The possibility that ingestion of chocolate can reduce the risk of cardiovascular disease was reported in recent meta-analyses [4, 5]. Several other meta-analyses have also demonstrated that cocoa products rich in flavan-3-ols have multiple effects on cardiovascular risk factors such as hypertension, dyslipidemia, and glucose intolerance. However, the effective component in chocolate and the mechanism of these multiple effects remains unclear. In the present study, we found that mean resting energy expenditure was reduced significantly by xanthine derivatives free flavan-3-ol fraction derived from cocoa powder (Figure 1c), and that this decrease was not affected by locomotor activity (Figure 1d). We also showed mean blood pressure and fasting blood glucose level were significantly lower in mice treated with flavan-3-ols compared with controls (Table 2). Our series of experiments on flavan-3-ols treatment showed a slight induction of MCAD in skeletal muscle a rate-controlling enzyme in β-oxidation, and a significant increase in gastrocnemius and CPT-2 in soleus muscles, a mitochondrial enzyme. In contrast, no increase in CPT-2 was observed in the liver (Figure 2b). UCP-1 in brown adipose tissue was also induced significantly by flavan-3-ols treatment, whereas UCP-2 in liver and white adipose tissue and UCP-3 in skeletal muscle remained largely unchanged (Figure 3). In addition, we confirmed that biogenesis of mitochondria was increased significantly in skeletal muscle, such as the soleus and gastrocnemius muscles (Figure 4). In contrast, no significant change was observed in mitochondria copy number in liver and white adipose tissue. Skeletal muscle is known to contribute largely to whole body energy expenditure through its mitochondrial oxidative phosphorylation and thermogenesis . The promotion activity of flavan-3-ols on the skeletal muscle mitochondrial biosynthesis suggested the possibility of improvement of metabolic syndrome by additional energy expenditure.
On the other hand, several systematic reviews have confirmed that consumption of dark chocolate rich in flavan-3-ols decreases blood pressure [14–19]. The current study confirmed the reduction in blood pressure. The mechanism of this hypotensive effect has been attributed to orally-administrated flavan-3-ols being distributed in vascular endothelial cells, resulting in direct stimulation of endothelium nitric oxide synthetase . As described above, flavan-3-ols other than monomers are poorly absorbed, and are unlikely to have a direct influence on endothelial cells. Further studies focusing on the circulation are therefore needed to elucidate the hypotensive mechanism of flavan-3-ols. Nevertheless, it was confirmed that the effective component in chocolate that improved metabolic syndrome risk factors was flavan-3-ols according to the results of the present study.
Numerous authors have demonstrated an increase in lipolysis following intake of polyphenols. For instance, catechins in green tea have been reported to enhance energy expenditure by inhibiting catechol O-methyl transferase . These results indicate green tea does not enhance energy expenditure or lipolysis by itself. Catechins in green tea showed a significant effect on energy metabolism at a high level of blood catecholamines, such as after either caffeine ingestion or during exercise [29–33]. Chocolate also contains caffeine found in green tea. An intervention trial of caffeine demonstrated that they may contribute to alterations in energy metabolism . There was limited information about the interaction between theobromine and polyphenols in cocoa. Etherton et al. reported that the xanthine derivatives unlikely to have significant impact on the reducing activity of LDL oxidative susceptibility of cocoa in randomized control study using theobromine capsule . As the present study investigated xanthin drivatives-free fraction of flavan-3-ol derived from cocoa powder, the results suggest that flavan-3-ols enhance fat oxidation independent of prolonged increases in blood catecholamines levels induced by xanthine derivatives. Further evidence for this ability of polyphenols to directly modify energy metabolism was provided in a recent study by Goto et al., which showed repeated ingestion of tiliroside, a glycosidic flavonoid, reduced RER and elevated AMP-activated protein kinase in both the liver and skeletal muscle . It has also been shown in mice that RER is reduced significantly by ingestion of coffee polyphenols .
Several reports have suggested that polyphenols inhibit glucose and fat absorption and/or digestion by inhibiting gastrointestinal enzymes [38, 39]. Similarly, flavan-3-ols derived from cocoa powder have been demonstrated to inhibit dietary fat absorption [40, 41]. In these studies, it was suggested that at least 300 to 800 mg/kg flavan-3-ol fraction was needed to inhibition of glucose or fat absorption and/or digestion. According to these previous results, it was unlikely suggested that the administration of 50 mg/kg flavan-3-ol fraction had a significant influence on digestion and absorption of carbohydrate or fat.
We observed an elevation in mitochondria copy number in skeletal muscle following supplementation of flavan-3-ols. A similar increase in mitochondrial biogenesis in skeletal muscle was also reported in patients with type 2 diabetes or heart failure following ingestion of cocoa . According to the previous reports, peroxisome proliferator-activated receptor γ coactivator α (PGC-1α) is recognized as a master regulator of mitochondrial biogenesis [43–45] by activating respiratory chain and fatty acid oxidation genes, increasing mitochondrial number, and enhancing mitochondrial respiratory capacity. It has been shown that PGC-1α exerts these effects through direct or coactivation with peroxisome proliferator-activated receptors (PPARs), estrogen-related receptors (ERRs), and nuclear respiratory factors (NRFs). Transcription factors such as myocyte enhancer factor 2 (MEF2), forkhead box class-O (FoxO1), activating transcription factor 2 (ATF2), and cAMP response element-binding protein (CREB) enhance PGC-1α transcription during physiological stress such as exercise, cold, fasting, and increased cytokine production . For UCP gene expression, PGC-1α interacts with different nuclear hormone receptors depending on the stimuli, with PPARγ being one of the key transcription factors. For activation of fatty acid oxidation enzymes, PGC-1α uses different transcription factors such as PPARα and estrogen-related receptors (ERRs), that are expressed at high levels in brown adiposities . In the present study, we observed an elevation in mitochondrial copy number, and induction of UCPs and β-oxidation enzymes in several tissues. These results suggested that ingestion of flavan-3-ols augmented PGC-1α transcription.