The generation of 2KR mice on a C57BL/6 genetic background was described previously (Kraakman et al., 2018). The Adipsin KO mouse line was obtained from Dr. James Lo (Weill Cornell Medical College). The mouse lines of C3 KO (Stock No: 029661), Adipoq-Cre (Stock No: 028020), and Pparg^loxp (Stock No: 004584) were purchased from Jackson Laboratory. All the lines were bred and maintained on the C57BL/6J background. Mice were housed at room temperature (RT, 23 ± 1°C) on a 12 hr light/12 hr dark cycle with access to food and water ad libitum. The High Fat Diet (HFD) contained 60% calories from fat, 20% from protein, and 20% from carbohydrates (Research Diets: D12492). Rosi maleate (Avandia) (Abcam, ab142461) was mixed into HFD at 100 mg/kg at Research Diets (New Brunswick, NJ) to achieve a dose of approximately 5 mg/kg body weight (BW). In the CR diet, mice received 70% of their daily food intake (chow) twice a day for 4 weeks. For the glucose tolerance test (GTT), mice were fasted overnight in cages with fresh bedding and intraperitoneally (i.p.) injected with glucose (2 g/kg BW). Blood glucose was measured with a Breeze2 glucometer (Bayer) at indicated time points. For the insulin tolerance test (ITT), mice were fasted for 4 hr and injected i.p. with insulin (0.75 U insulin/kg BW). Body compositions were determined by EchoMRI. All animal protocols used in this study were reviewed and approved by the Columbia University Animal Care and Utilization Committee.

We downloaded the gene expression dataset of whole-tissue BM in mice fed ad libitum or on CR for 3 weeks from GEO (GSE124063) (Collins et al., 2019). Differentially expressed genes (DEGs) were assessed by a fold change of 1 in the gene expression difference between control and CR. The secreted proteins were obtained from the UniProt with the keyword Secreted [KW-0964] (Bateman and UniProt Consortium, 2019). Only the manually annotated secretin proteins were retained for further analysis and experiments. We employed DAVID 6.8 to perform the functional annotation (Cellular Component [CC]) for the DEGs (Huang et al., 2009). The enriched CC was evaluated using a false discovery rate (FDR, Benjamini–Hochberg method) of 0.1 and DEG number ≥ 10.

Femurs were collected and fixed in 10% neutral buffered formalin overnight at 4°C, and subsequently used for bone micro-architecture analysis and lipid quantification. For micro-architecture analysis, a Quantum FX μCT Scanner (Perkin-Elmer) was used for scanning. For lipid quantification, a 14% EDTA solution was used to decalcify bone for at least 2 weeks with frequent changes. The bones were then stained for 48 hr in a 1% osmium tetroxide, 2.5% potassium dichromate solution at RT, washed in tap water for at least 2 hr, and imaged by μCT. The software Analyze 12.0 was used to quantify lipid volume and μCT scan parameters and performed according to their bone micro-architecture add-on. Marrow adipose sections were determined based on consistent 250 slice intervals measured from the identified growth plate of the femur.

BMSCs were isolated from 4-week-old mice. Briefly, the mice were euthanized, and bones were dissected and washed in sterile PBS. BM was flushed out using a 25-gauge needle into α modification of Minimum Essential Medium (αMEM) supplemented with 10% fetal bovine serum (FBS) and 1% penicillin/streptomycin. Upon reaching 70–80% confluence, BMSCs were passaged and plated in 6-well or 12-well plates for experiments.

Inguinal fat pads with the lymph nodes removed were dissected from 5- to 6-week-old wild-type (WT) and Adipsin knock-out (KO) mice, followed by mincing and digesting in Liberase (Sigma-Aldrich, catalog # 5401127001) at 37°C for 30 min with gentle agitation. After passing through a 100 μM pore cell strainer, the ASC was pelleted by centrifuging at 400 g for 5 min at 4°C. The pellet was resuspended and plated in basic medium (DMEM supplemented with 10% FBS, 1% penicillin/streptomycin, 1% gentamycin). Upon reaching 70–80% confluence, ASCs were passaged and plated in 6-well plates for experiments.

