Natural Products and Diabetes: (-)-Epicatechin and Mechanisms Involved in the Regulation of Insulin Sensitivity.

Type 2 diabetes (T2D) is a disease that occurs when cells do not respond normally to insulin, a condition called insulin resistance, which leads to high blood glucose levels. Although it can be treated pharmacologically, dietary habits beyond carbohydrate restriction can be highly relevant in the management of T2D. Emerging evidence supports the possibility that natural products (NPs) could contribute to managing blood glucose or counteract the undesirable effects of hyperglycemia and insulin resistance. This chapter summarizes the relevant preclinical evidence involving the flavonoid (-)-epicatechin (EC) in the optimization of glucose homeostasis, reducing insulin resistance and/or diabetes-associated disorders. Major effects of EC are observed on (i) intestinal functions, including digestive enzymes, glucose transporters, microbiota, and intestinal permeability, and (ii) redox homeostasis, including oxidative stress and inflammation. There is still a need for further clinical studies to confirm the in vitro and rodent data, allowing recommendations for EC, particularly in prediabetic and T2D patients. The collection of similar data and the lack of clinical evidence for EC is also applicable to other NPs.

( -)-Epicatechin and cardiometabolic risk factors: a focus on potential mechanisms of action.

This review summarizes experimental evidence on the beneficial effects of ( -)-epicatechin (EC) attenuating major cardiometabolic risk factors, i.e., dyslipidemias, obesity (adipose tissue dysfunction), hyperglycemia (insulin resistance), and hypertension (endothelial dysfunction). Studies in humans are revised and complemented with experiments in animal models, and cultured cells, aiming to understand the molecular mechanisms involved in EC-mediated effects. Firstly, an assessment of EC metabolism gives relevance to both conjugated-EC metabolites product of host metabolism and microbiota-derived species. Integration and analysis of results stress the maintenance of redox homeostasis and mitigation of inflammation as relevant processes associated with cardiometabolic diseases. In these processes, EC appears having significant effects regulating NADPH oxidase (NOX)-dependent oxidant production, nitric oxide (NO) production, and energy homeostasis (mitochondrial biogenesis and function). The potential participation of cell membranes and membrane-bound receptors is also discussed in terms of direct molecular action of EC and EC metabolites reaching cells and tissues.

A Decreased Response to Resistin in Mononuclear Leukocytes Contributes to Oxidative Stress in Nonalcoholic Fatty Liver Disease.

BACKGROUND: Deregulation of immune response and oxidative stress contribute to nonalcoholic fatty liver disease (NAFLD) pathogenesis. Resistin is a physiological modulator of inflammation and redox homeostasis of different cell types. Increased resistin serum concentration and the direct association between resistin hepatic expression and NAFLD severity suggest that resistin participates in NAFLD pathogenesis. AIMS: To evaluate resistin-induced regulation of redox homeostasis in mononuclear leukocytes from NAFLD patients and controls. METHODS: We evaluated basal and resistin-mediated modulation of reactive oxygen species (ROS) and glutathione content by flow cytometry, and antioxidant enzyme activities by spectrophotometry. RESULTS: Peripheral blood mononuclear cells (PBMC) from NAFLD patients showed higher ROS content and glutathione peroxidase activity and lower glutathione content, superoxide dismutase and glutathione reductase activities than control PBMC. Resistin decreased ROS levels and superoxide dismutase activity and increased glutathione reductase and catalase activities in PBMC from controls but not from patients. Resistin decreased glutathione content in PBMC from control and NAFLD patients, with greater effect on patient cells. Basal and resistin-modulated ROS levels were directly associated with obesity-related risk factors for NAFLD. Hepatic myeloid cells and T-lymphocytes from NAFLD patients showed higher basal ROS content than cells from controls. Resistin decreased ROS levels in hepatic T-lymphocytes from controls but not from patients. CONCLUSIONS: Resistin regulates redox homeostasis in mononuclear leukocytes. A decreased response to resistin in leukocytes from NAFLD patients is associated with an impaired redox homeostasis.

(-)-Epicatechin protects thoracic aortic perivascular adipose tissue from whitening in high-fat fed mice.

