Urinary Metabolomics Study on the Protective Role of Cocoa in Zucker Diabetic Rats via 1H-NMR-Based Approach.

Cocoa constitutes one of the richest sources of dietary flavonoids with demonstrated anti-diabetic potential. However, the metabolic impact of cocoa intake in a diabetic context remains unexplored. In this study, metabolomics tools have been used to investigate the potential metabolic changes induced by cocoa in type 2 diabetes (T2D). To this end, male Zucker diabetic fatty rats were fed on standard (ZDF) or 10% cocoa-rich diet (ZDF-C) from week 10 to 20 of life. Cocoa supplementation clearly decreased serum glucose levels, improved glucose metabolism and produced significant changes in the urine metabolome of ZDF animals. Fourteen differential urinary metabolites were identified, with eight of them significantly modified by cocoa. An analysis of pathways revealed that butanoate metabolism and the synthesis and degradation of branched-chain amino acids and ketone bodies are involved in the beneficial impact of cocoa on diabetes. Moreover, correlation analysis indicated major associations between some of these urine metabolites (mainly valine, leucine, and isoleucine) and body weight, glycemia, insulin sensitivity, and glycated hemoglobin levels. Overall, this untargeted metabolomics approach provides a clear metabolic fingerprint associated to chronic cocoa intake that can be used as a marker for the improvement of glucose homeostasis in a diabetic context.

Aging in Male Wistar Rats Associates With Changes in Intestinal Microbiota, Gut Structure, and Cholecystokinin-Mediated Gut-Brain Axis Function.

Aging in mammals is characterized by failure of the homeostatic mechanisms that regulate energy balance. Several mechanisms have been proposed such as the presence of a low-grade chronic inflammation in different tissues, as well as leptin and insulin resistance, but the primary alteration is not fully elucidated. The gut microbiota has recently emerged as a key player in a variety of metabolic and neurological disorders. A main concept in this context is the gut-brain axis that refers to alterations in the gut that mediate effects in the central nervous system, including those related with the control of energy balance. Using 16S rRNA analysis, we demonstrate that aged male Wistar rats have increased presence of mucin-degrading and lipopolysaccharide (LPS)-producing bacteria. In addition, old animals exhibit a lower number of neutral mucin secreting goblet cells, and a decrease of tight junctions and adherens junctions marker proteins, zonula occludens protein-1 (ZO-1) and ß-catenin, respectively. These data are compatible with a thinner mucus layer and a weaker gut barrier in older animals that likely facilitate LPS leakage. Our data also show that cholecystokinin (CCK) satiating effect is impaired in aged rats, one of the expected effects of increased LPS leakage. In contrast, no overt signs of gut or systemic inflammation are observed. Changes in microbiota in old male Wistar rats present features of situations of increased adiposity, but different from those of obese animals. These could partly explain the increased adiposity and fat deposition in liver and heart as observed here.

Preventive effect of cocoa flavanols against glucotoxicity-induced vascular inflammation in the arteria of diabetic rats and on the inflammatory process in TNF-α-stimulated endothelial cells.

Hyperglycaemia induces a vascular inflammatory process that is a critical event in cardiovascular disease in type 2 diabetes. Cocoa and its flavanols have been widely investigated for its antioxidant and anti-inflammatory properties, and several clinical and pre-clinical studies support their vascular benefits. However, the effects of cocoa flavanols on vascular inflammation in diabetes remains to be elucidated. Herein, we evaluated the anti-inflammatory effect of a cocoa-rich diet on the aortas of Zucker diabetic fatty (ZDF) rats. Moreover, the potential role of flavanol-derived colonic metabolites to modulate the adhesion and inflammatory processes were also evaluated using TNF-α-stimulated endothelial cells. Results demonstrate that cocoa attenuates the levels of phospho-p65-nuclear factor-kappaB (NF-κB) and the expression of inflammatory factors including intercellular adhesion molecule-1 (ICAM-1), vascular adhesion molecule-1 (VCAM-1) and inducible nitric oxide synthase in the aortas of ZDF rats. Experiments with endothelial cells further confirm that a mix of flavanol-derived colonic metabolites effectively down-regulate the levels of p-p65-NF-κB and the cell adhesion molecules ICAM-1 and VCAM-1, preventing thus the increase of monocyte-endothelial adhesion induced by TNF-α. These novel data provide the first evidence of the relevant role of cocoa and their flavanol-derived metabolites to avoid the development of endothelial inflammation and diabetic complications.

