CD146 is a Novel ANGPTL2 Receptor that Promotes Obesity by Manipulating Lipid Metabolism and Energy Expenditure

Figures

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  Figure 1. CD146 expression is upregulated in adipose tissues from obese mice and humans. Western blot A) and RT‐qPCR B) showing CD146 levels in various mouse tissues. SVF, stromal vascular fraction. Western blot showing CD146 expression in white adipose tissue (WAT) C) and brown adipose tissue (BAT) D) from mice fed a normal diet (ND) or high‐fat diet (HFD) (n = 5 per group). Western blot showing CD146 expression in WAT E) and BAT F) from 12 week old (12w) ob/ob or control mice (n = 5 per group). Western blot showing CD146 expression in WAT G) and BAT H) from 4 week old (4w) or 12w ob/ob mice (n = 5 per group). I) Correlation between CD146 levels in WAT and the age of mice (n = 14). J) Correlation between CD146 levels in WAT and the body weight of mice (n = 14). K) Western blot showing CD146 expression in WAT from lean and obese humans. L) Correlation between CD146 levels in human WAT and body mass index (n = 36). Data represent the mean ± SD in (B) and the mean ± SEM in (C)–(H) and (K). P values were determined using Student's t‐tests (C–H,K).

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  Figure 2. CD146 knockout protects mice from obesity. A) Body weight of wild‐type (WT), CD146 heterozygous knockout (Het), and knockout (KO) mice fed a normal diet (ND) or high‐fat diet (HFD) (n = 7 per group). B) Representative images of WT, Het, and KO mice fed an HFD. C) Body length of WT, Het, and KO mice fed an HFD (n = 7 per group). D) Representative images and weight of liver and fat pads (epididymal, inguinal, and renal) from WT, Het, and KO mice fed an HFD (n = 7 per group). E) Lean and fat mass of WT, Het, and KO mice fed an HFD, determined by micro‐CT (n = 7 per group). F) Representative H&E images (left) and triglyceride (TG) content (right) of livers from WT, Het, and KO mice fed an HFD (n = 7 per group). Scale bar, 100 µm. G) Glucose tolerance test (GTT) and insulin tolerance test (ITT) in WT, Het, and KO mice fed an HFD (n = 6 per group). H) Serum insulin, cholesterol, triglyceride, and free fatty acid levels in fasting WT, Het, and KO mice (n = 7 per group). I) Representative H&E images and adipocyte cell size measurements of epididymal fat pads from WT and KO mice fed an HFD. Scale bar, 100 µm. J) Representative F4/80 staining images and quantification of F4/80 positive…

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  Figure 3. Adipocyte‐specific CD146 knockout (CD146AT‐KO) protects mice from obesity. A) Body weight of CD146AT‐KO and CD146flox/flox control mice fed an HFD (n = 6 per group). B) Representative images of CD146AT‐KO and CD146flox/flox control mice fed an HFD. C) Representative images and weight of liver and fat pads (epididymal and inguinal) from CD146AT‐KO and CD146flox/flox control mice fed an HFD (n = 6 per group). D) Serum cholesterol, triglyceride, and FFA levels in fasting CD146AT‐KO and CD146flox/flox control mice. E) Representative H&E images and adipocyte cell size measurements of epididymal fat pads from CD146AT‐KO and CD146flox/flox control mice fed an HFD. Scale bar, 50 µm. F) GTT and ITT measured in CD146AT‐KO and CD146flox/flox control mice fed an HFD (n = 6 per group). G) RT‐qPCR analysis of F4/80 expression in epididymal fat pads from CD146flox/flox and CD146AT‐KO mice fed an HFD (n = 6 per group). H) RT‐qPCR analysis of the expression of the inflammatory factors Il‐6, Il‐1β, Tnf‐α, and Ccl2 in epididymal fat pads from CD146flox/flox and CD146AT‐KO mice fed an HFD (n = 6 per group). Data represented the mean ± SEM. P values were determined using Student's t‐tests.

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  Figure 4. Adipocyte‐specific CD146 knockout (CD146AT‐KO) enhances energy expenditure and BAT function. A) Food intake of CD146AT‐KO and CD146flox/flox control mice fed an HFD measured daily (n = 5 per group). Oxygen consumption B), carbon dioxide production C), respiratory quotient D), and energy expenditure E) of CD146AT‐KO and CD146flox/flox control mice fed an HFD (n = 4 per group). F) Representative images (top) and H&E staining (bottom) of BAT from CD146AT‐KO and CD146flox/flox control mice fed an HFD. Scale bar, 200 µm. BAT weight G) and mass H) of CD146AT‐KO and CD146flox/flox control mice fed an HFD (n = 3 per group). I) Representative images of BAT mitochondria from CD146AT‐KO and CD146flox/flox control mice fed an HFD. Scale bar, 1 µm. J) Ratio of mitochondria to cytosol area in CD146AT‐KO and CD146flox/flox control mice fed an HFD. K) Western blot showing UCP1 expression in BAT from CD146AT‐KO and CD146flox/flox control mice fed with HFD. L) RT‐qPCR showing the expression of thermogenesis and fatty acid oxidation (FAO)‐related genes in BAT from CD146AT‐KO and CD146flox/flox control mice fed an HFD (n = 3 per group). M) Oxygen consumption rate (OCR) in mitochondria from…