HEK293T (ATCC CRL-3216) and C3H10T1/2 (ATCC CCL-226) were obtained from ATCC along with thorough reports of analysis confirming the cell line identification, authentication, and confirmed negative testing for mycoplasma contamination. The PPARγ cDNAs, including PPARγ2-WT and PPARγ2-2KR, were cloned into a doxycycline-inducible lentiviral plasmid, pTRIPZ (Thermo Open Biosystems) (Li et al., 2016), and were stably overexpressed into PPARγ^-/- mouse embryonic fibroblasts (MEFs) (Rosen et al., 2002) with a selection of puromycin (2.5 μg/mL). Cells were grown in high-glucose DMEM (Corning, 10-017) supplemented with 10% FBS (heat inactivated; Corning, 35-011-CV) and 1% penicillin/streptomycin. These PPARγ cell lines have been previously validated and tested (Kraakman et al., 2018).

To induce osteogenic differentiation, we used mineralization-inducing αMEM containing 100 μM/mL ascorbic acid and 2 mM β-glycerophosphate. Two days after induction, the cells were maintained in the same medium changed every 2–3 days. Approximately 50 mM Alizarin Red S (Sigma-Aldrich) in water was used to stain calcium deposition after 21 days. To induce adipogenic differentiation, we cultured BMSCs in DMEM containing 1 μM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 10 μg/mL insulin, and 5 μM rosiglitazone (Rosi). Two days after induction, the cells were maintained in medium containing 10 μg/mL insulin and 5 μM Rosi until fully differentiated. Media were changed every 2–3 days. Recombinant Adipsin (Sino Biological, 1 μg/mL) and C3aR inhibitor, SB290157 (R&D Systems, 1 μM), were added to the medium 2 days before induction and included in the maintenance media when necessary. Recombinant Adipsin, SB290157, and recombinant Wnt3a (R&D Biosystems, 20 ng/mL) were added to the media of C3H10T1/2 cells 24 hr before harvesting. Oil Red O staining was performed to detect the lipid droplets using an Oil Red O staining kit according to the manufacturer’s instruction (Electron Microscopy Sciences).

Tissues and cells were lysed with 1 mL TRI reagent (Sigma-Aldrich). After phase separation through the addition of 250 μL chloroform, RNA was isolated using the NucleoSpin RNA Kit (Macherey-Nagel, Inc). The High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems) was used to synthesize cDNA from 1 μg total RNA. qPCR was performed on a Bio-Rad CFX96 Real-Time PCR system with the GoTaq qPCR Master Mix (Promega). Relative gene expression levels were calculated using the ΔΔCt method with CPA as the reference gene.

Cells were lysed and tissues were homogenized by Polytron homogenizer immediately after dissection in western extraction buffer (150 mmol/L NaCl, 10% glycerol, 1% NP-40, 1 mmol/L EDTA, 20 mmol/L NaF, 30 mmol/L sodium pyrophosphate, 0.5% sodium deoxycholate, 0.05% SDS, 25 mmol/L Tris-HCl: pH 7.4) containing protease inhibitor cocktail (Roche). The lysate was then sonicated, and debris was removed by centrifugation. SDS-PAGE and western blotting were performed and detected with ECL (Thermo Scientific). Antibodies used for western blot analysis were as follows: anti-Adipsin (R&D Systems, catalog# AF5430), anti-adiponectin (Invitrogen, catalog# PA1-054), anti-C3 (Proteintech, catalog# 21337-1-AP), anti-FABP4 (Cell Signaling Technology, catalog# 2120), anti-phospho-GSK-3β (Cell Signaling Technology, catalog# 9336), anti-HSP90 (Proteintech Group, Inc, catalog# 1371-1-AP), and anti-β‐Catenin (Cell Signaling Technology, catalog# 9562).