High adipose tissue (AT) accumulation in the body increases the risk for many metabolic and chronic diseases. This work investigated the capacity of the flavonoid (-)-epicatechin to prevent undesirable modifications of AT in mice fed a high-fat diet. Studies were focused on thoracic aorta perivascular AT (taPVAT), which is involved in the control of blood vessel tone, among other functions. Male C57BL/6J mice were fed for 15 weeks a high-fat diet with or without added (-)-epicatechin (20 mg per kg body weight per d). In high-fat diet fed mice, (-)-epicatechin supplementation: (i) prevented the expansion of taPVAT, (ii) attenuated the whitening of taPVAT (according to the adipocyte morphology, diameter, and uncoupling-protein 1 (UCP-1) levels) and (iii) blunted the increase in plasma glucose and cholesterol. The observed taPVAT modifications were not associated with alterations in the aorta wall thickness, aorta tumor necrosis factor-alpha (TNF-α) and NADPH-oxidase 2 (NOX2) expression, and endothelial nitric oxide synthase (eNOS) phosphorylation levels. In summary, our results indicate (-)-epicatechin as a relevant bioactive protecting from the slow and silent development of metabolic and chronic diseases as they are associated with excessive fat intake.

Ellagic acid protects Caco-2 cell monolayers against inflammation-induced permeabilization.

Chronic intestinal inflammation involves a cycle of oxidative stress, activation of redox sensitive transcription factors, and barrier permeabilization. The latter can lead to systemic inflammation and its associated co-morbidities. Diet can play a major role in the modulation of intestinal inflammation. Among plant bioactives, ellagic acid (EA) was reported to inhibit inflammatory bowel disease in animal models. This work investigated the mechanisms by which EA inhibits tumor necrosis factor alpha (TNFα)-induced inflammation, oxidative stress, and loss of barrier integrity. Caco-2 cells differentiated into an intestinal epithelial cell monolayer were incubated with TNFα (10 ng/ml), in the presence of different EA concentrations. TNFα triggered interleukin (IL) 6 and 8 release into the medium, which was inhibited by EA in a dose-dependent manner (IC50 = 17.3 µM for IL-6). TNFα also led to: i) increased ICAM-1 and NLRP3 expression; ii) loss of epithelial barrier function; iii) increased oxidant production from NOX and mitochondrial origin; iv) NF-κB and ERK1/2 activation; and v) increased MLCK gene expression and MLC phosphorylation. EA (10-40 µM) inhibited all these adverse effects of TNFα. EA mainly acted through NF-κB and ERK1/2 inhibition, breaking the cycle of inflammation, oxidative stress, redox-sensitive pathway (e.g. NF-κB, ERK1/2) activation and intestinal permeabilization. This suggests that consumption of EA, via foods or supplements, may afford a strategy to mitigate intestinal inflammation and its associated co-morbidities.

(-)-Epicatechin administration protects kidneys against modifications induced by short-term l-NAME treatment in rats.

The aim of this work was to evaluate the protective effects of (-)-epicatechin on the kidneys of NO-deprived rats. Male Sprague Dawley rats were divided into three groups: control (C), receiving water and standard diet; l-NAME (L), receiving a solution of N(ω)-nitro-l-arginine methyl ester (l-NAME) (360 mg l-1 in water) as a beverage and standard diet; and l-NAME-(-)-epicatechin (LE), receiving l-NAME solution as a beverage and standard diet supplemented with (-)-epicatechin (4 g kg-1 diet). The L-group showed altered kidney function parameters, evaluated based on plasma urea and creatinine. In parallel, kidney oxidative stress markers, i.e. superoxide anion production, malondialdehyde content, and 3-nitrotyrosine protein adducts, were significantly increased in the L group. In addition, l-NAME treatment induced modifications in kidney NO bioavailability determinants: increased expression of NOX subunits (p47phox, gp91phox, NOXO1, and NOX4) and lowered NOS activity. (-)-Epicatechin administration restored kidney function parameters, oxidative stress markers, expression of p47phox, gp91phox, and NOX4 and NOS activity to control values. These results indicate that (-)-epicatechin can mitigate NO-mediated impairment of kidney function, in part due to its capacity to modulate NOXs, NOSs, and consequently oxidative stress, and NO bioavailability.

