Adipocyte-derived shed Syndecan-4 suppresses lipolysis contributing to impaired adipose tissue browning and adaptive thermogenesis

Figures

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  Figure 1. Sdc4 expression and shedding are increased in adipocytes of obese mice. (A) Representative immunoblots of protein abundance of Sdc4 and HSP90 in stromal vascular fraction (SVF) and mature adipocytes (MA) in sWAT and eWAT of lean C57BL/6N male mice. The right panel is the quantification of Sdc4 band intensity normalized to HSP90 (n = 3). (B–I) Six-week-old C57BL/6N male mice were subjected to standard chow (STC) or high-fat diet (HFD) feeding for 14 weeks. (B–C) The relative mRNA abundance of Sdc4 in SVF and MA isolated from mouse (B) sWAT and (C) eWAT (n = 4). (D) The relative mRNA abundance of Sdc4 in mouse sWAT and eWAT (n = 6). (E) The concentration of shed Sdc4 in mouse serum (n = 8). (F–G) Representative immunoblots of protein abundance of full-length intact Sdc4, intra Sdc4 (the remaining part after shedding) and HSP90 in mouse (F) sWAT and (G) eWAT. The right panel is the quantification of Sdc4 band intensity normalized to HSP90 (n = 3). (H–I) The relative mRNA abundance of Sdc4 sheddase (Mmp9, Adamts1, and Adamts4) in (H) sWAT and (I) eWAT (n = 6). Data is presented as mean ± SEM. Statistical significance was analyzed by the Mann–Whitney U test (A, F, and G), two-way…

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  Figure 2. Adipocyte-specific Sdc4 knockout mice are resistant to diet-induced obesity. Six-week-old male adipose tissue-specific Sdc4 knockout (AT-Sdc4 KO) mice and relative wild-type (WT) littermates were subjected to HFD or STC feeding for 14 weeks. (A–D) The (A) body weight, (B) representative images (scale bar = 1 cm), (C) fat mass percentage, and (D) lean mass percentage of AT-Sdc4 KO and WT mice (n = 6). (E–F) The (E) representative images of mouse sWAT, eWAT, and BAT (scale bar = 1 cm) and (F) relative weights of various fat depots (n = 6). (G–H) The representative images of H&E staining of mouse (G) sWAT and (H) eWAT (Scale bar = 100 μm). (I–J) The distribution of adipocytes with different sizes in (I) sWAT and (J) eWAT shown in (G) and (H), respectively (n = 6). (K) Glucose tolerance test (left) and calculated areas under the curve (AUC, right) (n = 6). (L) The representative images of Oil red O staining of mouse liver. The right panel is the quantification of Oil red O positive area of HFD groups (n = 6). (M–N) The relative mRNA abundance of genes related to (M) lipid synthesis and (N) inflammation in mouse liver (n = 6). Data is presented as mean ± SEM. Statistical significance…

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  Figure 3. Adipocyte-Sdc4 deletion elevates energy expenditure in mice in response to diet-induction. Six-week-old male AT-Sdc4 KO mice and relative WT littermates were subjected to HFD or STC for 14 weeks, followed by metabolic cage assessment at 48 h. (A–B) The light (day) and dark (night) rate of (A) oxygen consumption (VO2) and (B) carbon dioxide production (VCO2) in HFD-fed mice. The right panels are the quantification of VO2 consumption and VCO2 production shown in (A) and (B), respectively (n = 5). (C–D) The calculated (C) energy expenditure and (D) RER of HFD-fed mice (n = 5). (E) Rectal temperature of HFD-fed mice measured at the 48 h endpoint (n = 6). (F) The oxygen consumption rate (OCR) of sWAT and BAT from HFD-fed AT-Sdc4 KO and WT mice (n = 6). (G–H) The (G) food intake and (H) locomotive activity of HFD-fed mice (n = 5). (I–J) The relative mRNA abundance of thermogenic genes (Ucp1, Dio2, Cidea, Prdm16, and Pgc1α) in (I) sWAT and (J) BAT of STC- and HFD-fed mice (n = 6). Data is presented as mean ± SEM. Statistical significance was analyzed by unpaired two-tailed Student's t test (A–H) or two-way ANOVA (I and J). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗∗p < 0.0001.

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  Figure 4. Sdc4 deficiency in adipocytes promotes lipolysis. (A) Gene set enrichment analysis (GSEA) of ontology-biological process pathways preferentially activated in the eWAT of HFD-fed AT-Sdc4 KO mice compared to WT mice (n = 4). Terms were ranked based on the normalized enrichment score (NES). (B) The representative GSEA-scoring plots of activated GO-BP pathways in (A). The reported NES values and adjusted p values (p. adjust) were calculated with 1,000 permutations in the GSEA software. (C-E) The relative mRNA abundance of genes-related to (C) lipolysis, (D) fatty acid oxidation, and (E) adipogenesis in eWAT from AT-Sdc4 KO mice and WT mice subjected to STC or HFD feeding for 14 weeks (n = 6). (F–G) Representative immunoblots of the protein abundance of p-HSL, t-HSL, and HSP90 in (F) eWAT and (G) sWAT of HFD-fed AT-Sdc4 KO mice and WT littermates subjected to STC or HFD feeding for 14 weeks following fasting for 16 h. The right panel is the quantification of p-HSL band intensity normalized to t-HSL (n = 3). (H–L) Six-week-old male AT-Sdc4 KO mice and WT mice fed with 14 weeks HFD were injected with isoproterenol (Iso, 10 mg/kg body weight). (H) The serum FFAs and glycerol levels were…