(-)-Epicatechin and the colonic metabolite 2,3-dihydroxybenzoic acid protect against high glucose and lipopolysaccharide-induced inflammation in renal proximal tubular cells through NOX-4/p38 signalling.

Chronic hyperglycaemia and inflammation are present in diabetes and both processes have been related to the pathogenesis of diabetic kidney disease. Epicatechin (EC) and main colonic phenolic acids derived from flavonoid intake, such as 2,3-dihydroxybenzoic acid (DHBA), 3,4-dihydroxyphenylacetic acid (DHPAA) and 3-hydroxyphenylpropionic acid (HPPA), have been suggested to exert beneficial effects in diabetes. This study was aimed at investigating whether the mentioned compounds could prevent inflammation in renal proximal tubular NRK-52E cells induced by high glucose and lipopolysaccharide (LPS). Pre-treatment of cells with EC and DHBA (5 µM) reverted the enhanced levels of pro-inflammatory cytokines, such as tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1), activated by high glucose and LPS. Additionally, EC and DHBA pre-incubation reduced the increased values of adhesion molecules, namely, intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), as well as those of mitogen-activated protein kinases (MAPKs) [extracellular signal-regulated kinase (ERK), -c-jun N-terminal kinase (JNK) and -p38 protein kinase (p38)] activated by the high glucose and LPS challenge. Thus, in EC and DHBA pre-treated cells ICAM-1, p-ERK and p-JNK were returned to control values, and VCAM-1 and p-p38 levels were reduced by ∼20 and 25%, respectively, when compared to high glucose plus LPS-stimulated cells. Likewise, pre-treatment with EC and DHBA protected against high glucose plus LPS-triggered oxidative stress by preventing increased ROS and NADPH oxidase 4 (NOX-4) levels (∼25 and 45% reduction, respectively). By using specific inhibitors of p38 and NOX-4, the participation of both proteins in EC- and DHBA-mediated protection against inflammation and associated oxidative stress was shown. Taken together, EC and DHBA exert beneficial effects in renal proximal tubular cells, as they contribute to preventing the inflammatory-induced milieu and the accompanying redox imbalance, playing NOX-4/p38 a crucial role.

Cocoa diet modulates gut microbiota composition and improves intestinal health in Zucker diabetic rats.

Cocoa supplementation improves glucose metabolism in Zucker diabetic fatty (ZDF) rats via multiple mechanisms. Furthermore, cocoa rich-diets modify the intestinal microbiota composition both in humans and rats in healthy conditions. Accordingly, we hypothesized that cocoa could interact with the gut microbiota (GM) in ZDF rats, contributing to their antidiabetic effects. Therefore, here we investigate the effect of cocoa intake on gut health and GM in ZDF diabetic rats. Male ZDF rats were fed with standard (ZDF-C) or 10% cocoa-rich diet (ZDF-Co) during 10 weeks. Zucker Lean animals (ZL) received the standard diet. Colon tissues were obtained to determine the barrier integrity and the inflammatory status of the intestine and faeces were analysed for microbial composition, short-chain fatty acids (SCFA) and lactate levels. We found that cocoa supplementation up-regulated the levels of the tight junction protein Zonula occludens-1 (ZO-1) and the mucin glycoprotein and reduced the expression of pro-inflammatory cytokines such as tumour necrosis factor-α (TNF-α), interleukin-6 (IL-6) and monocyte chemoattractant protein 1 (MCP-1) in the colon of ZDF diabetic animals. Additionally, cocoa modulated the microbial composition of the ZDF rats to values similar to those of the lean group. Importantly, cocoa treatment increased the relative abundance of acetate-producing bacteria such as Blautia and prevented the increase in the relative amount of lactate-producing bacteria (mainly Enterococcus and Lactobacillus genera) in ZDF diabetic animals. Accordingly, the total levels of SCFA (mainly acetate) increased significantly in the faeces of ZDF-Co diabetic rats. Finally, modified GM was closely associated with improved biochemical parameters related to glucose homeostasis and intestinal integrity and inflammation. These findings demonstrate for the first time that cocoa intake modifies intestinal bacteria composition towards a healthier microbial profile in diabetic animals and suggest that these changes could be associated with the improved glucose homeostasis and gut health induced by cocoa in ZDF diabetic rats.