Femurs were collected and fixed in 10% neutral buffered formalin overnight at 4°C and kept long term in 70% ethanol at 4°C. They were subsequently decalcified in a 20% EDTA solution for 4 weeks before becoming embedded with paraffin. Allocated sections were stained with Harris hematoxylin and eosin (H&E). Immunohistochemistry for β‐Catenin (Cell Signaling Technology, catalog# 9562) was performed using a 1:100 dilution in PBST.

BMAd RNA-seq analysis was performed on samples from a previous clinical trial of fasting and high-calorie diet in healthy human volunteers (Fazeli et al., 2021). The study was approved by the Partners HealthCare Institutional Review Board and complied with the Health Insurance Portability and Accountability Act guidelines. Written informed consent was obtained from all subjects. BMAds from those volunteers were separated by centrifugation from marrow sera and the RNA was extracted and frozen. RNA-seq analysis was performed at the Vermont Institute of Genomics Research. Samples were de-identified, and the analysis was blinded to assignment. Data analyses were performed using STRING (Search Tool for the Retrieval of Interacting Genes/Proteins). Protein coding genes were assessed with p<0.05, FDR < 0.05. Morpheus software (Broad Institute) was also utilized with hierarchical clustering done by Euclidean distance.

Values are presented as mean ± SEM. We used unpaired two-tailed Student’s t-test and two-way ANOVA to evaluate statistical significance with a p<0.05 considered to be statistically significant.

CR is a broadly adapted intervention for metabolic improvements. It induces the shrinkage of most adipose tissue depots, but, paradoxically, expands MAT as in the femur (Figure 1A). Transcriptomic analyses of whole BM revealed that the most pronounced changes during CR are associated with the secretome (Figure 1—figure supplement 1A). Interestingly, Cfd (encoding Adipsin) is among the top induced genes by CR in the BM and is the most abundant among all secretory genes (Figure 1B). Adipsin is a highly expressed adipokine in peripheral fat pads, such as subcutaneous white adipose tissue (SWAT) and epididymal white adipose tissue (EWAT) (Min and Spiegelman, 1986). However, its expression and regulation have not been examined in MAT. qPCR analysis validated an approximately ninefold induction of Cfd in the BM by CR compared to ad libitum-fed mice, accompanied by increased adipocyte genes (Pparg2, Fabp4, Plin1) and decreased osteoblast markers (Osteocalcin, Col1a1) (Figure 1C), whereas its induction in EWAT and SWAT was milder or unchanged, respectively (Figure 1—figure supplement 1B, C). In the bone, protein levels of Adipsin are also notably increased after CR (Figure 1D). Of note, CR increases circulating Adiponectin levels, attributed to its production by BMAds (Cawthorn et al., 2014). However, the upregulation of BM Adipsin, even though it is more pronounced and at a higher abundance than Adiponectin, is unlikely to contribute significantly to the circulating levels, as plasma Adipsin was not significantly increased in CR mice (Figure 1D, E). This data suggests that, unlike other adipokines, Adipsin is uniquely regulated in the BM, highlighting its potential role in regulating the BM microenvironment.

To examine whether the induction of BM Adipsin is restricted to CR, we employed a distinct model to induce MAT expansion in vivo using a PPARγ agonist (TZD) known as Rosi in DIO mice. This condition could induce profound MAT expansion, as indicated by a significant increase of osmium tetroxide staining of lipid droplets (Figure 1F). In this model, Cfd expression in the BM was upregulated >12-fold, more than other adipocyte markers (e.g., Pparg2 and Fabp4) and Adiponectin (Adipoq) (Figure 1G). Increased expression of Perilipin 1 (Plin1) was also robustly induced by Rosi treatment in the BM, indicating abundant MAT expansion (Styner et al., 2017; Li et al., 2019). In contrast, the upregulation of Cfd expression occurred to a lesser extent in the EWAT and SWAT (Figure 1—figure supplement 1D, E). The concentrations of bone and plasma Adipsin were elevated only moderately, differing from a significant increase in Adiponectin levels (Figure 1H, I). Again, Adipsin appears to be the most responsive adipokine to MAT expansion in the BM specifically.