Dietary (-)-epicatechin affects NF-κB activation and NADPH oxidases in the kidney cortex of high-fructose-fed rats.

Inflammation involves the activation of redox-sensitive transcription factors, e.g., nuclear factor κB (NF-κB). Administration of (-)-epicatechin to high-fructose-fed rats prevented NF-κB activation and up-regulation of the NADPH oxidase 4 (NOX4) in the kidney cortex. These results add mechanistic insights into the action of (-)-epicatechin diminishing inflammatory responses.

Effects of quercetin on heart nitric oxide metabolism in l-NAME treated rats.

This study investigated the effects of a quercetin-supplemented diet on the biochemical changes installed in the heart of NO-deficient rats in terms of oxidants production and NO bioavailability determinants. Sprague-Dawley rats were subjected to Nω-nitro-l-arginine methyl ester (l-NAME) treatment (360 mg/L l-NAME in the drinking water, 4 d) with or without supplementation with quercetin (4 g/kg diet). l-NAME administration led to increased blood pressure (BP) (30%), decreased nitric oxide synthase (NOS) activity (50%), and increases in NADPH oxidase (NOX)-dependent superoxide anion production (60%) and p47phox protein level (65%). The co-administration of quercetin prevented the increase in BP and the activation of NOX but did not modify the decrease in NOS activity caused by l-NAME. In addition, quercetin affected oxidative stress parameters as glutathione oxidation, and the activities of oxidant detoxifying enzymes superoxide dismutase, glutathione peroxidase, and catalase. Thus, quercetin administration counteracts l-NAME effects on NO bioavailability determinants in vivo, essentially through controlling NOX-mediated superoxide anion production.

Fructose increases corticosterone production in association with NADPH metabolism alterations in rat epididymal white adipose tissue.

Metabolic syndrome is an array of closely metabolic disorders that includes glucose intolerance/insulin resistance, central obesity, dyslipidemia, and hypertension. Fructose, a highly lipogenic sugar, has profound metabolic effects in adipose tissue, and has been associated with the etiopathology of many components of the metabolic syndrome. In adipocytes, the enzyme 11 ß-HSD1 amplifies local glucocorticoid production, being a key player in the pathogenesis of central obesity and metabolic syndrome. 11 ß-HSD1 reductase activity is dependent on NADPH, a cofactor generated by H6PD inside the endoplasmic reticulum. Our focus was to explore the effect of fructose overload on epididymal white adipose tissue (EWAT) machinery involved in glucocorticoid production and NADPH and oxidants metabolism. Male Sprague-Dawley rats fed with a fructose solution (10% (w/v) in tap water) during 9 weeks developed some characteristic features of metabolic syndrome, such as hypertriglyceridemia, and hypertension. In addition, high levels of plasma and EWAT corticosterone were detected. Activities and expressions of H6PD and 11 ß-HSD1, NAPDH content, superoxide anion production, expression of NADPH oxidase 2 subunits, and indicators of oxidative metabolism were measured. Fructose overloaded rats showed an increased potential in oxidant production respect to control rats. In parallel, in EWAT from fructose overloaded rats we found higher expression/activity of H6PD and 11 ß-HSD1, and NADPH/NADP+ ratio. Our in vivo results support that fructose overload installs in EWAT conditions favoring glucocorticoid production through higher H6PD expression/activity supplying NADPH for enhanced 11 ß-HSD1 expression/activity, becoming this tissue a potential extra-adrenal source of corticosterone under these experimental conditions.

Seasonal and ontogenetic changes modulate oxygen consumption and antioxidant defenses in the cutlassfish Trichiurus lepturus (Pisces, Trichiuridae).