Cocoa Flavanols Protect Human Endothelial Cells from Oxidative Stress.

Oxidative stress may cause functional disorders of vascular endothelia which can lead to endothelial apoptosis and thus alter the function and structure of the vascular tissues. Plant antioxidants protect the endothelium against oxidative stress and then become an effective option to treat vascular diseases. Cocoa flavanols have been proven to protect against oxidative stress in cell culture and animal models. In addition, epidemiological and interventional studies strongly suggest that cocoa consumption has numerous beneficial effects on cardiovascular health. The objective of this study was to test the chemo-protective effect of realistic concentrations of a cocoa phenolic extract and its main monomeric flavanol epicatechin on cultured human endothelial cells submitted to an oxidative challenge. Both products efficiently restrained stress-induced reactive oxygen species and biomarkers of oxidative stress such as carbonyl groups and malondialdehyde, and recovered depleted glutathione, antioxidant defences and cell viability. Our results demonstrate for the first time that a polyphenolic extract from cocoa and its main flavonoid protect human endothelial cells against an oxidative insult by modulating oxygen radical generation and antioxidant enzyme and non-enzyme defences.

Cocoa ameliorates renal injury in Zucker diabetic fatty rats by preventing oxidative stress, apoptosis and inactivation of autophagy.

Redox balance, autophagy and apoptosis are main processes involved in the development of diabetic nephropathy. Epidemiological and animal studies suggest that cocoa might reduce the risk of diabetic complications. However, the molecular mechanisms responsible for these potential preventive activities and whether cocoa exerts beneficial effects on dysregulated signalling pathways involved in cellular antioxidant defence, autophagy and apoptosis in the diabetic kidney remain largely unknown. Therefore, this work investigated the effect of a cocoa-rich diet on the mentioned processes in the renal cortex of Zucker Diabetic Fatty (ZDF) rats. Male ZDF rats were fed either a control or cocoa-rich diet (10%), and Zucker lean animals received the control diet (10-20 weeks-of-life). ZDF rats fed with cocoa decreased body weight and glucose and insulin levels, and improved renal function. Cocoa intake further prevented the enhanced renal cortical oxidative stress in diabetic rats by regulating the antioxidant defence system and close-related proteins to cytoprotection and cell response; thus, cocoa diminished oxidative markers (reactive oxygen species and carbonyl groups) and NADPH-oxidase-4 levels, and restored key enzymatic antioxidant activities (superoxide dismutase and catalase), nuclear-erythroid-2-related factor-2, and ERK-MAPK levels, as well as sirtuin-1/5'-AMP-activated-protein kinase signalling. Moreover, in ZDF rats cocoa-rich diet contributed to alleviation of the renal cortical injury through autophagy activation (p62 upregulation, and downregulation of beclin-1 and LC3), and inhibition of apoptosis (Bcl-xL stimulation and suppression of Bax and caspases-9 and -3). These findings provide the first in vivo evidence on the molecular mechanisms of cocoa to circumvent renal cortical damage that involve improvement of antioxidant competences, stimulation of autophagy and suppression of apoptosis in ZDF rats.