MAT expansion is generally believed to occur concurrently with bone loss. However, a short 3-week Rosi treatment was sufficient to stimulate a fivefold expansion of MAT in the femurs (Figure 1J). In contrast, neither the trabecular nor the cortical bone mineral density (BMD) was decreased by short-term Rosi treatment (Figure 1K, L). This data suggests that BM adipogenesis precedes the significant bone loss by TZD and that BMAd-derived factors, such as Adipsin, may participate in regulating downstream changes in the bone.

To obtain direct evidence of that Adipsin could influence BM plasticity, we utilized Adipsin KO mice. Upon CR, Adipsin KO mice displayed significantly lower bone marrow adiposity (BMA) detected by osmium tetroxide staining for lipid droplets (Figure 2A, B), though H&E staining revealed minimal lipid droplet accumulation in both CR conditions and chow-fed controls (Figure 2—figure supplement 1E). The inhibited MAT was underlined by decreased expression of adipogenesis makers in the BM, including Pparg, Adipoq, Fabp4, Fasn, Gdp1, and Perilipin (Figure 2C). Interestingly, in contrast to their reduced BMA, Adipsin KO mice preserved more fat mass, despite the similar response to CR on overall body weight, insulin sensitivity, and glucose tolerance (Figure 2—figure supplement 1A–D). Additionally, there were no observed differences in plasma insulin levels (Figure 2—figure supplement 1H). In line with their lower BMA, Adipsin KO mice showed higher BMD in the cortical and trabecular regions (Figure 2D, E), without significant changes between WT and Adipsin KO in trabecular number, cortical thickness (Figure 2—figure supplement 1F, G), or cortical bone volume (BV)/total volume (TV) but a decrease in trabecular BV/TV (Figure 2F, G). It should be noted that no significant changes of MAT or bone structure were observed in adult Adipsin KO mice on ad libitum chow diet feeding (Figure 2A–F) and that, given these parameters, CR was successful in inducing bone loss in WT mice.

In the parallel TZD model, following the induction of obesity and insulin resistance with 12 weeks of HFD feeding, Adipsin KO and WT mice were treated with Rosi to promote MAT expansion. Adipsin KO mice showed comparable metabolic phenotypes regarding obesity and insulin sensitivity (Figure 2—figure supplement 1I, J), but worse glucose tolerance (Figure 2—figure supplement 1K) as expected (Lo et al., 2014; Gómez-Banoy et al., 2019). H&E staining revealed fewer lipid droplets accumulated in the Adipsin KO mouse BM (Figure 2H). Furthermore, ablation of Adipsin improved skeletal health as shown by higher BMD in both the cortical and trabecular femoral regions without affecting BV (Figure 2I–L), trabecular number, or cortical thickness (Figure 2—figure supplement 1L, M) upon Rosi treatment. As expected, these differences in the bone microarchitecture of WT and Adipsin KO mice were not observed in the HFD-only cohort, implying that Adipsin is crucial to the response and plasticity of BMAs in conditions that are known to induce BM adipogenesis. Thus, Adipsin deficiency appears to consistently alter the balance between MAT expansion and bone homeostasis in distinct bone loss models, leading to an overall bone protection effect.

