Theobromine  (Synonyms: 3,7-Dimethylxanthine)

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].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

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].

MCE has not independently confirmed the accuracy of these methods. They are for reference only.

• [1]. Mitani T, et al. Theobromine suppresses adipogenesis through enhancement of CCAAT-enhancer-binding protein β degradation by adenosine receptor A1.  [Content Brief]

  [2]. Martín-Peláez S, et al. Effect of cocoa's theobromine on intestinal microbiota of rats. Mol Nutr Food Res. 2017 Oct;61(10).  [Content Brief]