Fig 1: S1P induces lipolysis through a PKCzeta/MAPK/HSL pathway in differentiated 3T3‐L1 cells. (A) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with vehicle (0.02% BSA), 10 μM S1P (in 0.02% BSA) in the presence or absence of 10 μM PD98059 or 25 μM small molecular HSL inhibitor (smHSLi). (n = 4 per group). (B) Representative Western blots for p‐p44/42 MAPK and p‐HSL Ser660 in differentiated T3‐L1 adipocytes treated with 10 μM S1P or vehicle (0.02% BSA) in the presence or absence of 3 μM bisindolylmaleimid I (Bis) for 15 min (n = 4–5 per condition). Quantification of p‐p44/42 MAPK and p‐HSL Ser660 normalized to GAPDH. (C) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with vehicle or 1 μM S1P lyase inhibitor VD‐78. (n = 5 per group). (D) Glycerol release per mg protein after 16 h treatment of primary mouse adipocytes with vehicle or 1 μM S1P lyase inhibitor VD‐78 (n = 6 per group). Data are presented as mean ± SEM. A one‐way ANOVA with Tukey's multiple‐comparisons test was used for statistical analysis. (E) Gylcerol release per mg protein after 16h treatment of differentiated 3T3‐L1 cells with vehicle (0.02% BSA) vs 10 µM S1P or vehicle (0.1% DMSO) versus 1 µM Isoproterenol in the presence or absence of 100 µM of the adenylate cyclase inhibitor SQ22536. (n = 4 per group). (F) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle control (0.02% BSA) in presence or absence of the 100 nM PI3K Inhibitor Wortmannin. (n = 3–8 per group). (G) Relative kinase activity of recombinant PKC zeta treated with 10 µM S1P or vehicle (MeOH) for 10 min as determined by in vitro phosphorylation assay. (n = 3 per group). (H) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle control (0.02% BSA) in presence or absence of the 3 µM PKC inhibitor bisindolylmaleimid I (Bis). (n = 3–8 per group). (I) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle (0.02% BSA) in the presence or absence of 10 µM PKC zeta pseudo‐substrate inhibitor (PSI). (n = 4 per group). (J) Representative Western blots for T410 phosphorylated PKC zeta, total PKC zeta and GAPDH in differentiated 3T3‐L1 adipocytes treated with 10 µM S1P or vehicle (0.02% BSA) for 15 min. (n = 4 per condition). Quantification of phospho‐PKC zeta T410 normalized GAPDH. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 2: S1P lyase inhibition increases free fatty acids and reduces gWAT mass in HFD‐fed mice due to PKC zeta/HSL activation. (A) Absolute S1P concentrations and relative S1P increase in plasma and gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 5–11 mice per group). (B) Body weight (left) and gWAT weight (right) of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 11–13 mice per group). (C) Representative H&E staining of perigonadal WAT from male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment (left). Quantification of adipocyte diameter and area (right). (n = 11–13 mice per group). (D) Representative H&E staining of inguinal WAT (iWAT) from male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment (left) and quantification of adipocyte diameter and area (right). (n = 4–5 mice per group). (E) Plasma free fatty acids (FFA) of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 10–12 mice per group). (F) FFA in gWAT of male C57BL/6J mice after 12 weeks of HFD versus 6 weeks HFD + 6 weeks HFD/DOP treatment. (n = 5–11 mice per group). (G) Representative Western blots of p‐PKC zeta T410 in gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. Quantification of p‐PKC zeta T410 to GAPDH. (n = 4–5 mice per group). (H) PKC zeta kinase activity as measured by in vitro phosphorylation assay with immunoprecipitated PKC zeta in gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 6–7 mice per group). (I) Representative Western blots of p‐HSL Ser660 in gWAT of male C57BL/6J mice after 12 weeks of HFD versus 6 weeks HFD + 6 weeks HFD/DOP treatment. Quantification of p‐HSL Ser660 to total beta‐actin. (n = 5 mice per group). Data are presented as mean ± SEM. A two‐tailed t‐test was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 3: S1P lyase inhibition increases free fatty acids and reduces gWAT mass in