Adipsin, also called Complement Factor D (Rosen et al., 1989; White et al., 1992), is an established activator of Complement Component 3 (C3), the central player of the complement system (Xu et al., 2001; Sahu and Lambris, 2001). C3 deficiency has been shown to protect mice from ovariectomy-induced bone loss (MacKay et al., 2018). Therefore, we speculated that the complement pathway could be the downstream effector through which Adipsin regulates MAT expansion. To investigate this, we challenged C3 KO mice to CR. Despite no discernible differences in body weight or composition and worse insulin sensitivity and glucose tolerance but similar fasting insulin levels (Figure 3—figure supplement 1A–E), C3 KO mice displayed a pronounced inhibition of MAT expansion (Figure 3A, B), though with minimal lipid droplets detected by H&E staining (Figure 3—figure supplement 1F). In contrast to MAT, the effects on bone were overall mild (Figure 3C–F), with increases observed only in cortical BMD (Figure 3C) and cortical thickness (Figure 3—figure supplement 1G, H). These data indicate that complement activity is critical to BM adipogenesis in response to CR.

To further establish the role of complement activation in MAT expansion, we analyzed C3 KO mice subjected to HFD to induce obesity followed by Rosi treatment. In baseline HFD-fed, obese mice C3 knockout restrained the development of MAT as shown by osmium tetroxide staining (Figure 3G, H) without affecting BMD (Figure 3J, K). This uncoupling of BMA from bone loss suggests that complement activation has a direct impact on BM adipogenesis. Strikingly, C3 deficiency prevented Rosi-induced MAT expansion by an approximately sixfold reduction (Figure 3G, H), further supported by observed differences in H&E staining (Figure 3I). Additionally, C3 KO mice displayed strong protection of cortical BMD (Figure 3J) and BV (BV/TV) in both the cortical and trabecular regions (Figure 3L, M), despite no changes in the trabecular number and cortical thickness (Figure 3—figure supplement 1L, M). The bone protection effect of C3 deficiency was associated with slightly improved insulin sensitivity but significantly worsened glucose tolerance (Figure 3—figure supplement 1I–K), in line with Adipsin KO mice (Figure 2—figure supplement 1K). Together, the inhibition of MAT expansion and bone loss in Adipsin KO mice is amplified in C3 KO mice, suggesting that Adipsin plays a role in regulating marrow homeostasis through complement activity.

Aging is associated with MAT expansion and decline in bone integrity (Krings et al., 2012). Interestingly, Adipsin in the circulation was gradually decreased during aging (Figure 4A), raising the question whether BM Adipsin is similarly altered with age. Indeed, Cfd was markedly upregulated in the BM at 78 weeks of age compared to middle-aged (26-week-old) mice (Figure 4B), in striking contrast to the decrease in the peripheral fat (Figure 4C), which potentially underlies the decrease in circulation. These data emphasize that BM Adipsin is distinct from peripheral Adipsin and is uniquely correlated with BMA and bone health during aging.

To understand the functional significance of BM Adipsin, we assessed bone health in aging (60-week-old) Adipsin KO and WT control mice. Similar to the CR model, the osmium tetroxide staining in the femur revealed an approximately 50% decrease in BMA in Adipsin KO mice (Figure 4D, E), despite no discernible lipid droplet appearance in H&E staining (Figure 4—figure supplement 1D). Furthermore, Adipsin KO mice displayed a significantly higher BMD in the femoral trabecular region (Figure 4G). Though the cortical region BMD, trabecular number, and cortical thickness did not change significantly (Figure 4F, Figure 4—figure supplement 1E, F), the associated cortical BV/TV was increased (Figure 4H), indicating better bone quality overall. These bone protective effects in aging Adipsin KO mice were independent of metabolic changes as their body weight, composition, insulin sensitivity, and glucose tolerance remained comparable to those of WT (Figure 4—figure supplement 1A–C). These results reinforce our hypothesis that BM-derived Adipsin is favorable to BMAd development but detrimental to bone health.

