | ab207799 | IF (1:100) |
Antibody | Anti-GFP antibody (chicken polyclonal) | Abcam | ab13970 | IF (1:500) |
Antibody | Anti-RFP Antibody Pre-adsorbed (rabbit polyclonal) | Rockland | 600-401-379 | IF (1:200) |
Antibody | Goat anti Rabbit IgG (H+L) Secondary Antibody, Alexa Fluor 594 (goat polyclonal) | Invitrogen | A11012 | IF (1:100) |
Antibody | Alexa Fluor 488 goat anti-chicken IgG (H+L) (goat polyclonal) | Invitrogen | A11039 | IF (1:100) |
Antibody | Anti-Ly6G FITC (rat monoclonal) | BD Biosciences | 551460 | FACS (1:200) |
Antibody | Anti-CD11b APC (rat monoclonal) | Invitrogen | 17-0112-82 | FACS (1:200) |
Antibody | Anti-CD115 APC-Cy7 (rat monoclonal) | Biolegend | 135531 | FACS (1:100) |
Antibody | Anti-CD3e PE-Cy7 (Armenian Hamster monoclonal) | Biolegend | 100319 | FACS (1:200) |
Antibody | Anti-CD19 Pacific Blue (rat monoclonal) | Invitrogen | 48-0193-82 | FACS (1:200) |
Antibody | Anti-CD45 AlexaFluor700 (rat monoclonal) | BD Biosciences | 560510 | FACS (1:200) |
Antibody | Anti-Gr-1 Biotin (rat monoclonal) | Biolegend | 79750 | FACS (1:200) |
Antibody | Anti-CD11b Biotin (rat monoclonal) | Biolegend | 79749 | FACS (1:200) |
Antibody | Anti-B220 Biotin (rat monoclonal) | Biolegend | 79752 | FACS (1:200) |
Antibody | Anti-CD3e Biotin (Armenian Hamster monoclonal) | Biolegend | 79751 | FACS (1:200) |
Antibody | Anti-TER119 Biotin (Armenian Hamster monoclonal) | Biolegend | 79748 | FACS (1:200) |
Antibody | Anti-Sca1 PE-Cy7 (rat monoclonal) | Invitrogen | 25-5981-82 | FACS (1:200) |
Antibody | Anti-cKit (CD117) APC-Cy7 (rat monoclonal) | Biolegend | 105826 | FACS (1:100) |
Antibody | Anti-CD150 BrilliantViolet 421 (rat monoclonal) | Biolegend | 115925 | FACS (1:200) |
Antibody | Anti-CD48 FITC (Armenian Hamster monoclonal) | Invitrogen | 11-0481-85 | FACS (1:100) |
Antibody | Anti-CD16/32 PerCP-Cy5.5 (rat monoclonal) | Biolegend | 101324 | FACS (1:200) |
Antibody | Anti-CD105 APC (rat monoclonal) | Biolegend | 120413 | FACS (1:200) |
Antibody | Anti-CXCR2 PE (rat monoclonal) | Biolegend | 149304 | FACS (1:200) |
Antibody | Anti-CXCR4 PE-Dazzle (rat monoclonal) | Biolegend | 146514 | FACS (1:100) |
Antibody | Anti-CD62L BrilliantViolet 421 (rat monoclonal) | Biolegend | 104435 | FACS (1:200) |
Commercial assay or kit | Acid Phosphatase Leukocyte (TRAP) Kit | Sigma-Aldrich | 387A-1KT | |
Commercial assay or kit | Free Glycerol Determination Kit | Sigma-Aldrich | FG0100 | |
Commercial assay or kit | NEFA Reagent (NEFA-HR(2)) | FUJIFILM Wako Diagnostics | NC9517309 | |
Commercial assay or kit | BCA Protein Assay Kit | Thermo Fisher Scientific | 23225 | |
Commercial assay or kit | RAT/MOUSE P1NP ELISA KIT | Immunodiagnostic Systems Inc | NC9666468 | |
Commercial assay or kit | Mouse TRANCE/RANK L/TNFSF11 Quantikine ELISA Kit | R&D Systems | MTR00 | |
Commercial assay or kit | MOUSE TRAP ASSAY | Immunodiagnostic Systems Inc | NC9360739 | |
Commercial assay or kit | MethoCult GF M3534 | ATEMCELL | 03534 | |
Commercial assay or kit | DNA-free Kit | Life Technologies | AM1906 | |
Chemical compound, drug | Calcein | Sigma-Aldrich | C0875 | |
Chemical compound, drug | EDTA | DOT Scientific Inc | dse57020 | |
Chemical compound, drug | Tetroxide Osmium | Electron Microscopy Sciences | 19190 | |
Chemical compound, drug | Forskolin | Cayman Chemical Company | 11018 | |
Chemical compound, drug | Bovine Serum Albumin (BSA), Fraction V | Gold Biotechnology | A-421–250 | |
Chemical compound, drug | qPCRBio SyGreen Mix Hi-ROX Blue | Innovative Solutions | 4SPB20.16 | |
Chemical compound, drug | PCRBio HS Taq Mix Red | Innovative Solutions | 4SPB10.23 | |
Chemical compound, drug | Agarose | Thermo Fisher Scientific | BP160-500 | |
Chemical compound, drug | RNA STAT-60 | AMSBIO | CS-502 | |