HFD‐fed mice due to PKC zeta/HSL activation. (A) Absolute S1P concentrations and relative S1P increase in plasma and gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 5–11 mice per group). (B) Body weight (left) and gWAT weight (right) of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 11–13 mice per group). (C) Representative H&E staining of perigonadal WAT from male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment (left). Quantification of adipocyte diameter and area (right). (n = 11–13 mice per group). (D) Representative H&E staining of inguinal WAT (iWAT) from male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment (left) and quantification of adipocyte diameter and area (right). (n = 4–5 mice per group). (E) Plasma free fatty acids (FFA) of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 10–12 mice per group). (F) FFA in gWAT of male C57BL/6J mice after 12 weeks of HFD versus 6 weeks HFD + 6 weeks HFD/DOP treatment. (n = 5–11 mice per group). (G) Representative Western blots of p‐PKC zeta T410 in gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. Quantification of p‐PKC zeta T410 to GAPDH. (n = 4–5 mice per group). (H) PKC zeta kinase activity as measured by in vitro phosphorylation assay with immunoprecipitated PKC zeta in gWAT of male C57BL/6J mice after 16 weeks of HFD versus 10 weeks HFD + 6 weeks HFD/DOP treatment. (n = 6–7 mice per group). (I) Representative Western blots of p‐HSL Ser660 in gWAT of male C57BL/6J mice after 12 weeks of HFD versus 6 weeks HFD + 6 weeks HFD/DOP treatment. Quantification of p‐HSL Ser660 to total beta‐actin. (n = 5 mice per group). Data are presented as mean ± SEM. A two‐tailed t‐test was used for statistical analysis. *p < 0.05; **p < 0.01; ***p < 0.001.
Fig 4: S1P induces lipolysis through a PKCzeta/MAPK/HSL pathway in differentiated 3T3‐L1 cells. (A) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with vehicle (0.02% BSA), 10 μM S1P (in 0.02% BSA) in the presence or absence of 10 μM PD98059 or 25 μM small molecular HSL inhibitor (smHSLi). (n = 4 per group). (B) Representative Western blots for p‐p44/42 MAPK and p‐HSL Ser660 in differentiated T3‐L1 adipocytes treated with 10 μM S1P or vehicle (0.02% BSA) in the presence or absence of 3 μM bisindolylmaleimid I (Bis) for 15 min (n = 4–5 per condition). Quantification of p‐p44/42 MAPK and p‐HSL Ser660 normalized to GAPDH. (C) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with vehicle or 1 μM S1P lyase inhibitor VD‐78. (n = 5 per group). (D) Glycerol release per mg protein after 16 h treatment of primary mouse adipocytes with vehicle or 1 μM S1P lyase inhibitor VD‐78 (n = 6 per group). Data are presented as mean ± SEM. A one‐way ANOVA with Tukey's multiple‐comparisons test was used for statistical analysis. (E) Gylcerol release per mg protein after 16h treatment of differentiated 3T3‐L1 cells with vehicle (0.02% BSA) vs 10 µM S1P or vehicle (0.1% DMSO) versus 1 µM Isoproterenol in the presence or absence of 100 µM of the adenylate cyclase inhibitor SQ22536. (n = 4 per group). (F) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle control (0.02% BSA) in presence or absence of the 100 nM PI3K Inhibitor Wortmannin. (n = 3–8 per group). (G) Relative kinase activity of recombinant PKC zeta treated with 10 µM S1P or vehicle (MeOH) for 10 min as determined by in vitro phosphorylation assay. (n = 3 per group). (H) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle control (0.02% BSA) in presence or absence of the 3 µM PKC inhibitor bisindolylmaleimid I (Bis). (n = 3–8 per group). (I) Glycerol release per mg protein after 16 h treatment of differentiated 3T3‐L1 cells with 10 µM S1P or vehicle (0.02% BSA) in the presence or absence of 10 µM PKC zeta pseudo‐substrate inhibitor (PSI). (n = 4 per group). (J) Representative Western blots for T410 phosphorylated PKC zeta, total PKC zeta and GAPDH in differentiated 3T3‐L1 adipocytes treated with 10 µM S1P or vehicle (0.02% BSA) for 15 min. (n = 4 per condition). Quantification of phospho‐PKC zeta T410 normalized GAPDH. *p < 0.05; **p < 0.01; ***p < 0.001.
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