PDGFRα/PDGFRβ signaling balance modulates progenitor cell differentiation into white and beige adipocytes

Abstract

The relative abundance of thermogenic beige adipocytes and lipid-storing white adipocytes in adipose tissue underlie its metabolic activity. The roles of adipocyte progenitor cells, which express PDGFRα or PDGFRβ, in adipose tissue function have remained unclear. Here, by defining the developmental timing of PDGFRα and PDGFRβ expression in mouse subcutaneous and visceral adipose depots, we uncover depot specificity of pre-adipocyte delineation. We demonstrate that PDGFRα expression precedes PDGFRβ expression in all subcutaneous but in only a fraction of visceral adipose stromal cells. We show that high-fat diet feeding or thermoneutrality in early postnatal development can induce PDGFRβ⁺ lineage recruitment to generate white adipocytes. In contrast, the contribution of PDGFRβ⁺ lineage to beige adipocytes is minimal. We provide evidence that human adipose tissue also contains distinct progenitor populations differentiating into beige or white adipocytes, depending on PDGFRβ expression. Based on PDGFRα or PDGFRβ deletion and ectopic expression experiments, we conclude that the PDGFRα/PDGFRβ signaling balance determines progenitor commitment to beige (PDGFRα) or white (PDGFRβ) adipogenesis. Our study suggests that adipocyte lineage specification and metabolism can be modulated through PDGFR signaling.

Keywords: Adipocyte; Adipose tissue; PDGFR; Progenitor; White and brown adipogenesis.

Fig. 1.

Distinct lineages generate white adipocytes in SAT and VAT. (A) Schematic for lineage tracing of PDGFRβ⁺ cells and their derivatives (mG⁺) among other cells (mT⁺). pCa, chicken actin promoter; triangle, LoxP; pA, polyadenylation site. (B) A whole mount of SAT from a 10-week-old Pdgfrb-Cre; mT/mG mouse showing mT⁺ and mG⁺ adipocytes (a) and mT⁺ vasculature (v). (C) Confocal microscopy analysis of SAT from a 10-week-old Pdgfrb-Cre; mT/mG mouse (whole mount) showing a large blood vessel (v) with perivascular mG⁺ ASCs (arrows). (D) Adherent mT⁺ and mG⁺ ASCs in the cell culture of SVF from SAT of a 10-week-old Pdgfrb-Cre; mT/mG mouse. (E) SVF from D subjected to white adipogenesis induction. There are lipid droplets in mT⁺ and mG⁺ adipocytes (a). (F) Confocal immunofluorescence on paraffin wax-embedded sections of SAT and VAT from 6-week-old and 8-week-old Pdgfrb-Cre; mT/mG mice subjected to perilipin 1 (red) in adipocytes/GFP (green) in ASC. Yellow indicates mG/perilipin 1 colocalization in adipocytes (a) at week 8. Arrows indicate GFP⁺ and RFP⁺ cells. (G) Paraffin wax-embedded sections of VAT and SAT from 6-week-old and 1-year-old Pdgfra-Cre; mT/mG mice subjected to PDGFRβ (red)/GFP (green) immunofluorescence. Yellow arrows indicate mG/PDGFRβ colocalization. Graph shows the quantification of PDGFRβ and GFP colocalization in the images. (H) Paraffin wax-embedded sections of SAT and VAT from 12-week-old Pdgfrb-Cre; mT/mG mice fed chow or high-fat diet (HFD) for 1 month subjected to RFP (red)/GFP (green) immunofluorescence. mT⁺ and mG⁺ adipocytes (a) are indicated. (I) Time-course of an increase in the frequency of mG⁺ adipocytes from experiment in H. Data are mean±s.e.m. for multiple fields (n=10); *P<0.05 (Student's t-test). In all panels, colocalization is indicated with yellow arrows; nuclei are blue. Scale bars: 50 µm. Experiments were repeated at least three times with similar results.

Fig. 2.

The PDGFRβ⁺ lineage contribution to the beige adipocyte pool is minimal. (A) Cultured SVF from SAT of Pdgfrb-Cre; mT/mG mouse were subjected to white or brown adipogenesis induction. Fixed cells were analyzed by immunofluorescence with RFP (red), GFP (green) and UCP1 (blue) antibodies. Arrows indicate expression of UCP1 in mT⁺ (purple) but not in mG⁺ adipocytes (a). Separate channels are shown on the right; phase-contrast image shows adipocyte lipid droplets. (B,C) Paraffin wax-embedded sections of SAT from Pdgfrb-Cre; mT/mG mice 7 days after injection with PBS (control) or the β3AR activator CL 316,243. Nuclei are blue. (B) Immunofluorescence with RFP (red) and GFP (green) antibodies. (C) Immunofluorescence with UCP1 (red) and GFP (green) antibodies. UCP1 expression is present in multilocular mT⁺ adipocytes (arrows) but not in unilocular mG⁺ adipocytes (a). (D) Quantification of data from B. (E) Quantification of data from C. (F) Quantification of 30°C data from G. (G) Paraffin wax-embedded sections of VAT, SAT and BAT from 6-week-old Pdgfrb-Cre; mT/mG mice after 7 days of housing at 30°C or at room temperature subjected to anti-RFP (red)/GFP (green) immunofluorescence showing mG⁺ ASCs (arrows) at room temperature and mG⁺ adipocytes (a) at 30°C. In all panels, nuclei are blue. Scale bars: 50 µm. Data are mean±s.e.m. for multiple fields (n=10). *P<0.05 (Student's t-test). Experiments were repeated at least three times with similar results.