Several oxidative stress markers and liver oxygen consumption were measured in different tissues of the marine fish Trichiurus lepturus in late summer and late winter, as well as in juveniles and adult females. Oxygen consumption in liver, superoxide dismutase (SOD) and catalase (CAT) activity in liver, red cells, lens and roe, vitamin E, ubiquinol10, ß-carotene in liver, red cells, and roe, as well as contents of reduced glutathione (GSH) and lipoperoxidation (TBARS) in red cells were evaluated. Regarding ontogeny, compared to adult fish, juveniles showed significant higher SOD activity in liver and lens, as well as higher liver contents of vitamin E. In contrast, adult females showed higher contents of vitamin E in roe, ubiquinol10 in liver and roe, and higher GSH levels in red cells, while the other markers remained unchanged. Regarding seasonal changes, no differences were detected in adult females for liver CAT and ubiquinol10, CAT in roe, vitamin E in roe and in red cells, liver and red cell ubiquinol10, and in GSH in red cells. However, and coinciding with the spawning period of late summer, liver oxygen consumption, SOD and CAT activity and ubiquinol10 contents in roe and SOD activity in red cells, and red cell TBARS contents were higher compared to late winter. These temporal antioxidant adjustments of Trichiurus lepturus seem to be parallel to the higher oxygen consumption typical of juvenile forms and also to the intense spawning and foraging activities of adult females in late summer.

LPS-induced renal inflammation is prevented by (-)-epicatechin in rats.

This work investigated the capacity of (-)-epicatechin to prevent the renal damage induced by LPS administration in rats. Male Sprague Dawley rats were fed for 4 days a diet without or with supplementation with (-)-epicatechin (80mg/kg BW/d), and subsequently i.p. injected with lipopolysaccharide (LPS). Six hours after injection, LPS-treated rats exhibited increased plasma creatinine and urea levels as indicators of impaired renal function. The renal cortex of the LPS-treated rats showed: i) increased expression of inflammatory molecules (TNF-α, iNOS and IL-6); ii) activation of several steps of NF-κB pathway; iii) overexpression of TLR4, and iv) higher superoxide anion production and lipid peroxidation index in association with increased levels of gp91phox and p47phox (NOX2) and NOX4. Pretreatment with dietary (-)-epicatechin prevented the adverse effects of LPS challenge essentially by inhibiting TLR4 upregulation and NOX activation and the consequent downstream events, e.g. NF-kB activation.

Modifications in nitric oxide and superoxide anion metabolism induced by fructose overload in rat heart are prevented by (-)-epicatechin.

Fructose overload promotes functional and metabolic derangements in humans and in animal experimental models. Evidence suggests that dietary flavonoids have the ability to prevent/attenuate the development of metabolic diseases. In this work we investigated the effects of (-)-epicatechin on the modifications induced by fructose overload in the rat heart in terms of nitric oxide and superoxide metabolism. Male Sprague Dawley rats received 10% (w/v) fructose in the drinking water for 8 weeks, with or without (-)-epicatechin (20 mg per kg body weight per day) in the rat chow diet. These conditions of fructose overload did not lead to overt manifestations of heart hypertrophy or tissue remodeling. However, biochemical and molecular changes were observed and could represent the onset of functional alterations. (-)-Epicatechin prevented a compromised NO bioavailability and the development of oxidative stress produced by fructose overload essentially acting on superoxide anion metabolism. In this line, the increase in superoxide anion production, the overexpression of NOX2 subunit p47phox and of NOX4, the decrease in superoxide dismutase activity, and the higher oxidized/reduced glutathione ratio installed by fructose overload were absent in the rats receiving (-)-epicatechin. These results support the hypothesis that diets rich in (-)-epicatechin could prevent the onset and progression of heart dysfunctions associated with metabolic alterations.

Dietary (-)-epicatechin mitigates oxidative stress, NO metabolism alterations, and inflammation in renal cortex from fructose-fed rats.

High fructose consumption has been associated to deleterious metabolic conditions. In the kidney, high fructose causes renal alterations that contribute to the development of chronic kidney disease. Evidence suggests that dietary flavonoids have the ability to prevent/attenuate risk factors of chronic diseases. This work investigated the capacity of (-)-epicatechin to prevent the renal damage induced by high fructose consumption in rats. Male Sprague Dawley rats received 10% (w/v) fructose in the drinking water for 8 weeks, with or without supplementation with (-)-epicatechin (20mg/kg body weight/d) in the rat chow diet. Results showed that, in the presence of mild proteinuria, the renal cortex from fructose-fed rats exhibited fibrosis and decreases in nephrin, synaptopodin, and WT1, all indicators of podocyte function in association with: (i) increased markers of oxidative stress; (ii) modifications in the determinants of NO bioavailability, i.e., NO synthase (NOS) activity and expression; and (iii) development of a pro-inflammatory condition, manifested as NF-κB activation, and associated with high expression of TNFα, iNOS, and IL-6. Dietary supplementation with (-)-epicatechin prevented or ameliorated the adverse effects of high fructose consumption. These results suggest that (-)-epicatechin ingestion would benefit when renal alterations occur associated with inflammation or metabolic diseases.

