Fig. 4.

CRACE reduced lipogenesis and induced lipolysis in mature 3T3-L1 adipocytes by increasing cAMP level and PKA activation. a. mRNA expression analysis of lipogenesis related genes in CRACE treated and untreated mature adipocytes. b. mRNA expression analysis of major players of TAG lipolysis in CRACE treated and untreated mature adipocytes. In panel a and b mature adipocytes received CRACE (500 μg/mL equivalent) for 4 days. Bars represent mean ± SEM. n = 3. * p < 0.05 for untreated vs. CRACE treated. c. Treatment with CRACE (500 μg/mL equivalent) for 3 h increased lipolysis in mature 3T3-L1 adipocytes as measured by glycerol release. Isoproterenol (10 μM) was used as positive control. CRACE also increased lipolysis in cells where basal lipolysis was normalised with 100 nM adenosine (A) and 1 U/mL of adenosine deaminase (AD) treatment. Bars represent mean ± SEM. n = 3. * indicates p < 0.05 for untreated vs. different treatments and # indicates p < 0.05 for A + AD vs. A + AD + different treatments. d. CRACE increased PLN1 phosphorylation and decreased Akt phosphorylation (S473). Mature 3T3-L1 adipocytes were treated with CRACE (500 μg/mL equivalent) for 24 h followed by western blot analysis. Bars represent mean ± SEM, n = 3. * p < 0.05 for untreated vs. CRACE treated adipocytes. e. CRACE (500 μg/mL equivalent) increased intracellular cAMP level in mature 3T3-L1 adipocytes. 0.5 mM IBMX was added in all the cells for cAMP sustenance. Bars represent mean ± SEM, n = 3. * p < 0.05 for untreated vs. treated adipocytes. f. CRACE stimulated lipolysis by PKA activation in mature 3T3-L1 adipocytes. Glycerol release induced in mature adipocytes by CRACE (500 μg/mL equivalent) or isoproterenol in presence or absence of PKA inhibitor H89 was measured. CRACE induced lipolysis was significantly reduced by H89. Bars represent mean ± SEM, n = 3. * p < 0.05 for ‘given treatments’ vs. ‘treatment + H89’ (i.e. Untreated vs H89; CRACE vs CRACE + H89; ISO + CRACE vs ISO + CRACE + H89). Unpaired t-test (for panel 4a, 4b, 4d and 4e) and one-way ANOVA followed by Bonferroni post-test (for panel 4c and 4f) was performed to evaluate the statistical significance