Fig. 3.

Adipocyte-derived stromal cells in remodeling SAT. (A) Scheme for lineage tracing of adiponectin (Adn)⁺ cells and their derivatives (mG⁺) among other cells (mT⁺). pCa, chicken actin promoter; triangle, LoxP; pA, polyadenylation site. (B) Cultured SVF from SAT of a 12-week-old Adn-Cre; mT/mG mouse pre-fed HFD to develop DIO and then grafted with RM1 tumor cells for 1 week (lipolysis induction). mG⁺ cells with ASC morphology emerge among mT⁺ cells (arrows). (C) Increased frequency of mG⁺ cells with ASC morphology in culture-plated SVF from SAT of Adn-Cre; mT/mG mice in which lipolysis was induced as in B, compared with untreated 4-week-old mice (control). Data are mean±s.e.m. for 10 view fields; *P<0.05 (Student's t-test). (D) Culture-plated SVF (P0) from SAT of a mouse in B were subjected to four-color anti-PDGFRβ immunofluorescence (yellow). Arrow indicates an mG⁺ cell with ASC morphology expressing PDGFRβ. (E) Paraffin wax-embedded sections of SAT from mice described in C subjected to PDGFRα or PDGFRβ (red) and GFP (green) immunofluorescence. Arrows indicate mG/PDGFR colocalization. (F) Paraffin wax-embedded sections of SAT of a mouse in B subjected to PLN1 (red) and GFP (green) immunofluorescence. Arrows indicate PLN1 in GFP⁺ stromal cells next to a blood vessel (b). a, adipocytes. (G) SVF from a mouse in B subjected to white adipogenic conditions for 3 days. The phase-contrast image indicates quick lipid droplet (arrows) accumulation in Adn⁺ lineage stromal cells; nuclei are blue. Scale bars: 50 μm.

Fig. 4.

Depletion of the PDGFRβ⁺ lineage results in AT beiging. (A) Four-week-old Pdgfrb-tk mice (n=6/group) were injected intreperitoneally (i.p.) daily with 50 mg/kg or 100 mg/kg ganciclovir (GCV) or PBS control for 10 days. Data are fat and lean body mass measured using EchoMRI, along with whole body mass at the three time points post-treatment. (B) Paraffin wax-embedded sections of SAT from wild-type (control) and Pdgfrb-tk mice 1 month post-treatment with 100 mg/kg GCV were subjected to anti-PDGFRα or anti-PDGFRβ immunofluorescence (red) and endothelium-specific isolectin B4 (IB4) staining (green). Arrows indicate the comparatively high density of PDGFRβ⁺ or PDGFRα⁺ cells. (C) Body temperature of Pdgfrb-tk mice 1 month post GCV or PBS treatment measured over 240 min at 4°C. There is increased cold tolerance of PDGFRβ⁺ lineage-depleted mice. Data are mean±s.e.m. for multiple mice; *P<0.05 (Student's t-test). (D) Paraffin wax-embedded sections of SAT from Pdgfrb-tk or C57BL/6 (control) mice 1 month post-treatment with PBS (control) or 100 mg/kg GCV were subjected to Hematoxylin and Eosin staining (top) and anti-UCP1 immunofluorescence (red, bottom). Scale bars: 50 µm. Nuclei are blue. Experiments were repeated at least three times with similar results.

Fig. 5.

