BMPR2 promotes fatty acid oxidation and protects white adipocytes from cell death in mice

Figures

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  Fig. 1. BMPR2 knockout mice lose white fat at weaning. a Representative pictures of WT and KO mice. b Body weight of WT and KO mice at different age (n = 6). c Representative pictures of WAT and BAT in WT and KO mice. d Fat index (percentage of fat pad weight to the whole body weight) of inguinal WAT (iWAT), gonadal WAT (gWAT), BAT in WT and KO mice (n = 6). e Survival curve of male WT and KO mice. f Representative images of H&E-stained sections of adipose tissues at different ages. Scale bar: 25 μm. g Quantification of adipocyte diameter of iWAT, gWAT and BAT from WT and KO mice. (Data were collected by using ImageJ software from H&E staining sections of three individual mice, five fields per mouse, and 10−15 cells per field in each group.) h, i Levels of glucose (h) and triglyceride (i) in serum of 2-week-old mice. Data in b, d, g, h, i are expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001.

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  Fig. 2. BMPR2 deficiency increases adipocyte death in white adipose tissue. a TUNEL (FITC) assay shows apoptosis in WT and BMPR2 knockout iWAT performed on tissue slides. Scale bar: 20 μm. b TUNEL (FITC) assay shows apoptosis in smeared mature adipocytes isolated from iWAT of 16-day-old mice. Scale bar: 20 μm. c Western blot analysis of caspase 3 in pooled mature adipocytes isolated from iWAT of four individuals in each group. d Precursor cells were isolated from iWAT of WT and KO mice and induced to differentiation. Oil red O staining for mature adipocytes showed the differentiation rate, and Western blots showed levels of BMPR2, FABP4, and HSP90 (loading control) in the two groups of cells. e Representative images showed the appearance of adipocytes of WT BMPR2 KO and cells at days 7 and 11 after induced to differentiation. Scale bar: 25 μm. f Western blot for cleaved caspase 3 showed the level of apoptosis of adipocytes in e. g Western blot analysis of caspase 1 in pooled mature adipocytes isolated from iWAT of four individuals in each group. h Immunochemistry showed positive staining for caspase 1 in iWAT of 19-day-old mice. Scale bar: 20 μm. i Quantitation of caspase 1-positive area…

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  Fig. 3. BMPR2-deficient adipocytes are sensitive to TNFα-induced apoptosis. a Levels of TNFα in serum of 14-day-old mice were determined using ELISA. n = 6. Data are expressed as means ± SD. ***p < 0.001. b Representative image of Western blot for TNFα in iWAT of two pairs of littermates at 14 days old. c Statistical analysis for the gray density of TNFα bands after normalized with HSP90 in Western blot assay described in c (n = 4). Data are expressed as means ± SD. *p < 0.05. d Western blot showed levels of cleaved caspase 3 and total caspase 3 in differentiated adipocytes from precursor cell in culture. e Image of mature adipocytes differentiated from isolated precursor cells in subcutaneous WAT at hip region of four human individuals. Scale bar: 25 μm. f Western blot showed levels of BMPR2 and cleaved caspase 3 (Cl-Caspase 3) in cells of d. g Western blot showing the levels of BMPR2 and cleaved caspase 3 in human subcutaneous adipose tissues from hip region (left panel). h Levels of BMPR2 and cleaved caspase 3 in g were normalized by HSP90 and analysis for linear regression to show their correlation (right panel).

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  Fig. 4. BMPR2-deficiency impairs fatty acid metabolism and oxidative phosphorylation. a Gene ontology analysis for downregulated genes in KEGG pathways, pathways with FDR < 0.1 and p value < 0.00015 were listed and grouped designated with different color. b Pathways with FDR < 0.1 and p value < 0.00015 were visualized in Cytoscape v.3.4.0, the node sizes and color presented p value and FDR, respectively. c Downregulated genes identified by sequencing involved fatty acid metabolism and oxidative phosphorylation. d Real-time PCR validation of genes selected from pathways in c. Total RNA were isolated from mature adipocyte isolated from inguinal fat (n = 6). Data are expressed as means ± SD. *p < 0.05, **p < 0.01. e−g Correlation between mRNA level of BMPR2 and indicated genes of human adipose tissue samples determined by quantitative PCR.

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  Fig. 5. Dysregulation of perilipin in BMPR2 knockout adipocytes. a Western blot for perilipin in mature adipocytes isolated from iWAT of BMPR2 knockout mice and their littermates (1#, 2#, 3# represents three different litters). b Relative gray intensity of the bands normalized with HSP90 was quantitated using ImageJ software. c Representative image of IHC staining for perilipin on iWAT sections. Scale bar: 20 μm. d The positive staining area of perilipin in c was quantitated using ImageJ software. Data were collected from sections of three individual mice, 5–6 fields per mouse. e Western blot showed phosphorylated p38/MAPK and total p38 in adipocytes differentiated from precursor cells with or without 20 μg/ml TNFα treatment for 1 h. f The lysate of cultured cells was separated by a phosphate affinity SDS-page and blotting for mobility shift detection of phosphorylated perilipin. The arrow head pointed bands were those affected by BMPR2 level. g Western blot showed phosphorylated p38/MAPK and total p38 in adipocytes differentiated from precursor cells after treatment with 20 μg/ml TNFα with or without 10 ng/ml BMP4 for 1 h. SB203580 (10 μM) was used to pretreat cells for 1 h to inhibit…

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  Fig. 6. Reduced lipolysis induces apoptosis through mitochondrial pathway. a Glycerol in culture medium of adipocytes was determined by colorimetry and normalized to total amount of neutral lipid (stained Oil red O rinsed with methanol and absorbance measured at 490 nm). b Quantitative PCR determined expression of genes for fatty acid oxidation in cultured adipocytes differentiated from SVF, which were treated with 20 μg/ml TNFα for 1 h. c Adipocytes were treated with BMP4 or with P-p38 inhibitor SB283058 pretreated and subjected to 20 μg/ml TNFα-stimulated lipolysis; glycerol in culture medium was determined. d Adipocytes in culture were treated with 20 ng/ml TNFα for 16 h; OCR was determined using a seahorse XF24 analyzer. Data were from four replications and expressed as mean ± SD. e Quantification of data in d. Data were expressed as means ± SD. *p < 0.05, **p < 0.01, ***p < 0.001. f, g Representative Western blot determined the level of phosphorylated AMPK. Adipocytes were treated with 20 ng/ml TNFα for 16 h. Atglistatin (20 μM) pretreated cells to inhibit lipolysis. h, i TUNEL staining (FITC positive) of differentiated adipocytes treated and stimulated in the presence and absence…

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  Fig. 7. Schematic model of the role of BMPR2 in the inhibition of fatty acid metabolism and induction of cell death. BMPR2 signaling facilitates lipolysis by activating phosphorylation and dynamics of perilipin, thus guaranteeing the availability of fatty acid for β-oxidation (FAO) and oxidative phosphorylation (OXPHOS) through electron transport chain (ETC). BMPR2 deficiency disrupts this action, leading to the failure of OXPHOS, and activates mitochondria-mediated apoptosis in adipocytes when adipocytes were stimulated by TNFα. Meanwhile, deficiency of lipolysis leads to hypertrophy of adipocytes and subsequent pyroptosis and inflammation, among which elevation of TNFα is prominent. TNFα in turn poses more pressure on adipocyte, accelerating the process of cell death. BMPR2 deficiency thus renders the white adipocytes vulnerable to cell death. FFA free fatty acid, TAG triacylglyceride.