DOI: 10.5061/dryad.dz08kps7c
title: Adipose-derived leptin and complement factor D mediate osteoarthritis severity and pain

description: Obesity is a risk factor for osteoarthritis (OA), and leptin is among the
 adipokines implicated in obesity-induced OA. However, the specific role of
 leptin in OA severity and pain is not known. Using lipodystrophic (LD)
 mice, we show fat-secreted factors are required for knee OA development,
 implicating a fat-cartilage crosstalk. Fat pad implantation or systemic
 leptin restoration in LD mice reintroduced structural OA and pain, whereas
 implantation of leptin-deficient fat pad did not change OA susceptibility.
 Isochronic parabiosis and spatial transcriptomics confirmed that fat-joint
 crosstalk likely occurred via soluble mediators. Global unsupervised
 multiomics of conditioned media from fat implants revealed that leptin
 exerts a regulatory effect on adipsin (or complement factor D), the
 activity of which modulates contrastive OA structural and pain phenotype.
 These findings suggest adipokines influence OA pathogenesis, providing
 conclusive evidence of a fat-joint crosstalk and implicating OA as a
 systemic disease of adipose tissue.
description: <strong><em>Animal
 Studies</em></strong> All experimental
 procedures were approved by the Washington University School of Medicine
 Department of Comparative Medicine Institutional Animal Care and Use
 Committee (WUSTL IACUC 22-0306) and were conducted in accordance with
 ARRIVE guidelines. LD mice and WT (DTA/+) littermate controls were
 generated for these studies by crossing adiponectin-Cre (Jackson Labs:
 028020) mice with homozygous lox-stop-lox-ROSA-diphtheria toxin A (Jackson
 Labs: 010527) mice on a C57BL/6 background. To breed at mendelian
 frequency, mice were bred and maintained at thermoneutrality throughout
 their lifespan (30 °C). Mixed genotype mice were group-housed such
 that <em>n</em> = 3 to 5 per cage. Male LD
 mice received either a MEF transplant (MEF-rescue) from WT, leptin
 heterozygous (MEF-HET) or leptin homozygous knockout (MEF-KO) pups as
 previously described, which developed into adipose-like tissue between 3
 and 5 weeks of age. The injection was delivered subcutaneously to the
 sternal aspect of a donor LD mouse (3–5 weeks old) under 2% isoflurane
 with a 27-gauge needle. Animal numbers used in each experiment can be
 found in the figure legend.
 <strong><em>Exogenous Leptin Delivery
 </em></strong> Osmotic pumps were
 surgically implanted to the dorsal aspect of mice at 15 weeks of age, one
 week prior to DMM. Leptin or saline was delivered to LD mice using Azlet
 2006 (Cupertino, CA) at a release rate of 0.15 to deliver a
 total of 1mg of leptin over 6 weeks, and were replaced with new, sterile
 implants until 28 weeks of age. Leptin concentration was confirmed after
 implantation. <strong><em>OA induction by
 DMM </em></strong> At 16 weeks of age,
 surgical animals underwent destabilization of the medial meniscus surgery
 in their left knee joints, and the right limb served as a nonsurgical
 contralateral control. Behavioral and metabolic assays were conducted
 throughout the course of this study. Mice were sacrificed at 28 weeks of
 age to assess OA severity, bone microstructure, and serum and SF
 inflammatory profiles. Knee joints were fixed in 4% paraformaldehyde and
 decalcified in 10% formic acid  (Cal-Ex II) for 48-72 hrs. Histological
 assessment was performed by Modified Mankin Scoring, Synovitis Scoring,
 and Osteophyte scoring on formalin fixed paraffin embedded 5um sections as
 previously described. Bone marrow adipocytes were counted in ImageJ on
 histological sections. <strong><em>Body
 Composition</em></strong><strong>, <em>Tissue
 Harvest and Storage</em></strong> Mice were
 weighed weekly throughout the course of the study using a standard scale
 (g). Body fat was measured by DXA (Lunar Piximus) at 27 weeks of age to
 quantify body fat. Animals were euthanized at 28 weeks of age in
 accordance with the timelines in each figure. Body mass, liver mass, body
 fat, blood, L3-L5 dorsal root ganglia sensory neurons from
 <em>Cfd<sup>-/-</sup></em> and WT mice and
 synovial fluid was harvested.