PDGFRα/PDGFRβ signaling balance directs adipogenesis. (A) Quantitative RT-PCR analysis of Pdgfra, Pdgfrb and Zfp423 mRNA expression, normalized to 18S RNA, in immortalized brown preadipocytes with knockout (KO) of Pdgfra or Pdgfrb compared with control cells transduced with empty CRISPR/Cas9 vector. Cells were induced to undergo white adipogenesis for 8 days. Below, knockout of PDGFRα and PDGFRβ is confirmed by immunoblots with the corresponding antibody prior to adipogenesis induction. Images show differentiated Pdgfra KO and Pdgfrb KO adipocytes. (B) Quantitative RT-PCR analysis of Ucp1 mRNA expression, normalized to 18S RNA, in immortalized brown preadipocytes lacking Pdgfra or Pdgfrb compared with control cells transduced with empty CRISPR/Cas9 vector. Cells were induced to undergo adipogenesis for 8 days prior to analysis. Baseline control is non-differentiated cells (N.D.). Changes in UCP1 protein expression are confirmed by immunoblotting. (C) Quantitative RT-PCR analysis of Ucp1 gene expression, normalized to 18S RNA, in 3T3-L1 cells transduced with an ectopic Pdgfra expression vector or empty lentiviral vector. Cells were induced to undergo white or brown adipogenesis for 8 days prior to analysis. Baseline control is non-differentiated cells (N.D.). Changes in UCP1 protein expression are confirmed by immunoblotting. (D) Quantitative RT-PCR analysis of Pdgfrb and Zfp423 gene expression, normalized to 18S RNA, in 3T3-L1 cells transduced with an ectopic Pdgfrb expression vector or empty lentiviral vector. In A-D, data are mean±s.e.m. for two qPCR reactions; *P<0.05 (Student's t-test). (E) UCP1 immunoblotting of extracts from immortalized brown preadipocytes transduced with doxycycline-inducible Pdgfrb expression lentivirus. Cells were induced to undergo white or brown adipogenesis for 8 days prior to analysis and not treated (control) or treated (Pdgfrb) with doxycycline prior to differentiation induction. Baseline control is non-differentiated cells (N.D.). (F) Images of adipocytes differentiated from 3T3-L1 cells overexpressing Pdgfrα or Pdgfrb. (G) Immunoblotting of cells from C and D prior to adipogenesis induction showing the extent of Pdgfra and Pdgfrb overexpression. In all blots, β-actin immunoblotting is a protein-loading control. (H,I) Paraffin wax-embedded sections of SAT from mice treated with PDGF-AA or PDGF-DD; control mice were injected with PBS. (H) Hematoxylin and Eosin staining reveals smaller adipocyte in PDGF-AA-treated mice. (I) Anti-UCP1 immunofluorescence/anti-perilipin 1 immunofluorescence reveals UCP1 expression in PDGF-AA-treated mice. Scale bars: 50 µm. Nuclei are blue.

Fig. 6.

Human ASC heterogeneity and PDGFRα/PDGFRβ signaling balance. (A) Paraffin wax-embedded sections of VAT from a bariatric surgery patient subjected to PDGFRα (green)/PDGFRβ (red) immunofluorescence. Mutually exclusive PDGFRβ expression is found on perivascular cells and PDGFRα expression is found on cells further away from the lumen of blood vessels (v). (B) ASCs isolated from SAT of a bariatric surgery patient were treated with a PDGFRα-blocking antibody or a PDGFRβ-blocking antibody and then either not differentiated (N.D.) or subjected to adipogenesis induction (2-4). Phase-contrast images demonstrate formation of adipocytes with smaller lipid droplets upon PDGFRβ blockade (3). Immunoblotting of cell extracts shows that PDGFRβ blocking suppresses expression of adipogenesis markers aP2 and CD36. Equal protein loading is confirmed by β-actin immunoblotting. Data are mean band intensity± s.e.m. for three samples; *P<0.05 (Student's t-test). (C) 3T3-L1 cells transduced with human PDGFRA expression vector or control lentiviral vector and subjected to PDGFRα (green) immunofluorescence. Quantitative RT-PCR analysis of human PDGFR gene expression, normalized to 18S RNA are plotted. Data are mean±s.e.m. for two reactions; *P<0.05 (Student's t-test). (D) 3T3-L1 cells transduced with human PDGFRA, PDGFRB or the control lentiviral vector were induced to undergo white adipogenesis; adipocyte extracts were subjected to UCP1 immunoblotting. (E) Cells from C were induced to undergo white adipogenesis and stained with Mitotracker (red), indicating abundance of active mitochondria in adipocytes differentiated from PDGFRA-expressing 3T3-L1 cells. (F) Quantitative RT-PCR analysis of mouse Pdgfrb mRNA expression normalized to 18S RNA and of human PDGFRB mRNA expression, normalized to GAPDH. For mouse Pdgfrb, cDNA was prepared from samples of total SAT and VAT of mice raised for 5 months on chow (lean) or HFD (obese). For human PDGFRB, cDNA was prepared from SVF of SAT and VAT samples from lean (BMI<30) or obese (BMI>30) patients. Data are mean±s.e.m. for the multiple samples analyzed. *P<0.05 (Student's t-test). Experiments were repeated at least three times with similar results.

Fig. 7.

A model of SAT and VAT adipocyte lineage specification. In SAT, all progenitor cells (gray) are of the PDGFRα⁺ lineage and give rise to PDGFRβ⁻ and PDGFRβ⁺ preadipocytes (orange) that can differentiate into beige adipocytes rich in mitochondria (brown) or unilocular white adipocytes. SAT adipocytes can transiently acquire lipid-free ASC morphology and drift from mitochondria-rich beige to white as a function of PDGFRβ expression in de-differentiated preadipocytes. In VAT, some adipocytes are also derived from the PDGFRα⁺ progenitor lineage without PDGFRβ expression in preadipocytes, whereas other adipocytes are derived from a distinct PDGFRα-negative progenitor cells expressing only PDGFRβ.