Dietary Cocoa Prevents Aortic Remodeling and Vascular Oxidative Stress in Diabetic Rats.

SCOPE: The aim of the present study is to investigate the potential protective effect of a cocoa-rich diet on functional and structural vascular alterations in diabetes and the mechanism involved. METHODS AND RESULTS: Male Zucker diabetic fatty (ZDF) rats are fed on a standard (ZDF-C) or cocoa-rich diet (ZDF-Co) from week 10 to 20 of life. Diabetic ZDF-C rats showed increased blood pressure and enhanced aortic stiffness, as demonstrated by the increased pulse pressure and the augmented aortic medial thickness with loss and disruption of elastic fibres. Interestingly, cocoa intake strongly avoided all these adverse effects and reduced aortic oxidative stress. Mechanistically, cocoa diet prevented sirtuin-1 (SIRT-1) depletion and increased NADPH oxidases (NOXs) and reactive oxygen species production as well as reduced active nuclear factor E2 related factor 2 (Nrf2) and their antioxidant products. CONCLUSION: The results demonstrate for the first time that a cocoa-rich diet strongly prevents aortic stiffening and remodeling in diabetic animals and avoids aortic oxidative stress. It is suggested that this effect could be mediated via its effects on SIRT-1, NOXs, and Nrf2.

Cocoa intake attenuates renal injury in Zucker Diabetic fatty rats by improving glucose homeostasis.

Glucotoxicity (high levels of glucose) is a major factor in the pathogenesis of diabetic kidney disease. Cocoa has anti-diabetic effects by lowering glucose levels. However, whether cocoa exerts beneficial effects on the renal cortex glucose homeostasis and the molecular mechanisms responsible for this possible protective activity remain largely unknown. Thus, the potential anti-diabetic properties of cocoa on insulin signalling, glucose transporters and gluconeogenic enzymes were evaluated in the renal cortex of Zucker Diabetic fatty (ZDF) rats. Male ZDF rats were fed a control or cocoa-rich diet (10%), and Zucker Lean animals received the control diet. ZDF rats supplemented with cocoa (ZDF-Co) showed decreased body weight gain, glucose and insulin levels, improved glucose tolerance, insulin resistance and structural alterations in renal cortex. Moreover, cocoa-rich diet ameliorated insulin resistance by reverting decreased tyrosine-phosphorylated-insulin receptor levels and by preventing the inactivation of glycogen synthase kinase-3/glycogen synthase pathway (GSK-3/GS) in the renal cortex of ZDF-Co rats. Cocoa antihyperglycaemic effect also appeared to be mediated through the diminution of phosphoenolpyruvate-carboxykinase (PEPCK), glucose-6-phosphatase (G-6-Pase), sodium-glucose-co-transporter-2 (SGLT-2), and glucose-transporter-2 (GLUT-2) levels in ZDF-Co rat's renal cortex. These findings demonstrate that cocoa alleviates renal injury by contributing to maintain the glucose homeostasis in type 2 diabetic ZDF rats.

Protective effects of (-)-epicatechin and the colonic metabolite 3,4-dihydroxyphenylacetic acid against glucotoxicity-induced insulin signalling blockade and altered glucose uptake and production in renal tubular NRK-52E cells.