Osteoblasts and BMAds share a common progenitor, BMSCs. Given the inhibited bone loss and MAT expansion observed in Adipsin and C3 deficiency, we asked whether the differentiation of BMSCs is affected. We isolated BMSCs from WT and Adipsin KO mice and stimulated them to differentiate into adipocytes or osteoblasts. Interestingly, even with Rosi activation of PPARγ, the adipogenic capacity of Adipsin KO BMSCs was diminished, shown by decreased Oil Red O staining for lipid droplets and mRNA expression of adipocyte markers (Figure 5A, B). On the other hand, these cells were more readily differentiated into osteoblasts as determined by increased Alizarin Red staining for mineralization and increased expression of osteoblast markers, including Runx2, Atf4, Osx, Alpl, Ptfhr, Col1a1, and Osteocalcin (Figure 5C, D). To assess whether this impaired adipogenesis was a generalized feature of Adipsin deficiency in adipocyte precursor cells, we isolated the ASCs from the SWAT of WT and Adipsin KO mice and stimulated their differentiation into adipocytes. However, the adipogenic capacity for the ASCs from Adipsin KO mice was not hindered indicated by comparable Oil Red O staining and adipogenic gene expression (Figure 5—figure supplement 1A, B). The difference in differentiation capability between BMSCs and ASCs highlights the specific effect of Adipsin on the BM.

Given the overlapping effects of Adipsin and C3 deficiencies on BM homeostasis in vivo, we further suspected that Adipsin drives changes in BM through its activation of C3 in the alternative complement pathway. Similar to Adipsin KO cells, C3 KO BMSCs had impaired adipogenesis, as shown by lower Oil Red O staining and modest downregulation of markers related to adipogenesis (Figure 5E, F). Conversely, the ablation of C3 enhanced osteoblast differentiation capacity, indicated by increased Alizarin Red staining and upregulation of mRNA expression of osteoblastogenesis markers (Figure 5G, H). Together, these results suggest that Adipsin deficiency directly modulates BMSC fate by hindering their differentiation into adipocytes and favoring osteoblastogenesis through complement activity.

Due to the previously observed influence of Adipsin on the overall BM microenvironment, we sought to determine whether Adipsin KO BMSCs were primed toward osteoblastogenesis prior to induction. Confluent, undifferentiated Adipsin KO and WT BMSCs were treated with 1 μM dexamethasone, a glucocorticoid commonly used in vitro to stimulate both adipogenesis and osteoblastogenesis (Chen et al., 2016). We reasoned that the short-term dexamethasone treatment would initiate differentiation but not fully determine lineage preference. Interestingly, at 48 hr post-treatment, Adipsin KO cells displayed a higher expression of genes associated with osteoblastogenesis, including Runx2, Atf4, and Opn, whereas adipocyte markers Adipoq, Fabp4, Fasn, and Cd36 were significantly repressed (Figure 6A). These data suggest that Adipsin KO BMSCs may be primed toward osteoblastogenic differentiation. As nascent wild-type BMSCs barely express adipogenic genes such as Adipsin, these results further imply that the presence of Adipsin in the BM microenvironment primes BMSC fate toward adipogenesis in vivo.

To obtain direct evidence of the priming of BMSCs by Adipsin, we treated BMSCs isolated from Adipsin KO mice, which are naïve to Adipsin exposure, with recombinant Adipsin. Adipsin treatment repressed the induction of osteoblast markers during osteoblastogenic differentiation (Figure 6B). On the other hand, exogenous Adipsin had a mild effect on promoting adipocyte differentiation, as shown by increased lipid accumulation and upregulation of PPARγ2, the master regulator of adipogenesis (Figure 6—figure supplement 1A–C). Conversely, inhibiting the downstream C3 complement activity by SB290157, a C3aR inhibitor, prevented lipid accumulation and repressed adipogenic gene expression during adipogenesis (Figure 6C, Figure 6—figure supplement 1D, E) while promoting the upregulation of osteoblast transcription factor Osx during osteoblastogenesis (Figure 6—figure supplement 1F) in BMSCs. Moreover, treating BMSCs with SB290157 blunted the effects of exogenous Adipsin on their differentiation into adipocytes and osteoblasts (Figure 6—figure supplement 1G, H). Together, these data suggest that complement activity suppression is optimal for osteoblast differentiation and inhibitory of adipogenesis. These data support our conclusion that Adipsin functions in the BM microenvironment to prime BMSCs toward adipocyte differentiation and inhibit osteoblastogenesis.