 <strong><em>Serum and Synovial Fluid Profiling
 </em></strong> All mice were fasted
 overnight prior to sacrifice. Serum was collected in a BD Microtainer STT
 serum separator tube and allowed to clot at room temperature. Synovial
 fluid was collected using an alginate pad that was digested in alginate
 lyase until a stop solution was applied. Both fluids were stored at −80 °C
 until analysis by Luminex multiplex 18-plex chemokine/cytokine array assay
 (Eve Technologies, Calgary). Enzyme-linked immunosorbent assay (ELISA) for
 serum leptin was conducted using a mouse/rat Leptin Quantikine ELISA Assay
 kit (MOB00; R&D Systems) at a dilution of 1:2 in LD mice, and all
 other groups were evaluated at the manufacturer’s recommended 1:20
 dilution. <strong><em>Bone Microstructure
 Analysis </em></strong> Whole knee joints
 were scanned by microcomputed tomography (Bruker SkyScan1176) at a
 resolution of 18-μm isotropic voxel resolution according to previously
 reported methods. To reduce beam hardening, a 0.5-mm aluminum filter was
 used during scanning. Hydroxyapatite calibration phantoms were scanned to
 calibrate bone density. Scans were reconstructed to three-dimensional (3D)
 images using NRecon software, and CTAn software was used to segment
 subchondral and trabecular regions from the medial tibial plateau, lateral
 tibial plateau, medial femoral condyle, and lateral femoral condyle for
 analysis. The tibial epiphysis was identified using the subchondral plate
 and growth plate as references. The tibial metaphysis was defined as the
 1-mm area directly below the growth plate. The main outcomes reported from
 microCT images are BMD, BV/TV (%), trabecular number, and trabecular
 thickness. <strong><em>Insulin and glucose
 tolerance tests </em></strong> Insulin- and
 glucose-tolerance tests were performed 4 weeks post-DMM surgery (20 weeks
 of age) after fasting mice for a minimum of 4 hours. Each test was
 conducted at least 1 week apart in all animals. For both tests, fasting
 glucose levels were measured by tail bleed at time point 0 to establish a
 baseline. For glucose-tolerance tests, animals were administered 1 mg/kg
 (10% dextrose: 1% volume/body mass) by intraperitoneal (IP) injection, and
 for insulin-tolerance tests, 0.75 U/kg body mass of insulin (Humulin R
 diluted to 75 mU/mL, 1% volume/body mass) was administered by IP
 injection. Serial blood glucose measurements were taken via tail vein at
 20, 40, 60, and 120 min after injection with a glucose meter (Contour;
 Bayer). <strong><em>Pain Assessments and
 Behavioral Testing </em></strong> All
 animals were acclimatized to all equipment 1 d prior to the onset of
 testing. Two measures for pain were conducted at 27 weeks of age. To
 assess tactile allodynia in the DMM limb, an Electronic Von Frey assay was
 used as previously described(<em>8</em>). Hind paws of DMM
 limbs were stimulated three to five times. The intensity of the stimulus
 (grams) was recorded by the tester when the paw was withdrawn. To assess
 mechanical hyperalgesia, pressure-pain tests were conducted using a Small
 Animal Algometer (SMALGO) (Bioseb). Three to five trials of the surgical
 and nonsurgical limb were collected by applying a steadily increasing
 force to the lateral aspect of each limb until the limb was withdrawn. The
 average of three trials for each limb was reported, and maximum value of
 450 g was employed to avoid tissue damage to the knee joint. 
 <strong><em>Isochronic Parabiosis
 </em></strong> Female LD and WT littermates
 were housed as a pair from weaning until 8-10 weeks of age, where we
 performed isochronic parabiosis. At 16 weeks, the LD mouse was challenged
 with DMM surgery unilaterally on their L outside limb. Mice were monitored
 for 12 weeks and euthanized at 28 weeks of age. Serum and hindlimbs were
 collected for further analysis.
