SDCCAG3 inhibits adipocyte hypertrophy and improves obesity-related metabolic disorders via SDCCAG3/SMURF1/PPARγ axis

Figures

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  Fig. 1. Determining SDCCAG3 as a regulator of adipocyte metabolism. A: Protein expression levels of SDCCAG3 in IngWAT of mice on NCD or HFD. B: Protein expression levels of SDCCAG3 in EpiWAT of mice on NCD or HFD. C: SDCCAG3 mRNA levels in IngWAT and EpiWAT of mice on NCD or HFD. D and E: Changes in SDCCAG3 protein and mRNA levels during the differentiation of 3T3-L1 cells into mature adipocytes. F: Protein and mRNA levels of SDCCAG3 in differentiated 3T3-L1 cells cultured under normal or high-fat conditions. G: Protein expression levels of PPARγ, C/EBPα and SDCCAG3 with knockdown or overexpression of SDCCAG3 on differentiated 3T3-L1 cells cultured under high-fat conditions. H: SDCCAG3, PPARγ, C/EBPα, FABP4, LPL and ADIPOQ mRNA levels with knockdown or overexpression of SDCCAG3 on differentiated 3T3-L1 cells cultured under high-fat conditions. I and K: Oil red O staining of differentiated 3T3-L1 cells and relative area with knockdown or overexpression of SDCCAG3, scale bar = 100 μm. J and L: BODIPY staining of differentiated 3T3-L1 cells and relative intensity with knockdown or overexpression of SDCCAG3, scale bar = 75 μm. Data were presented as mean ± standard deviation (S.D.). ∗P <…

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  Fig. 2. SDCCAG3 promoted the protein stability of PPARγ. A: Co-immunoprecipitation of SDCCAG3 and PPARγ in 3T3-L1 adipocytes was detected using SDCCAG3 antibody binding. B: Co-immunoprecipitation of SDCCAG3 and PPARγ in HEK293T cells exogenously expressing HA-Sdccag3 and Flag-Pparγ. C: Luciferase activity was measured following the transfection of HEK293T cells with PPARγ luciferase reporter gene plasmid, renilla luciferase gene plasmid and SDCCAG3 plasmid. D and E: Protein expression levels of Flag-PPARγ and HA-SDCCAG3 in HEK293T cells overexpressing HA-Sdccag3 and Flag-Pparγ after treated with CHX for 0, 2, 4 and 6h. F and G: Protein expression levels of Flag-PPARγ and SDCCAG3 in HEK293T cells of knockdown SDCCAG3 and overexpression Flag-Pparγ after treated with CHX for 0, 2, 4 and 6h. H: In HEK293T cells with SDCCAG3 knockdown, Flag-PPARγ and SDCCAG3 protein expression levels were detected after treatment with CHX or dual treatment with MG132 and CHQ for 4h. I: After SDCCAG3 knockdown or overexpression in HEK293T cells treated with UB and Flag-Pparγ plasmids, Flag-PPARγ protein was pulled down by magnetic beads, and ubiquitination levels were detected by Western blot. Data were…

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  Fig. 3. SMURF1 mediated the regulation of SDCCAG3 on PPARγ protein stability and adipocyte. A: Co-immunoprecipitation of SDCCAG3 and SMURF1 in 3T3-L1 cells. B: Co-immunoprecipitation of SDCCAG3 and SMURF1 in HEK293T cells exogenously expressing HA-Sdccag3 and Smurf1. C: The mRNA levels of SMURF1 with knockdown or overexpression of SDCCAG3 on differentiated 3T3-L1 cells. D: Protein expression levels of SMURF1 and SDCCAG3 with knockdown or overexpression of SDCCAG3 on differentiated 3T3-L1 cells. E: The HEK293T cells overexpressing Flag-Pparγ were co-transfected with HA-Sdccag3 or Smurf1, and the protein expression levels of Flag-PPARγ, SMURF1 and SDCCAG3 were detected after CHX treatment for 0, 2, 4, and 6h. F: HA-Sdccag3 or Smurf1 were co-transfected with UB and Flag-Pparγ plasmids into HEK293T cells. Flag-PPARγ protein was pulled down by magnetic beads, and the ubiquitination levels were detected by Western blot. G: Protein expression levels of PPARγ, C/EBPα, SDCCAG3 and SMURF1 with co-transfection of Smurf1 and HA-Sdccag3 on differentiated 3T3-L1 cells cultured under high-fat conditions. H: PPARγ, C/EBPα, FABP4, LPL and ADIPOQ mRNA levels with co-transfection of Smurf1 and HA-Sdccag3…