To understand the mechanism underlying the priming of BMSCs, we assessed whether intrinsic transcriptional differences already existed in undifferentiated Adipsin KO BMSCs. We found a notable upregulation in genes associated with the canonical (Wwtr1, Lrp5) and non-canonical Wnt pathways (Wnt5a) (Figure 6D). In the canonical Wnt pathway, Wnt ligand inactivates GSK3 via phosphorylation to inhibit the phosphorylation of β-catenin, leading the latter to stabilize and translocate into the nucleus in order to transcribe downstream target genes (Wu and Pan, 2010). This pathway is well established to inhibit adipogenesis (Park et al., 2019; Kim et al., 2010) and promote bone formation (Gong et al., 2001; Mani et al., 2007; Glass and Karsenty, 2007); while upregulation of non-canonical-associated proteins induces Runx2 expression (Takada et al., 2007; Tu et al., 2007), the main osteoblastogenic transcriptional factor. Thus, Adipsin likely inhibits Wnt signaling in order to determine the differentiation fate of BMSCs. Consistently, Adipsin treatment inhibited both the canonical (Lrp5) and non-canonical (Wnt5a) Wnt signaling genes, whereas SB290157 treatment abolished this inhibition in Adipsin KO BMSCs (Figure 6E). In support of this hypothesis, Adipsin treatment blunted Wnt3a-induced phosphorylation of GSK3β and prevented the subsequent accumulation of β-catenin in C3H10T1/2 mesenchymal stem cells (Figure 6F). Furthermore, the activation of canonical Wnt signaling was recapitulated in Adipsin KO mice, which displayed increased GSK3β phosphorylation in the bone (Figure 6G). Though β-catenin signal was too low to be detected by western blotting, immunohistochemical staining revealed an increase in β-catenin in the BM of Adipsin KO mice (Figure 6H). Additionally, Wnt/β-catenin signaling pathway genes Wnt3a, Fzd9, and Ctnnb1 (Rudloff and Kemler, 2012), but not the β-catenin-repressive Tcf3 and Tcf7l2, were upregulated in the BM of Adipsin KO mice after Rosi treatment (Figure 6I). There were no significant changes of p-GSK3β and β-catenin observed in the EWAT (Figure 6—figure supplement 2A), emphasizing a bone-specific role for Adipsin. We also observed increases in GSK3β phosphorylation, β-catenin, and expression of Wnt pathway genes in the bone of C3 KO mice after Rosi treatment (Figure 6J–L), despite no discernible differences in the EWAT (Figure 6—figure supplement 2B). The Adipsin KO and C3 KO mice on CR recapitulated these changes to Wnt signaling markers as well (Figure 6—figure supplement 2C-F). Together, these data imply that Adipsin inhibits Wnt signaling through its complement activity to prime BMSCs toward adipocyte differentiation.