 <strong><em>Spatial Transcriptomics and Knee Joint
 Assessment  </em></strong> Samples were
 fixed in 4% PFA for 24h from mice that were joined by isochronic
 parabiosis. A reduced decalcification time of 10h at 4C on an orbital
 shaker was used for knee joints. Joint tissues were processed according to
 previous protocols and cut on a Leica Microtome at 5uM. Serial sections
 were used for staining with hematoxylin & eosin, toluidine
 blue/oil red O, and an unstained slide onto Fisher superfrost plus slides
 or Dako Flex IHC adhesive slides, and prepared for spatial transcriptomics
 by 10x Visium using the standard CytAssist protocol. The concentration of
 each library was accurately determined through qPCR utilizing the KAPA
 library Quantification Kit according to the manufacturer’s protocol (KAPA
 Biosystems/Roche) to produce cluster counts appropriate for the Illumina
 NovaSeq6000 instrument.  Normalized libraries were sequenced on a NovaSeqX
 10B or NovaSeq6000 S4 Flow Cell using the 151x10x10x151 sequencing recipe
 according to manufacturer protocol.  Read 1 was trimmed to the 10x
 Genomics recommendation of 28bp. A median sequencing depth of 50,000
 reads/cell was targeted for each Gene Expression Library.  The data were
 then spatially analyzed using standard graph-based clustering methods and
 visualized using 10x Space Ranger and 10x Loupe browser version
 7. <strong><em>Metabolomics and
 Proteomics </em></strong> Fat was
 explanted, weighed, washed, filtered, and 100mg was prepared for bulk
 sequencing (n=3-5/group), while 250mg was cultured for 24h in 1%
 Dulbecco’s Modified Eagle’s Medium. The resulting conditioned media (CM)
 was collected and compared to CM derived from naïve WT fat of equal mass
 (250mg) (n=4-8/group). CM protein and metabolites were extracted and
 evaluated via liquid chromatography-mass spectrometry
 (LC-MS)(<em>62</em>). Significant metabolites and protein
 intensities were detected using clustering and false-discovery-rate
 corrections in MetaboAnalyst (p&lt;0.05). Data were log transformed
 and compared using hierarchical cluster analysis (HCA), volcano plot
 analysis, principal component analysis (PCA) partial least-squares
 discriminant analysis (PLS-DA), and variable importance in projection
 (VIP) scores.
description: # Adipose-derived leptin and complement factor D mediate osteoarthritis
 severity and pain
 [https://doi.org/10.5061/dryad.dz08kps7c](https://doi.org/10.5061/dryad.dz08kps7c) Co-Corresponding Author Information: Name: Kelsey H. Collins ORCID: 0000-0001-7348-7348 Institution: UCSF Address: 35 Medical Center Way, RMB Pod C, San Francisco, Ca, 94941 Email: [kelsey.collins@ucsf.edu](mailto:kelsey.collins@ucsf.edu) Name: Farshid Guilak ORCID: 0000-0001-7380-0330 Institution: Washington University in St. Louis Address: 660 S. Euclid Ave, St. Louis, Mo, 63110 Email: [guilak@wustl.edu](mailto:guilak@wustl.edu) ## Description of the data and file structure This study investigates the role of systemic leptin and complement factor D secreted by adipose tissue in osteoarthritis and musculoskeletal pain in mice. Enclosed are the data for all figures in the paper except for the bulk RNA sequencing datasets. Please address questions to F. Guilak and K. Collins. These data were generated, collected, and analyzed between January 2020 - January 2025. ### Files and variables Each file includes all of the raw data plotted for each subpanel for each figure. Each figure has an individual .csv file, and each tab indicates each sub figure. Files included:  * **Figure 1**: File name *Figure1Data_3Jan2025.xlsx*. Adipose tissue is necessary for OA, but ablating fat-derived leptin protects knee joints from the onset of structural damage and pain.  A .csv file containing the raw data for all graphs in the figure 1B-U.  *Included data:* serum assessments for leptin (B), and implant size (C) body mass, liver mass (D), body fat (E). Knee joint structural damage assessed by Modified Mankin Score a (F), synovitis score and osteophyte score (G). Pain measurements included pressure-pain hyperalgesia at the knee (H) and Electronic Von Frey (I). Insulin tolerance tests and area under the curve (AUC, J), and glucose tolerance tests and AUC (K). Serum levels of IL-1a (L), IL-6 (M), IL-10 (N), IL-17a (O), MCP-1 (P), and TNF-a (Q). Synovial fluid levels for IL-1a (R), IL-6 (S), IL-10 (T), and TNF-a (U). N= 4-16/group. Abbreviations: leptin knockout mice (Ob/Ob), heterozygous (Ob/+), lipodystrophic mice (LD) and control mice (WT); DMM (destabilization of the medial meniscus); MEF – mouse embryonic fibroblast, MEF-Ob (MEF with knockout of leptin) MEF-Ob/+ (MEF with het knockout of leptin). * **Figure 2**: File name *Figure2Data_3Jan2025.xlsx*. Reintroducing leptin systemically via osmotic pump reverses cartilage protection in LD mice. A .csv file containing the raw data for all graphs in the figure 2B-U *Included data:* Serum assessments for leptin (B), body mass, liver mass (C). Knee joint structural damage assessed by Modified Mankin Score and medial tibial plateau images for Saf-O/Fast Green and Hematoxylin & Eosin (E), synovitis score (G) and osteophyte score (H). Pain measurements included pressure-pain hyperalgesia at the knee (I) and Electronic Von Frey (J). Serum measurements for IL-4 (K), IL-6 (L), IL-10 (M), IL-17a (N), MCP-1 (O), and TNF-a (P). Synovial fluid levels for IL-1a (Q), IL-4 (R), IL-6 (S), IL-10 (T) and TNF-a (U). N= 8-14/group. Abbreviations: lipodystrophic mice (LD), saline osmotic pump in LD mouse (sal, saline), leptin osmotic pump in LD mouse (Lep) and control mice (WT); DMM (destabilization of the medial meniscus). * **Figure 3**: File names: * WTL.cloupe 61.28 MB (wildtype, inside knee from parabiosis)  * LDL.cloupe 65.08 MB (lipodystrophic fat free, DMM)  * LDR.cloupe 68.99 MB (lipodystrophic, inside knee from parabiosis) * LDL_2.cloupe 93.03 MB (lipodystrophic fat free, DMM)  * WTR.cloupe 87.81 MB (wildtype, no injury)  * WTR_2.cloupe 138.77 MB (wildtype, no injury)  LD mice demonstrate cartilage damage when joined by isochronic parabiosis with DTA littermates, but do not reconstitute the infrapatellar fat pad. **Loupe files corresponding to the knee joint spatial transcriptomics studies in Figure 3, to be analyzed in the 10x Loupe Browser. Abbreviations: lipodystrophic mice (LD), saline osmotic pump in LD mouse (sal, saline), leptin osmotic pump in LD mouse (Lep) and control mice (WT); DMM (destabilization of the medial meniscus). This includes whole genome transcriptomics.  **Methods**: Female LD and WT littermates were housed as a pair from weaning until 8-10 weeks of age, where we performed isochronic parabiosis(*58*). At 16 weeks, the LD mouse was challenged with DMM surgery unilaterally on their L outside limb. Mice were monitored for 12 weeks and euthanized at 28 weeks of age. Serum and hindlimbs were collected for further analysis.Samples were fixed in 4% PFA for 24h from mice that were joined by isochronic parabiosis. A reduced decalcification time of 10h at 4C on an orbital shaker was used for knee joints. Joint tissues were processed according to previous protocols and cut on a Leica Microtome at 5uM. Serial sections were used for staining with hematoxylin & eosin, toluidine blue/oil red O, and an unstained slide onto Fisher superfrost plus slides or Dako Flex IHC adhesive slides and prepared for spatial transcriptomics by 10x Visium using the standard CytAssist protocol. The concentration of each library was accurately determined through qPCR utilizing the KAPA library Quantification Kit according to the manufacturer’s protocol (KAPA Biosystems/Roche) to produce cluster counts appropriate for the Illumina NovaSeq6000 instrument.  Normalized libraries were sequenced on a NovaSeqX 10B or NovaSeq6000 S4 Flow Cell using the 151x10x10x151 sequencing recipe according to manufacturer protocol.  Read 1 was trimmed to the 10x Genomics recommendation of 28bp. A median sequencing depth of 50,000 reads/cell was targeted for each Gene Expression Library.  