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. 2022 Jun 24;63:101531. doi: 10.1016/j.molmet.2022.101531

Figure 5.

Figure 5

CLSTN3 overexpression suppresses stimulated lipolysis in vivo and ex vivo. (A) Representative H&E staining images of iWAT sections of AAV-CON and AAV-CLSTN3 mice after 4-week AAV injection, and its quantification of adipocyte area. Scale bar, 50 μm. (B) Quantitative PCR analysis of mRNA expression of adipogenesis (Pparg, Cebpa, and Fabp4), lipogenesis (Acaca, Fasn, and Scd1) and lipolysis (Lipe and Pnpla2) related genes in iWAT from both groups (n = 5). (C and D) Free glycerol (C) and non-esterified fatty acid (NEFA) (D) levels in serum of AAV-CON and AAV-CLSTN3 mice upon 4-hour cold stress (n = 10). (E) Representative immunoblot analysis for the expression of phosphorylated HSL Ser563 (P-HSLSer563) and total HSL (T-HSL) in iWAT from both groups upon cold stress, and the quantification for the ratio of P-HSLSer563 to T-HSL expression. (F and G) Free glycerol (F) and NEFA (G) levels at different time points in serum of AAV-CON and AAV-CLSTN3 mice after β3-adrenoceptor agonist CL-316,243 injection (n = 7–8). (H) Immunoblot analysis and its quantification for the ratio of P-HSLSer563 to T-HSL expression in iWAT from both groups from in vivo lipolysis assay. (I and J) Free glycerol (I) and NEFA (J) levels at different time points in medium released from iWAT explants isolated from AAV-CON and AAV-CLSTN3 mice upon 10 μmol/L isoproterenol stimulation (n = 10). (K) Immunoblot analysis and its quantification for the ratio of P-HSLSer563 to T-HSL expression in iWAT from both groups from ex vivo lipolysis assay. Data are shown as mean ± SEM of biologically independent samples, and ∗p < 0.05, ∗∗p < 0.01.