Chemical compound, drug | M-MLV Reverse Transcriptase | Thermo Fisher Scientific | 28025013 | |
Chemical compound, drug | 100bp DNA Ladder | NEB | N3231S | |
Software, algorithm | Microsoft Office | Microsoft | https://its.umich.edu/communication/collaboration/microsoft-office-365/getting-started | |
Software, algorithm | Adobe photoshop | Adobe | https://www.adobe.com/creativecloud/desktop-app.html | |
Software, algorithm | Prism 9 | GraphPad software | https://www.graphpad.com/ | |
Software, algorithm | Image J | Image J | https://imagej.nih.gov/ij/ | |
Software, algorithm | MetaMorph | BioVision Technologies | https://www.biovis.com/metamorph.html | |
Software, algorithm | Scano μCT 100 | SCANCO Medical AG | https://www.scanco.ch/ | |
Software, algorithm | BIOQUANT OSTEO | BIOQUANT Image analysis corporation | https://bioquant.com/ | |
Software, algorithm | STAR | PMID:23104886 | https://github.com/alexdobin/STAR; Dobin et al., 2013; Dobin, 2022 | |
Software, algorithm | DESeq2 | PMID:25516281 | https://bioconductor.org/packages/release/bioc/html/DESeq2.html | |
Software, algorithm | QualiMap-2 | PMID:26428292 | https://qualimap.conesalab.org | |
Software, algorithm | R | The R Foundation | https://www.r-project.org/ | |
Software, algorithm | Metascape | PMID:30944313 | https://metascape.org/gp/index.html | |
Software, algorithm | Dragonfly | ORS - OBJECT RESEARCH SYSTEMS | https://www.theobjects.com/dragonfly/index.html | |
Software, algorithm | FlowJo | BD Biosciences | https://www.flowjo.com/solutions/flowjo | |
Other | Streptavidin BrilliantViolet 510 | Biolegend | 405233 | FACS (1:200) |
Other | Element HT5 Veterinary Hematology Analyzer | Heska | https://www.heska.com/product/element-ht5/ | Service provided by UMICH In-Vivo Animal Core (IVAC) |
Other | FACSAria III cell sorter | BD Biosciences | N/A | Shared equipment in UMICH Flow Cytometry Core |
Other | LSRFortessa Cell Analyzer | BD Biosciences | N/A | Shared equipment in UMICH Flow Cytometry Core |
Other | Nikon A1 Confocal Microscope | Nikon | N/A | Shared equipment in UMICH Microscopy and Imaging Analysis Core (MIAC) Michigan Diabetes Research Center |
Other | Bayer Contour Next Test Glucose Strips | Diabetic Corner | ByrContournext | https://www.contournextone.com/siteassets/pdf/web85688006_cntrnxtone_ug_r01-17.pdf |
Other | Scanco μCT 100 micro-computed tomography system | SCANCO Medical AG | https://www.scanco.ch/ | Service provided by UMICH School of Dentistry MicroCT Core |
Other | Olympus BX51 | Olympus | N/A | https://www.olympus-lifescience.com/en/microscope-resource/primer/techniques/fluorescence/bx51fluorescence/ |
Other | StepOnePlus Real-Time PCR System | Thermo Fisher Scientific | 4376600 | https://www.thermofisher.com/order/catalog/product/4376600 |
Other | Microtome | Leica | RM2235 | https://www.leicabiosystems.com/en-br/histology-equipment/microtomes/leica-rm2235/ |
Other | Slide scanner | Nikon | N/A | Shared equipment in UMICH Orthopaedic Research Laboratories (ORL) Histology Core |
BMAd-lipolysis deficiency has subtle effects on circulating mature blood cells.
Whole blood from male mice at 24 weeks of age fed ad libitum (top) or a 30% CR diet for 6weeks (bottom) was submitted for complete blood counts (CBC). White- and red- blood cell related parameters are shown. 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.
BMAd-lipolysis deficiency causes mild changes in bone marrow hematopoietic cells.
Femoral bone marrow cells from male mice at 24 weeks of age fed ad libitum (top) or a 30% CR diet for 6 weeks (bottom) were collected and stained with antibodies for flow cytometry analyses. Mature blood cells and hematopoietic stem/progenitor cells (HSPCs) were counted. 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.
PCR primer list.