The data were then spatially analyzed using standard graph-based clustering methods and visualized using 10x Space Ranger and 10x Loupe browser version 7. * **Figure 5:** Untargeted metabolomics (file name: *Figure5_MetabolomicsData.xlsx*) and proteomic analysis (file name: *Figure5_ProteomicsData.xlsx*)  identify complement factor D as a consistent differentially secreted protein by fat in the reversal of cartilage protection. Untargeted metabolomics and proteomics data from adipose conditioned media presented in Figure 5 are available in .csv files. Abbreviations: MEF-KO (leptin KO), MEF-WT (wildtype fat implant).  **Methods:** Fat was explanted, weighed, washed, filtered, and 100mg was prepared for bulk sequencing (n=3-5/group), while 250mg was cultured for 24h in 1% Dulbecco’s Modified Eagle’s Medium. The resulting conditioned media (CM) was collected and compared to CM derived from naïve WT fat of equal mass (250mg) (n=4-8/group). CM protein and metabolites were extracted and evaluated via liquid chromatography-mass spectrometry (LC-MS). Significant metabolites and protein intensities were detected using clustering and false-discovery-rate corrections in MetaboAnalyst (p&lt;0.05). Data were log transformed and compared using hierarchical cluster analysis (HCA), volcano plot analysis, principal component analysis (PCA) partial least-squares discriminant analysis (PLS-DA), and variable importance in projection (VIP) scores. Median intensity heatmaps were calculated in MATLAB to determine features uniquely upregulated in each group. Integrated metabolomics and proteomics pathway impact analysis was conducted in MetaboAnalyst using UniProt and KEGG identifications. * **Figure 6:** File name *Figure6Data_3Jan2025.xlsx*. Complement factor D is a key mediator in the development of osteoarthritis and joint pain with DMM. The Figure 6B-U raw data for all graphs in the figure are attached in the figure 6 spreadsheet.  *Included data:* Knee joint structural damage assessed by Modified Mankin Score (C), synovitis score (D) and synovitis images stained with Hematoxylin & Eosin (E) and osteophyte scores (F). Pain measurements included pressure-pain hyperalgesia at the knee (G), which were not explained by synovitis (H), Electronic Von Frey (I). There were no differences in insulin tolerance (J) or glucose tolerance (K). Body fat (L), serum leptin (M), and body weight (N). Serum levels for IL-1a (O), IL-6 (P), IL-10 (Q), MCP-1 (R), and TNF-a (S). Synovial fluid levels for IL-6 (T), and IL-10 (U) N= 6-12/group, Abbreviations: Factor D knockout mice (FD-/-), FD-/- mice with mouse embryonic fibroblast correction to restore FD (FD-/-+ MEF), control mice (WT); DMM (destabilization of the medial meniscus) Figure 4 and 7 RNA seq data was uploaded to GEO (GSE287251), link to database can be found in the published paper.  * **Methods:** Total RNA integrity was determined using Agilent Bioanalyzer. Library preparation was performed with 500ng of total RNA. Ribosomal RNA was removed by an RNase-H method using RiboErase kits (Kapa Biosystems). mRNA was then fragmented in reverse transcriptase buffer and heating to 94 degrees for 8 minutes. mRNA was reverse transcribed to yield cDNA using SuperScript III RT enzyme (Life Technologies, per manufacturer's instructions) and random hexamers. A second strand reaction was performed to yield ds-cDNA. cDNA was blunt ended, had an A base added to the 3' ends, and then had Illumina sequencing adapters ligated to the ends. Ligated fragments were then amplified for 16 cycles using primers incorporating unique dual index tags. Fragments were sequenced on an Illumina NovaSeq-6000 using paired end reads extending 150 bases. Basecalls and demultiplexing were performed with Illumina’s bcl2fastq software with a maximum of one mismatch in the indexing read.  RNA-seq reads were then aligned to the Ensembl release 101 primary assembly with STAR version 2.7.9a1.  Gene counts were derived from the number of uniquely aligned unambiguous reads by Subread:featureCount version 2.0.32. Isoform expression of known Ensembl transcripts were quantified with Salmon version 1.5.23. Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected.  The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 4.04. * **Analysis of RNAseq:** RNA-seq data were processed as in previous study.