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  Figure 5. CD146 promotes adipogenesis and lipid accumulation. Western blot showing CD146, C/EBPα, and Fabp4 expression during the differentiation of stromal vascular fractions (SVF) A) and 3T3‐L1 cells B). C) Schematic diagram illustrating a putative CREB binding site in the CD146 promoter. D) ChIP‐qPCR analysis showing CREB binding to the CD146 promoter during DMI‐induced 3T3‐L1 differentiation. E) Schematic diagram of luciferase reporter constructs, wherein luciferase is driven by the CD146 promoter with a WT or deleted putative CREB binding site. F) Dual luciferase reporter assay confirming that CREB binds to the CD146 promoter during DMI‐induced 3T3‐L1 differentiation. CD146 expression G), oil‐red‐O staining H) and TG content I) of adipocytes differentiated from WT and CD146 KO SVF. J) Western blot showing CD146, C/EBPα, and Fabp4 expression during adipocyte differentiation from SVF. K) RT‐qPCR showing the expression of adipogenesis and lipogenesis‐related genes in adipocytes differentiated from SVF. L) Oil‐red‐O staining of adipocytes differentiated from 3T3‐L1 cells with ectopic CD146 expression. M) Triglyceride content in adipocytes differentiated from 3T3‐L1 cells with ectopic…

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  Figure 6. CD146 interacts with ANGPTL2. A) Analysis of CD146‐interacting proteins in the human proteome microarray. B) Fluorescent signal and signal‐noise ratio (SNR) of CD146‐interacting cytokines. C) Co‐immunoprecipitation (co‐IP) assay showing the interaction between CD146 and ANGPTL2 in mouse WAT. D) Co‐IP assay showing the enhanced interaction between CD146 and ANGPTL2 in WAT from obese mice. E,F) Pull‐down assay showing the direct interaction between CD146 and ANGPTL2. Purified His‐tagged ANGPTL2 and CD146 proteins were incubated in PBS. CD146 and ANGPTL2 were pulled down using anti‐CD146 antibodies E) and anti‐His tag antibodies F), respectively. G) Schematic diagram of the full‐length (FL) and CD146 truncates fused with Flag‐tag. H) Co‐IP assay showing that the fourth domain of CD146 is required for the ANGPTL2‐CD146 interaction. ANGPTL2 was coexpressed with different CD146 truncates in 293 cells. I) Pull‐down assay showing that anti‐CD146 AA98 antibodies blocked the CD146‐ANGPTL2 interaction.

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  Figure 7. ANGPTL2‐CD146 interaction regulates adipogenesis, lipid metabolism, and adipose inflammation. A) Oil‐red‐O staining and triglyceride (TG) content of adipocytes differentiated from WT and CD146 KO SVF treated with ANGPTL2 and anti‐CD146 AA98 or AA1 antibodies. B) 14C radioactivity (left) and lipogenesis‐related gene expression (right) in WT and CD146 KO adipocytes treated with ANGPTL2 (14C radioactivity, n = 3 per group; gene expression, n = 4 per group). C) 14C radioactivity (left) and lipogenesis‐related gene expression (right) in adipocytes treated with ANGPTL2 and anti‐CD146 AA98 or AA1 antibodies (14C radioactivity, n = 3 per group; gene expression, n = 4 per group). D) 14C radioactivity (left) and fatty acid oxidation‐related gene expression (right) in WT and CD146 KO adipocytes treated with ANGPTL2 (14C radioactivity, n = 3 per group; gene expression, n = 4 per group). E) 14C radioactivity (left) and fatty acid oxidation‐related gene expression (right) in adipocytes treated with ANGPTL2 and anti‐CD146 AA98 or AA1 antibodies (14C radioactivity, n = 3 per group; gene expression, n = 4 per group). F) RT‐qPCR analysis of proinflammatory factor Il‐6, Il1‐β, Tnf‐α, and Ccl2…

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  Figure 8. Anti‐CD146 AA98 antibody attenuates obesity development and obesity‐related inflammation. A) Triglyceride (TG) content of adipocytes differentiated from human SVF treated with ANGPTL2 or anti‐CD146 AA98 or AA1 antibodies. RT‐qPCR showing the expression of lipogenesis B) and fatty acid oxidation‐related genes C) in human adipocytes treated with ANGPTL2 or anti‐CD146 AA98 or AA1 antibodies. Body weight D) and WAT weight E) of db/db mice treated with AA98, AA1, or mIgG (n = 7 per group). F) Serum cholesterol, triglyceride, and free fatty acid (FFA) levels in db/db mice treated with AA98, AA1, or mIgG (n = 7 per group). G) Representative H&E images and quantification of adipocyte size in db/db mice treated with AA98, AA1, or mIgG. H) GTT (left) and the AUC of GTT (right) in db/db mice treated with AA98, AA1, or mIgG (n = 7 per group). I) ITT (left) and the AUC of ITT (right) in db/db mice treated with AA98, AA1, or mIgG (n = 7 per group). Macrophage number J) and macrophage gene F4/80 expression K) in the visceral fat pads of db/db mice treated with AA98, AA1, or mIgG (n = 7 each group). L) RT‐qPCR showing the expression of proinflammatory factors in visceral adipose tissue from…