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  Figure 5. Sdc4 deficiency in adipocytes promotes sWAT browning and BAT activation. Eight-week-old male AT-Sdc4 KO mice and their WT littermates were subjected to 30 °C acclimation for 7 days followed by 6 °C cold challenge or 30 °C thermoneutral conditions for 7 days. (A) Schematic diagram showing the cold challenge experiments. (B–C) The quantification of the (B) rate of O2 consumption and (C) energy expenditure of mice in light (day) and dark (night) (n = 6). (D) The dynamic rectal temperature of AT-Sdc4 KO mice and WT mice (n = 6) recorded during cold challenge for 8 h. (E) The representative images of H&E staining of sWAT (left panel) and BAT (right panel) (scale bar = 100 μm). (F) The oxygen consumption rate (OCR) of sWAT and BAT from mice subjected to 6 °C. (G–H) The serum concentration of (G) FFA and (H) glycerol of mice at indicated time points during cold exposure (n = 6). (I) Representative immunoblots of the protein abundance of p-HSL, t-HSL, and HSP90 in mouse eWAT. The right panel is the quantification of p-HSL band intensity normalized to t-HSL (n = 3). (J–K) The relative mRNA abundance of thermogenic genes in mouse (J) sWAT and (K) BAT (n = 5). (L) Representative immunoblots…

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  Figure 6. Sdc4 shedding is inhibited during cold exposure, and recombinant Sdc4 (RbSdc4) inhibits thermogenesis. (A–B) Eight-week-old C57BL/6N male mice were subjected to cold exposure for 7 days. (A) The concentration of shed Sdc4 in mouse serum during cold exposure (n = 6). (B) The relative mRNA abundance of Sdc4 sheddase in mouse sWAT (n = 6). (C–D) Beige adipocytes differentiated from SVFs isolated from C57BL/6N mice were treated with either recombinant Sdc4 extracellular domain protein (RbSdc4) or PBS for 24 h. (C) The relative mRNA abundance of thermogenic genes (Ucp1, Cidea, Prdm16, and Cox8b) and adipogenic markers (Pgc1α and Pparγ) in beige adipocytes (n = 6). (D) Representative immunoblots of the protein abundance of p-HSL, t-HSL, UCP1, and HSP90 in beige adipocytes. The right panels are the quantification of p-HSL band intensity normalized to t-HSL and UCP1 band intensity normalized to HSP90 (n = 4). Data is presented as mean ± SEM. Statistical significance was analyzed by one-way ANOVA (A), unpaired two-tailed Student's t test (B and C) or the Mann–Whitney U test (D). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001.

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  Figure 7. Shed Sdc4 and FGF2 synergistically inhibit HSL phosphorylation in a FGFR1-dependent manner. (A) The concentration of shed Sdc4 in serum from AT-Sdc4 KO and WT mice subjected to STC or HFD feeding for 14 weeks (n = 8). (B) Representative immunoblots of the protein abundance of p-HSL, t-HSL, and HSP90 in white adipocytes treated with RbSdc4 (0, 10, 50, 100 ng/ml) for 24 h. The right panel is the quantification of p-HSL band intensity normalized to t-HSL (n = 3). (C) The FFA release rate of white adipocytes treated with RbSdc4 in the presence or absence of HSL-IL-1 (1 μM) followed by FSK (10 μM) stimulation. The data was normalized by protein abundance (n = 4). (D) Representative immunoblot of immunoprecipitation (IP) of Sdc4 and FGF2 in the eWAT of AT-Sdc4 KO mice and WT mice fed with STC or HFD for 14 weeks. (E) Representative immunoblots of the protein abundance of p-HSL, t-HSL, ATGL, and HSP90 in Sdc4 KO or WT white adipocytes treated with FGF2 (5 ng/ml) for 24 h. The right panels are the quantification of p-HSL band intensity normalized to t-HSL and the ATGL band intensity normalized to HSP90 (n = 4). (F) Representative immunoblots of the protein abundance of p-HSL, t-HSL,…

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  Figure 8. Proposed summary. Adipocyte Sdc4 shedding increased in mice subjected to diet-induced obesity. Shed Sdc4 binds and concentrates FGF2, thus activating FGFR1 to suppress HSL phosphorylation. Impaired lipolysis reduces FFA release for FFA oxidation and, therefore, reduces energy expenditure. Accumulated lipid in adipocytes leads to adipose tissue dysfunction. Both of these exaggerate obesity and its related metabolic disorders. In AT-Sdc4 KO mice subjected to cold exposure, lack of Sdc4 shedding waives the suppressing effect on HSL-mediated lipolysis. FFA can, therefore, be provided as an energy substrate for thermogenesis.