Theobromine

Kinase experiment:

3T3-L1 preadipocytes are pre-incubated with MG132 (10 μM) for 30 min, followed by incubation with IBMX in the presence or absence of Theobromine (25 μM) for 8 h. The cells are lysed in denaturing cell extraction buffer (50 mM Tris-HCl, pH 7.5, containing 70 mM β-mercaptoethanol and 2% SDS) at 95°C for 10 min. The cell lysates are diluted 20 fold with dilution buffer (20 mM Tris-HCl, pH 7.5, containing 150 mM NaCl, 1 mM EDTA, 1mM EGTA, 1% TritonX-100, 2.5 mM sodium pyrophosphate and protease inhibitor cocktail) and centrifuged at 20,000 g for 30 s. The supernatant is incubated with rabbit polyclonal anti-C/EBPβ IgG, anti-FLAG IgG or control IgG at 4°C overnight, followed by incubation with 30 μL protein G-Sepharose resin at 4°C for 1 h. The resin is washed with lysis buffer three times and proteins bound to the resin are separated by SDS-PAGE and analyzed by western blotting[1].

Animal experiment:

Lewis rats (3 week old) are used in this study. The rats are randomly distributed into three dietary groups (n=7 per group): the reference (RF) group ingested standard diet AIN-93M, the cocoa (CC) group ingested a standard diet with 10% of natural Forastero cocoa containing 0.25% Theobromine, and the Theobromine (TB) group ingested a standard diet including 0.25% of Theobromine, i.e. the content of Theobromine presents in the CC diet[2].

References:

[1]. Mitani T, et al. Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.
[2]. Martín-Peláez S, et al. Effect of cocoa's theobromine on intestinal microbiota of rats. Mol Nutr Food Res. 2017 Oct;61(10).