(*59*) RNA-seq data of mouse ganglia were processed by Cutadapt (v2.7; --quality-cutoff=15,10 --minimum-length=36) to remove adapters and FastQC (v0.11.4) to estimate the sequencing quality. Trimmed reads were then aligned to the mouse genome mm10 with GENCODE annotation vM28 using STAR (v2.5.4) with default parameters. Transcript quantification was performed using featureCounts from the subread package (v1.6.3). Sequencing performance was assessed for the total number of aligned reads, total number of uniquely aligned reads, and features detected. The ribosomal fraction, known junction saturation, and read distribution over known gene models were quantified with RSeQC version 2.6.2. Principle component analysis and differential expression analysis were determined using DESeq2 in negative binomial mode using batch-corrected transcripts from featureCounts (&gt;2-fold expression change, &gt;1 count per million (CPM), Benjamini corrected p &lt; 0.05). Pairwise comparisons were made between groups to determine differentially expressed genes (DEGs) within each group. Gene ontology (GO) and KEGG analyses were performed using EnrichR for DEGs. The gene expression was plotted using ggplot2 and pheatmap. Statistical analysis was performed using R. ## Code/software Data were analyzed with the following software:  Figure 1, 2, 6 .csv files - Graphpad Prism  Adipose Conditioned Media Metabolomics dataset - Metaboanalyst ([https://www.metaboanalyst.ca/](https://www.metaboanalyst.ca/)) Adipose Conditioned Media Proteomics dataset - Spectronaut and Metaboanalyst  Spatial Transcriptomics of knee joints from parabiosis studies  - processed by SpaceRanger, Loupe files can be read by the 10x Loupe Browser ([https://www.10xgenomics.com/support/software/loupe-browser/latest](https://www.10xgenomics.com/support/software/loupe-browser/latest)) ## Methods  The authors are happy to answer any questions and request to be alerted before use of these data for further publication.  Key Methods:  ***Animal Studies*** All experimental procedures were approved by the Washington University School of Medicine Department of Comparative Medicine Institutional Animal Care and Use Committee (WUSTL IACUC 22-0306) and were conducted in accordance with ARRIVE guidelines. LD mice and WT (DTA/+) littermate controls were generated for these studies by crossing adiponectin-Cre (Jackson Labs: 028020) mice with homozygous lox-stop-lox-ROSA-diphtheria toxin A (Jackson Labs: 010527) mice on a C57BL/6 background. To breed at mendelian frequency, mice were bred and maintained at thermoneutrality throughout their lifespan (30 °C). Mixed genotype mice were group-housed such that *n* = 3 to 5 per cage. Male LD mice received either a MEF transplant (MEF-rescue) from WT, leptin heterozygous (MEF-HET) or leptin homozygous knockout (MEF-KO) pups as previously described, which developed into adipose-like tissue between 3 and 5 weeks of age. The injection was delivered subcutaneously to the sternal aspect of a donor LD mouse (3–5 weeks old) under 2% isoflurane with a 27-gauge needle. Animal numbers used in each experiment can be found in the figure legend. ***Exogenous Leptin Delivery*** Osmotic pumps were surgically implanted to the dorsal aspect of mice at 15 weeks of age, one week prior to DMM. Leptin or saline was delivered to LD mice using Azlet 2006 (Cupertino, CA) at a release rate of 0.15![](file:////Users/kelcollins/Library/Group%20Containers/UBF8T346G9.Office/TemporaryItems/msohtmlclip/clip_image002.png) to deliver a total of 1mg of leptin over 6 weeks, and were replaced with new, sterile implants until 28 weeks of age. Leptin concentration was confirmed after implantation. ***OA induction by DMM*** At 16 weeks of age, surgical animals underwent destabilization of the medial meniscus surgery in their left knee joints, and the right limb served as a nonsurgical contralateral control. Behavioral and metabolic assays were conducted throughout the course of this study. Mice were sacrificed at 28 weeks of age to assess OA severity, bone microstructure, and serum and SF inflammatory profiles. Knee