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  Fig. 4. Determining the functional fragment of SDCCAG3 and a SDCCAG3-PPARγ-SDCCAG3 lipid metabolism enhancement loop. A: Protein expression levels of SDCCAG3, PPARγ, C/EBPα in differentiated 3T3-L1 cells transfected with HA-Sdccag3 and treated with GW9662 under high-fat conditions. B: The mRNA levels of SDCCAG3, PPARγ, C/EBPα, FABP4, LPL and ADIPOQ in differentiated 3T3-L1 cells transfected with HA-Sdccag3 and treated with GW9662 under high-fat conditions. C: Differentiated 3T3-L1 cells transfected with HA-Sdccag3 were treated with GW9662 and stained with oil red O, scale bar = 50 μm. D: Differentiated 3T3-L1 cells transfected with HA-Sdccag3 were treated with GW9662 and stained with BODIPY, scale bar = 75 μm. E: Example plots of full-length Sdccag3 and its mutants lacking segments 1–145, 146–261, and 262–432, respectively. F: In HEK293T cells, full-length Sdccag3 and its mutants (mut1, mut2, and mut3) were transfected with Flag-Pparγ, and the co-immunoprecipitation of HA and Flag was detected. G: In HEK293T cells, full-length Sdccag3 and its mutants (mut1, mut2, and mut3) were transfected with Smurf1, and the co-immunoprecipitation of HA and SMURF1 was detected. H: Protein expression…

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  Fig. 5. Adipose-specific knockout of SDCCAG3 aggravated adipocyte hypertrophy and abnormal lipid-related indexes. A: Body weight changes in AKO-WT and AKO-Sdccag3 groups over the course of 16 weeks of NCD or HFD feeding, n = 5. B: Micro-CT scans of AKO-WT and AKO-Sdccag3 groups of NCD or HFD feeding to reconstruct subcutaneous fat and visceral fat maps. Orange, subcutaneous fat; Green, visceral fat. C: Photos of EpiWAT from AKO-WT and AKO-Sdccag3 groups of NCD or HFD feeding. D: EpiWAT weight of NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. E: Ratio of EpiWAT weight to body weight in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. F: HE staining of IngWAT and mean area of adipocytes from NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, scale bar = 50 μm. G: HE staining of EpiWAT and mean area of adipocytes from NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, scale bar = 50 μm. H and I: PPARγ, C/EBPα, FABP4, LPL and ADIPOQ mRNA levels in IngWAT and EpiWAT of NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups. J: Protein expression levels of PPARγ, C/EBPα and SDCCAG3 in IngWAT of NCD-fed AKO-WT and AKO-Sdccag3 groups. K: Protein expression levels of PPARγ, C/EBPα, SDCCAG3 and SMURF1 in…

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  Fig. 6. Adipose-specific knockout of SDCCAG3 aggravated insulin resistance and obesity-related metabolic disorders. A: Serum triglyceride levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. B: Serum cholesterol levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. C: Serum low density lipoprotein cholesterol levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. D: Serum high density lipoprotein cholesterol levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. E: Glucose tolerance test and area under curve in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. F: Insulin tolerance test and area under curve in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. G: Fasting serum glucose levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. H: Fasting serum insulin levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. I: Serum free fatty acid levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. J: Serum IL-6 levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. K: Serum TNF-α levels in NCD or HFD-fed AKO-WT and AKO-Sdccag3 groups, n = 5. Data were presented as mean ± standard deviation (S.D.). ∗P < 0.05, ∗∗P < 0.01,…

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  Fig. 7. Adipose-specific overexpression of SDCCAG3 inhibited adipocyte hypertrophy and increased the expression of lipid metabolism related indicators. A: Body weight changes in AAV-NC and AAV-Sdccag3 groups over the course of 16 weeks of NCD or HFD feeding, n = 6. B: Micro-CT scans of AAV-NC and AAV-Sdccag3 groups of NCD or HFD feeding to reconstruct subcutaneous fat and visceral fat maps. Orange, subcutaneous fat; Green, visceral fat. C: Photos of EpiWAT from AAV-NC and AAV-Sdccag3 groups of NCD or HFD feeding. D: EpiWAT weight of NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 6. E: Ratio of EpiWAT weight to body weight in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 6. F: HE staining of IngWAT from NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, scar bar = 50 μm. G: HE staining of EpiWAT from NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, scar bar = 50 μm. H: Protein expression levels of PPARγ, C/EBPα and SDCCAG3 in IngWAT of NCD-fed AAV-NC and AAV-Sdccag3 groups. I: Protein expression levels of PPARγ, C/EBPα, SMURF1 and SDCCAG3 in IngWAT of HFD-fed AAV-NC and AAV-Sdccag3 groups. J: Protein expression levels of PPARγ, C/EBPα and SDCCAG3 in EpiWAT of NCD-fed AAV-NC and AAV-Sdccag3…

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  Fig. 8. Adipose-specific overexpression of SDCCAG3 improved insulin sensitivity and resisted to obesity-related metabolic disorders. A: Serum triglyceride levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. B: Serum cholesterol levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. C: Serum low density lipoprotein cholesterol levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. D: Serum high density lipoprotein cholesterol levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. E: Glucose tolerance test and area under curve in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. F: Insulin tolerance test and area under curve in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. G: Fasting serum glucose levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. H: Fasting serum insulin levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. I: Serum free fatty acid levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. J: Serum IL-6 levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. K: Serum TNF-α levels in NCD or HFD-fed AAV-NC and AAV-Sdccag3 groups, n = 5. Data were presented as mean ± standard deviation (S.D.). ∗P <…

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  Supplementary Figure S1.

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  Supplementary Figure S2.

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  Supplementary Figure S3.

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  Supplementary Figure S4.