Next, we sought to understand how Adipsin is regulated. Adipsin is induced during adipogenesis by PPARγ, the master regulator of adipocyte biology (Tontonoz et al., 1994). Adipocyte-specific ablation of PPARγ leads to significantly increased trabecular bone density with an associated absence of BMA accompanied by a lipoatrophy phenotype (Wang et al., 2013). Notably, these knockouts displayed a complete absence of circulating Adipsin (Figure 7A), suggesting that PPARγ is essential for Adipsin production. Furthermore, we have observed that Cfd expression is sensitive to PPARγ acetylation changes in our previous studies (Qiang et al., 2012; Kraakman et al., 2018). We, therefore, asked whether Adipsin is a downstream target of PPARγ deacetylation. As predicted, Adipsin levels in the EWAT and in circulation were markedly decreased in the PPARγ deacetylation-mimetic 2KR mice, while other adipokines and adipocyte markers, such as Adiponectin and aP2, were not as significantly altered (Figure 7B). This decrease of circulating Adipsin was supported by prevalent repression of Cfd in three major adipose depots – EWAT, SWAT, and brown adipose tissue (BAT) (Figure 7—figure supplement 1A–C) as well as in the BM of 2KR mice (Figure 7—figure supplement 1D). This is in contrast to the largely normal expression of other adipocyte genes (Kraakman et al., 2018). Of note, the 2KR model largely recapitulates the bone protection and inhibited MAT expansion effect observed in adipocyte conditional PPARγ KO mice but without the previously observed lipoatrophy (Kraakman et al., 2018). The repression of Adipsin in these two PPARγ mutant mouse models is consistent with the inhibited BMA and improved skeletal health observed in Adipsin KO and C3 KO models.

We then investigated the mechanism whereby repression of Adipsin by PPARγ deacetylation occurs. In HEK293T cells, Adipsin promoter was activated >300-fold by WT PPARγ in a luciferase reporter assay, while the 2KR mutant showed a much weaker transcriptional activity, especially in response to activation by Rosi treatment (Figure 7C), recapitulating the in vivo results (Figure 7B) and indicating a direct regulation of PPARγ on the Adipsin promoter. We further showed that WT PPARγ directly bound to the Adipsin promoter while the 2KR mutation impaired this binding at multiple regions, particularly in the regions of −448 to −355 bp, −252 to −110 bp, and −114 to +33 bp identified by ChIP in comparably differentiated adipocytes (Figure 7D). Further manipulation of the promoter by truncated deletions revealed that the −281 to +35 bp region accounted for major promoter activation by PPARγ, with a sequence at −165 to −50 bp necessary for maximal promoter activity (Figure 7E–G). Thus, PPARγ deacetylation on Lys268 and Lys293, though localized to the ligand-binding domain (LBD), impairs the recruitment of PPARγ to the Adipsin promoter, underlying the repressed expression. Ultimately, the distinct regulation of Adipsin by PPARγ acetylation as well as the role of Adipsin as a secretory protein that functions on BM progenitor cells provides added emphasis for Adipsin as a mediator of BM homeostasis.

In human, fasting is a potent signal that can induce an increase in MAT. To understand the paradox that both fasting and high-calorie diet drive marrow adipogenesis, we examined gene expression patterns in BMAds isolated from BM aspirates in a previous study of high calorie and fasting in human volunteers (Fazeli et al., 2021). 11 volunteers (six females, five males) were fasted for 10 days and had pre- and post-fast marrow aspirate analyzed by MR spectroscopy and bone resorption markers among other phenotypic studies. By spectroscopy, vertebral MAT increased 8.1 ± 2.6% (p=0.01) at day 10 and bone resorption increased by 77% (p<0.0001). 5 (three females, two males) of the 11 individual marrow samples (with adequate both pre- and post-samples) had qualitative BMAd RNA that met criteria for subsequent RNA-seq analysis. The top 250 genes were selected based on the highest and lowest fold change and smallest p-value. Complement activation was the second most upregulated pathway from pre- to post-10 day fast (Figure 8A). Pairwise analysis for the five samples between pre- and post-fasted mRNA revealed that Adipsin was increased 2.8-fold (p=9.5×10⁻⁹) after fasting, in line with the fifth upregulated pathway of upstream PPAR signaling. 229 protein-coding genes were mapped by STRING with CFD at the center region (Figure 8B), suggesting an active role of Adipsin in the regulatory network. Additionally, heat maps were generated using GO terms for complement activation including 173 genes. 32 genes met the cutoff criteria, including Adipsin. The complement pathway genes were markedly upregulated, particularly CFD (Adipsin), which clustered with other complement-related genes (Figure 8C).