joints were fixed in 4% paraformaldehyde and decalcified in 10% formic acid  (Cal-Ex II) for 48-72 hrs. Histological assessment was performed by Modified Mankin Scoring, Synovitis Scoring, and Osteophyte scoring on formalin fixed paraffin embedded 5um sections as previously described(*8*). Bone marrow adipocytes were counted in ImageJ on histological sections. ***Body Composition, Tissue Harvest and Storage*** Mice were weighed weekly throughout the course of the study using a standard scale (g). Body fat was measured by DXA (Lunar Piximus) at 27 weeks of age to quantify body fat. Animals were euthanized at 28 weeks of age in accordance with the timelines in each figure. Body mass, liver mass, body fat, blood, L3-L5 dorsal root ganglia sensory neurons from *FD-/-* and WT mice and synovial fluid was harvested. ***Serum and Synovial Fluid Profiling*** All mice were fasted overnight prior to sacrifice. Serum was collected in a BD Microtainer STT serum separator tube and allowed to clot at room temperature. Synovial fluid was collected using an alginate pad that was digested in alginate lyase until a stop solution was applied. Both fluids were stored at −80 °C until analysis by Luminex multiplex 18-plex chemokine/cytokine array assay (Eve Technologies). Enzyme-linked immunosorbent assay (ELISA) for serum leptin was conducted using a mouse/rat Leptin Quantikine ELISA Assay kit (MOB00; R&D Systems) at a dilution of 1:2 in LD mice, and all other groups were evaluated at the manufacturer’s recommended 1:20 dilution. ***Bone Microstructure Analysis*** Whole knee joints were scanned by microcomputed tomography (Bruker SkyScan1176) at a resolution of 18-μm isotropic voxel resolution according to previously reported methods. To reduce beam hardening, a 0.5-mm aluminum filter was used during scanning. Hydroxyapatite calibration phantoms were scanned to calibrate bone density. Scans were reconstructed to three-dimensional (3D) images using NRecon software, and CTAn software was used to segment subchondral and trabecular regions from the medial tibial plateau, lateral tibial plateau, medial femoral condyle, and lateral femoral condyle for analysis. The tibial epiphysis was identified using the subchondral plate and growth plate as references. The tibial metaphysis was defined as the 1-mm area directly below the growth plate. The main outcomes reported from microCT images are BMD, BV/TV (%), trabecular number, and trabecular thickness. ***Insulin and glucose tolerance tests*** Insulin- and glucose-tolerance tests were performed 4 weeks post-DMM surgery (20 weeks of age) after fasting mice for a minimum of 4 hours. Each test was conducted at least 1 week apart in all animals. For both tests, fasting glucose levels were measured by tail bleed at time point 0 to establish a baseline. For glucose-tolerance tests, animals were administered 1 mg/kg (10% dextrose: 1% volume/body mass) by intraperitoneal (IP) injection, and for insulin-tolerance tests, 0.75 U/kg body mass of insulin (Humulin R diluted to 75 mU/mL, 1% volume/body mass) was administered by IP injection(*8, 12*). Serial blood glucose measurements were taken via tail vein at 20, 40, 60, and 120 min after injection with a glucose meter (Contour; Bayer). ***Pain Assessments and Behavioral Testing*** All animals were acclimatized to all equipment 1 d prior to the onset of testing. Two measures for pain were conducted at 27 weeks of age. To assess tactile allodynia in the DMM limb, an Electronic Von Frey assay was used as previously described. Hind paws of DMM limbs were stimulated three to five times. The intensity of the stimulus (grams) was recorded by the tester when the paw was withdrawn. To assess mechanical hyperalgesia, pressure-pain tests were conducted using a Small Animal Algometer (SMALGO) (Bioseb). Three to five trials of the surgical and nonsurgical limb were collected by applying a steadily increasing force to the lateral aspect of each limb until the limb was withdrawn. The average of three trials for each limb was reported, and maximum value of 450 g was employed to avoid tissue damage to the knee joint.