(-)-Epicatechin reduces blood pressure increase in high-fructose-fed rats: effects on the determinants of nitric oxide bioavailability.

This work investigated the blood pressure (BP)-lowering effect of the flavanol (-)-epicatechin in a model of metabolic syndrome. Rats were fed a regular chow diet without (Control) or with 10% (w/v) fructose in the drinking water (high fructose, HF) for 8 weeks. A subgroup of the HF-fed rats was supplemented with (-)-epicatechin 20 mg/kg body weight (HF-EC). Dietary (-)-epicatechin reverted the increase in BP caused by the fructose treatment. In aorta, superoxide anion production and the expression of the NADPH oxidase (NOX) subunits p47(phox) and p22(phox) were enhanced in the HF-fed rats. The increase was prevented by (-)-epicatechin. Similar profile was observed for NOX4 expression. The activity of aorta nitric oxide synthase (NOS) was increased in the HF group and was even higher in the HF-EC rats. These effects were paralleled by increased endothelial NOS phosphorylation at the activation site Ser1177. Among the more relevant mitogen-activated protein kinase pathways in vascular tissue, c-Jun-N-terminal kinase was shown to be activated in the aorta of the HF-fed rats, and (-)-epicatechin supplementation mitigated this activation. Thus, the results suggest that dietary (-)-epicatechin supplementation prevented hypertension in HF-fed rats, decreasing superoxide anion production and elevating NOS activity, favoring an increase in NO bioavailability.

Interactions of flavan-3-ols and procyanidins with membranes: mechanisms and the physiological relevance.

Flavonoids are a type of phenolic compound widely present in edible plants. A great number of health benefits have been ascribed to flavonoid consumption in the human population. However, the molecular mechanisms involved in such effects remain to be identified. The flavan-3-ols (-)-epicatechin and (+)-catechin, and their related oligomers (procyanidins) have been thoroughly studied because of their capacity to interact with cell membranes. Starting with these interactions, procyanidins could modulate multiple biochemical processes, such as enzyme activities, receptor-ligand binding, membrane-initiated cell signaling, and molecule transport across membranes. This review focuses on molecular aspects of procyanidin interactions with membrane lipid components, and the resulting protection of the membranes against mechanical and/or oxidative damage, resulting in the maintenance of cell functions.

(-)-Epicatechin prevents alterations in the metabolism of superoxide anion and nitric oxide in the hearts of L-NAME-treated rats.

The aim of this work was to evaluate the effects of (-)-epicatechin administration in the heart of a rat model with reduced NO production that follows a short-term treatment with L-NAME. Sprague-Dawley rats were treated for 4 d with L-NAME in the absence or presence of (-)-epicatechin in the diet. The redox status in cardiac tissue was improved by (-)-epicatechin administration. L-NAME treatment induced a decrease in NO synthase activity (-62%, p<0.05) and an increase in NADPH-dependent superoxide anion production (+300%, p<0.05) that were totally prevented by (-)-epicatechin administration. These effects of (-)-epicatechin were associated with a higher endothelial NO synthase phosphorylation at an activation site and a reduced expression of the regulatory subunit, p47(phox), suggesting the involvement of posttranslational mechanisms in (-)-epicatechin action. Thus, the (-)-epicatechin treatment would restore NO steady state levels in vivo through effects on both, its synthesis and degradation via the reaction with superoxide anion. The fact that (-)-epicatechin is commonly present in human diet makes this compound a reasonable explanation for the positive cardiovascular effects of a high consumption of fruits and vegetables.

Flavanols and vascular health: molecular mechanisms to build evidence-based recommendations.

