Modulation of Obesity-Related Inflammation by Cocoa (Theobroma Cacao L. Sterculiaceae)

Open Access
- Author:
- Gu, Yeyi
- Graduate Program:
- Food Science
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 06, 2013
- Committee Members:
- Joshua D Lambert, Dissertation Advisor/Co-Advisor
Ryan John Elias, Committee Member
Gregory Ray Ziegler, Committee Member
Sheila Grace West, Committee Member - Keywords:
- cocoa
Theobroma Cacao
procyanidin
obesity
inflammation
metabolic syndrome - Abstract:
- Obesity is associated with many metabolic diseases and it is now recognized as a state of chronic inflammation. Cocoa (Theobroma cacao L. Sterculiaceae) is a rich source of polyphenols, in particular oligomeric flavan-3-ols (i.e. procyanidins) which have received considerable attention due to potential health beneficial effects including anti-inflammatory activities. However, the modulation of inflammatory and metabolic effects by cocoa has not been fully elucidated and there are still gaps and inconsistencies in the literature. In my thesis research, I hypothesize that dietary cocoa and cocoa procyanidins (CPs) can exert anti-inflammatory activity and modulate obesity-related pathologies via inhibition of digestive enzymes, suppression of cytokine and eicosanoid production, regulation of nuclear factor-κB (NF-κB) target gene expression and modulation of metabolic endotoxemia. Results from this research would provide a comprehensive body of scientific evidence for the modulatory effects of cocoa on obesity-related inflammation, as well as mechanistic insights into the understanding of its in vivo anti-inflammatory actions. This research is significant to public health, and both the pharmaceutical and food industries. To investigate this hypothesis, I first investigated the in vitro inhibitory activities of three cocoa extracts and isolated CPs with degree of polymerization (DP) of 2-10 against three purified digestive enzymes: pancreatic α-amylase (PA), pancreatic lipase (PL) and phospholipase A2 (PLA2). These enzymes, all of which play an important role in starch or lipid digestion, represent attractive targets for reducing macronutrient absorption and the prevention of obesity and its co-morbidities. CPs dose-dependently inhibited PA, PL, and PLA2, and the inhibitory potency increased as a function of DP (R2 > 0.93). Extract of Lavado cocoa, which is non-fermented and non-alkalized, was the most potent enzyme inhibitor among the three extracts (IC50 = 8.5- 47 µg/mL). The type of inhibition was characterized by kinetic analysis. Results suggest that regular cocoa extract, the CP pentamer (DP=5) and decamer (DP=10) inhibited PL activity in a mixed mode. The pentamer and decamer non-competitively inhibited PLA2 activity, whereas the regular cocoa extracts inhibited PLA2 competitively. This study demonstrates that cocoa rich in procyanidins can inhibit digestive enzymes in vitro, which may provide a safe and cost-effective approach for weight management and glycemic control. Second, given the greater bioactive potential of CPs with higher DP and gaps in the literature regarding their anti-inflammatory activities, I examined the anti-inflammatory effects of high DP CPs on modulation of eicosanoid metabolism and cytokine production in vitro. Eicosanoids and pro-inflammatory cytokines are both important mediators in the establishment and maintenance of a state of obesity-related inflammation. In this study, a murine macrophage RAW 264.7 cell line stimulated by bacterial endotoxin (lipopolysaccharide, LPS) was used as a model of inflammation. Results indicate that CPs mixture with DP ≥ 7 dose-dependently inhibited eicosanoid-generating enzymes (PLA2 and cyclooxygenase-2 (COX-2)) in vitro, and significantly decreased the production of their metabolite prostaglandin E2 (PGE2) in LPS-stimulated RAW 264.7 macrophages. In addition, CP octamer suppressed pro-inflammatory cytokine production, including tumor necrosis factor-α (TNF-α), interleukin-6 (IL-6), and monocyte chemoattractant protein-1 (MCP-1). In particular, CP octamer (100 μM) without cytotoxicity significantly decreased the production of IL-6 and MCP-1 by 97% and 70%, respectively in macrophages pre-challenged with LPS. These results suggest that high-DP CPs may counteract the effect of LPS and facilitate the resolution of inflammation. Together, this study demonstrated an anti-inflammatory role and potential therapeutic value of procyandin-rich cocoa in the diet of patients with inflammatory conditions. Third, based on the observed modulatory effects of cocoa on digestive enzymes and inflammatory markers in vitro, I designed studies to examine the effect of cocoa supplementation on markers of obesity-related inflammation in a mouse model. In this study, male C57BL/6J mice were fed with either a low-fat (LF, 10% kcal from fat) or a high-fat (HF, 60% kcal from fat) diet. After 8 wk, HF-fed mice were randomized into two groups: half were maintained on the HF diet and half were fed with a HF diet supplemented with 8% unsweetened cocoa powder (HF-HFC group) for 10 wk. At the end of wk 18, blood and tissue samples were collected for biochemical analyses. Results show that cocoa supplementation significantly reduced the rate of body weight gain (15.8%), and this was accompanied by increased fecal lipid content (55.2%) compared to HF-fed control mice. Further, it counteracted obesity-induced insulin resistance and fatty liver disease compared to the HF group. Cocoa supplementation also significantly decreased plasma levels of IL-6 (30.4%) and MCP-1 (25.2%), and increased adiponectin (33.7%) compared to HF-fed mice. Expression of pro-inflammatory genes (Il6, Il12b, Nos2 and Emr1) by adipose tissue macrophages was significantly reduced (37-56%) in the cocoa-supplemented mice. Therefore, in this animal study, I’ve shown that short-term dietary supplementation with cocoa can ameliorate HF-induced body weight gain, systemic inflammation, insulin resistance and fatty liver disease in mice. Finally, I conducted another mouse study to examine the effects and mechanisms of cocoa on adipose tissue (AT) inflammation through the alteration of eicosanoid metabolism, regulation of NF-κB target gene expression, and modulation of metabolic endotoxemia in HF-fed mice. In HF-induced obesity, AT has a central role in lipid and glucose metabolism and produces a number of hormones and cytokines that predispose to the development of chronic inflammation. In this study, male C57BL/6J mice were fed a LF diet, a HF diet, and a HFC diet for 18 wk. Results demonstrate that cocoa supplementation significantly decreased pro-inflammatory gene expression (Tnfa, Il6, iNos and Emr1) in AT by 40-60% compared to the HF group, and this was accompanied by decreased protein expression of NF-κB. AT arachidonic acid (AA) levels, an inflammatory eicosanoid precursor, were positively correlated with adiposity (r = 0.57) and cocoa-treated mice had 33% lower levels of AA compared to HF controls. Moreover, protein expression of the eicosanoid-generating enzymes adipose-specific PLA2 (AdPLA) and COX-2 were also reduced in cocoa-treated mice by 53% and 55%, respectively. Finally, plasma levels of endotoxin were found to be 40% lower than that of HF-fed mice. This decrease in metabolic endotoxemia was in parallel with increased a gut barrier function marker (glucagon-like peptide -2, GLP-2). Overall, this study has shown for the first time that long-term cocoa supplementation reduces AT inflammation in HF-fed mice by down-regulating NF-κB target gene expression and modulating eicosanoid metabolism, which may be partly due to decreased metabolic endotoxemia by cocoa. In conclusion, cocoa exerted anti-inflammatory activity and modulated obesity-related pathologies in vitro and in vivo. These effects appear to be mediated in part by inhibition of digestive enzymes and modulation of metabolic endotoxemia. As a result, moderate consumption of cocoa could be part of a healthy diet to prevent and treat obesity-related inflammation and resultant co-morbidities.