Proceedings of The Physiological Society

Physiology 2016 (Dublin, Ireland) (2016) Proc Physiol Soc 37, PCA286

Poster Communications

Theobromine crosses the blood brain barrier in vivo resulting in increased phosphorylation of vasodilator-stimulated phosphoprotein in the mouse brain

N. Sugimoto1,2, M. Katakura2,3, K. Matsuzaki2, T. Ohno-Shosaku1, A. Yachie1, O. Shido2

1. Kanazawa University, Kanazawa, Ishikawa, Japan. 2. Shimane University, Izumo, Japan. 3. Johsai University, Sakado, Japan.


Theobromine, a caffeine derivative, is the primary methylxanthine in Theobroma cacao. Theobromine works as a phosphodiesterase inhibitor to increase intracellular cAMP and has been shown to have antitumor effects in vitro, inhibiting the growth of a cell line derived from a human malignant glioma (Sugimoto et al., 2014). As glioma is a cancer of the brain, to be an effective therapeutic, theobromine would need to cross the blood-brain barrier to exert its antitumor effect. In this study, we investigated whether orally administered theobromine could cross the blood-brain barrier in mice to act centrally. Vasodilator-stimulated phosphoprotein (VASP), a critical factor in regulating actin dynamics, is phosphorylated by cAMP-dependent protein kinases; thus, the level phosphorylated VASP (pVASP) was used as an index of theobromine activity. All animal experiments were performed according to the Guidelines for Animal Experimentation of Shimane University Faculty of Medicine. Mice were divided into two groups (n = 6/group), fed either a normal diet or a diet supplemented with 0.05% theobromine for 30 days. Mice were sacrificed at the end of the experiments, and brain and plasma were harvested from mice and kept at -80°C until processing. The concentration of theobromine in brain and plasma samples was measured by high-performance liquid chromatography in combination with ESI-MS, using a TSQ quantum mass spectrometer (Thermo Fisher Scientific K.K., Tokyo, Japan). Plasma glucose concentrations were determined using a Glucose CII-Test WAKO Kit (Wako Pure Chemical Industries, Osaka, Japan). The pVASP expression level in brain samples was analyzed by western blot (n = 6/group), using a phospho-specific VASP antibody (Cell Signaling Thechnology, USA). Data are presented as the mean ± S.E.M. Statistical analysis was performed using a Student's t-test. Body weights did not differ between the theobromine-fed mice (TB) (24.3 ± 0.4 g) and control mice (CN) (24.6 ± 0.4 g, p = 0.59). In TB mice, theobromine was detected in both plasma (2.20 ± 0.12 μg/mL) and brain (0.21 ± 0.02 μg/mL homogenate), whereas theobromine was not detected in either tissue from CN mice. Plasma glucose concentrations did not differ significantly between CN (254.6 ± 13.85 mg/dL) and TB (241.35 ± 12.91 mg/dL) mice (p = 0.52). The pVASP level was significantly elevated in TB mice in comparison to CN mice (p < 0.05). We interpret the increase in pVASP expression within the TB brain as indicative of an elevation in intracellular cAMP, which is generated as the result of theobromine activity. Thus, our results suggest that theobromine, taken orally, crosses the blood brain barrier in sufficient quantities to exert its effects as a phosphodiesterase inhibitor in the brain.

Where applicable, experiments conform with Society ethical requirements