Observational studies as well as public awareness and ancient medicine identify tea, wine and cocoa as healthy foods. Further compilations of epidemiological data reinforce the healthy properties of the grape, tea and cocoa derived foods and drinks made from, especially when considering cardiovascular disease, some cancers and other inflammation-related pathologies. Flavanols have emerged as bioactives responsible for such health effects, and flavanol-rich foods have been used in clinical studies. Results of these studies show a major participation of flavanols in mechanisms positively affecting endpoints of cardiovascular disease, i.e. hypertension and vascular function. In line, based on the chemistry (bioavailability and molecular structure of flavanol and target entities) several physiological mechanisms have been described backing the epidemiological and clinical studies. In summary, the discussion for defining evidence-based recommendations for flavanols is based on: a) the extensive research done and the positive results obtained support the incorporation of flavanol-rich foods as part of a healthy diet, this is a cost-effective action to ameliorate silent undesirable conditions as it is chronic inflammation; b) the fact that cardiovascular health seems especially sensitive to the beneficial effects of flavanols: based on clinical and mechanistic studies showing that certain flavanols, favor NO production; and c) the increasing technical possibilities to evaluate flavanols in foods and biological samples. Supported by UBACyT 20020120100177, CONICET PIP-20110100752, and ANPCyT PICT 2012/0765.

Procyanidins can interact with Caco-2 cell membrane lipid rafts: involvement of cholesterol.

Large procyanidins (more than three subunits) are not absorbed at the gastrointestinal tract but could exert local effects through their interactions with membranes. We previously showed that hexameric procyanidins (Hex), although not entering cells, interact with membranes modulating cell signaling and fate. This paper investigated if Hex, as an example of large procyanidins, can selectively interact with lipid rafts which could in part explain its biological actions. This mechanism was studied in both synthetic membranes (liposomes) and Caco-2 cells. Hex promoted Caco-2 cell membrane rigidification and dehydration, effects that were abolished upon cholesterol depletion with methyl-ß-cyclodextrin (MCD). Hex prevented lipid raft structure disruption induced by cholesterol depletion/redistribution by MCD or sodium deoxycholate. Supporting the involvement of cholesterol-Hex bonding in Hex interaction with lipid rafts, the absence of cholesterol markedly decreased the capacity of Hex to prevent deoxycholate- and Triton X-100-mediated disruption of lipid raft-like liposomes. Stressing the functional relevance of this interaction, Hex mitigated lipid raft-associated activation of the extracellular signal-regulated kinases (ERK) 1/2. Results support the capacity of a large procyanidin (Hex) to interact with membrane lipid rafts mainly through Hex-cholesterol bondings. Procyanidin-lipid raft interactions can in part explain the capacity of large procyanidins to modulate cell physiology.

(-)-Epicatechin reduces blood pressure and improves vasorelaxation in spontaneously hypertensive rats by NO-mediated mechanism.

Studies in humans have found consumption of certain flavanoid-containing foods to be associated with improvement in endothelial function and with reduction of blood pressure (BP). (-)-Epicatechin is a compound representative of the flavanols (a subfamily of flavonoids), abundant in cocoa seeds, which is preserved during the industrialization process to chocolate. The antihypertensive effect of dietary (-)-epicatechin was investigated on spontaneously hypertensive rats (SHRs). Consumption of (-)-epicatechin-supplemented diet (3 g (-)-epicatechin/kg diet) decreased BP in SHR by 27 and 23 mm Hg on days 2 and 6, respectively. On day 6, a 173% increase of nitric oxide synthase (NOS) activity was observed in the aorta of EPI-SHR as compared to nonsupplemented SHR (P < 0.05). Responses to acetylcholine (ACh) were then examined in femoral arteries in the absence and the presence of L-NAME, a nonselective NOS inhibitor, to assess the ACh-mediated relaxation ascribed to NO-dependent and -independent mechanisms. Acetylcholine-induced endothelium-dependent relaxation in the femoral artery was significantly higher in EPI-SHR than in SHR, with a predominance of the NO-dependent component of this relaxation. The endothelium-independent relaxation, assayed by using the NO donor sodium nitroprusside, resulted in nonsignificant difference in the three experimental groups, demonstrating an unaffected function of vascular smooth muscle cells. These results give further support to the concept that (-)-epicatechin can modulate BP in hypertension by increasing NO levels in the vasculature.