Glucotoxicity (high levels of glucose) is a major cause in the pathogenesis of diabetes. Evidences indicate that (-)-epicatechin (EC) and colonic metabolites derived from flavonoid intake could possess antidiabetic effects, but the mechanisms for their preventive activities related to glucose homeostasis and insulin signalling in the kidney remain largely unknown. This work is aimed to investigate the effect of EC and main colonic phenolic acids derived from flavonoid intake, i.e. 2,3-dihydroxybenzoic-acid, 3,4-dihydroxyphenylacetic-acid (DHPAA) and 3-hydroxyphenylpropionic-acid, on insulin signalling, and glucose production and uptake in renal tubular proximal NRK-52E cells treated with high glucose. Pre-treatment with EC or DHPAA prevented the decreased tyrosine-phosphorylated and total levels of IR caused by high glucose. EC and DHPAA pre-treatment also avoided the inactivation of the PI3K/AKT pathway and AMPK, and the elevation of PEPCK levels induced by high glucose. Additionally, EC and DHPAA pre-treatment alleviated the altered glucose uptake and production caused by high glucose, although this protective effect was abrogated when AKT and AMPK were inhibited. These results suggest EC and DHPAA prevent or delay a potential dysfunction of NRK-52E cells treated with high glucose through the attenuation of the insulin signalling blockade and the modulation of glucose homeostasis via AKT and AMPK.

Colonic metabolites from flavanols stimulate nitric oxide production in human endothelial cells and protect against oxidative stress-induced toxicity and endothelial dysfunction.

Oxidative stress is involved in endothelial dysfunction, the key player in the development of vascular events. Flavanols, the major antioxidants in cocoa have been related to vascular protection and lower cardiovascular risk. However, the bioavailability of cocoa flavanols is very low and their bioactivity in vivo seems to be greatly mediated by the derived phenolic metabolites formed by intestinal microbiota. Hence, we investigated whether microbial-derived flavanol metabolites 3,4-dihydroxyphenylacetic acid (DHPAA), 2,3-dihydroxybenzoic acid (DHBA), 3-hydroxyphenylpropionic acid (HPPA) and a mix of them could influence endothelial function and prevent oxidative stress in human endothelial cells (Ea.hy926). Our results revealed that a mixture of flavanol colonic metabolites significantly increased phosphorylation of endothelial nitric oxide synthase (eNOS) and nitric oxide (NO) production. By using specific inhibitors, we also established the participation of the adenosine monophosphate-activated protein kinase (AMPK) and protein kinase B (AKT) in eNOS activation. Likewise, flavanol metabolite mix protected against oxidative stress-induced endothelial dysfunction and cell death by preventing increased ROS generation and activation of signaling pathways related to oxidative stress. We concluded that flavanol colonic metabolites could exert beneficial effects in endothelial cells and prevent oxidative stress-induced vascular dysfunction.

(-)-Epicatechin and the Colonic 2,3-Dihydroxybenzoic Acid Metabolite Regulate Glucose Uptake, Glucose Production, and Improve Insulin Signaling in Renal NRK-52E Cells.

SCOPE: (-)-Epicatechin (EC) and main colonic phenolic acids derived from flavonoid intake, such as 2,3-dihydroxybenzoic acid (DHBA), 3,4-dihydroxyphenylacetic acid (DHPAA), 3-hydroxyphenylpropionic acid (HPPA), and vanillic acid (VA), have been suggested to exert beneficial effects in diabetes, although the mechanism for their actions remains unknown. In this study, the modulation of glucose homeostasis and insulin signaling by the mentioned compounds on renal proximal tubular NRK-52E cells is investigated. METHODS AND RESULTS: Levels of the glucose transporters SGLT-2 and GLUT-2, as well as glucose uptake, glucose production, and key proteins of the insulin pathways, namely insulin receptor (IR), insulin receptor substrate-1 (IRS-1), and PI3K/AKT pathway are analyzed. EC (5-20 µm) and DHBA (20 µm) reduced both renal glucose uptake and production. Interestingly, EC and DHBA did not modify the levels of SGLT-2 and GLUT-2, and modulated the expression of phosphoenolpyruvate carboxykinase via AKT leading to a diminished glucose production. EC and DHBA also enhanced the tyrosine phosphorylation and total IR and IRS-1 levels, and activated the PI3K/AKT pathway in NRK-52E cells. CONCLUSION: EC and DHBA regulate the renal glucose homeostasis by modulating both glucose uptake and production, and strengthen the insulin signaling by activating key proteins of that pathway in NRK-52E cells.