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• Incorporation of several aliphatic chains to (-)-Epicatechin contributed to increasing the affinity towards GPER. • Energetic studies showed that Epi, Epi-4-prop and Epi-5-prop showed better thermodynamic to GPER than Epi-Ms and Epi-prop. • Epi, Epi-4-prop and Epi-5-prop share a high number of common residues in GPER-ligand complex stabilization. • The coupling of Epi, Epi-4-prop and Epi-5-prop to GPER was linked to decreases in conformational flexibility. (-)-Epicatechin (Epi) has been demonstrated to activate pathways involved in GPER-stimulated nitric oxide (NO) production via endothelial NO synthase, known as the eNOS/NO pathway. Previous studies combining synthesis of four Epi derivatives demonstrated that Epi and Epi-prop, Epi-4-prop and Epi-5-prop were able to bind GPER by acting as GPER agonists, whereas docking studies allowed observation of structural details of the binding of these derivatives at the GPER binding site. However, due to the nature of past studies, the theoretical methods employed did not allow observation of structural and energetic details linked to ligand binding at the GPER binding site. In this contribution, we explore the structural and energetic changes coupling the binding of Epi and its four derivatives to GPER. To this end, MD simulations on the microsecond scale (1 μs) with an MMGBSA approach were used for each GPER-ligand complex. Energetic analysis demonstrated that incorporation of several aliphatic chains to Epi contributed to increasing the affinity towards the GPER binding site, thus helping to explain the experimental evidence. Structural analysis demonstrated that Epi, Epi-4-prop and Epi-5-prop share more similar interactions at GPER binding sites with similar conformational behavior than with Epi-prop and Epi-Ms. However, Epi-prop had additional residues that could explain its different but related biological effects. [ABSTRACT FROM AUTHOR]

To potentially identify proteins that interact (i.e. bind) and may contribute to mediate (−)-epicatechin (Epi) responses in endothelial cells we implemented the following strategy: 1) synthesis of novel Epi derivatives amenable to affinity column use, 2) in silico molecular docking studies of the novel derivatives on G protein-coupled estrogen receptor (GPER), 3) biological assessment of the derivatives on NO production, 4) implementation of an immobilized Epi derivative affinity column and, 5) affinity column based isolation of Epi interacting proteins from endothelial cell protein extracts. For these purposes, the Epi phenol and C3 hydroxyl groups were chemically modified with propargyl or mesyl groups. Docking studies of the novel Epi derivatives on GPER conformers at 14 ns and 70 ns demostrated favorable thermodynamic interactions reaching the binding site. Cultures of bovine coronary artery endothelial cells (BCAEC) treated with Epi derivatives stimulated NO production via Ser1179 phosphorylation of eNOS, effects that were attenuated by the use of the GPER blocker, G15. Epi derivative affinity columns yielded multiple proteins from BCAEC. Proteins were electrophoretically separated and inmmunoblotting analysis revealed GPER as an Epi derivative binding protein. Altogether, these results validate the proposed strategy to potentially isolate and identify novel Epi receptors that may account for its biological activity. [ABSTRACT FROM AUTHOR]

The production of nitric oxide (NO) by nitric oxide synthases (NOS) depends on the bioavailability of L -arginine as NOS competes with arginase for this common substrate. As arginase activity increases, less NO is produced and adverse cardiovascular consequences can emerge. (-)-Epicatechin (EPI), the most abundant flavonoid in cacao, has been reported to stimulate endothelial and neuronal NOS expression and function leading to enhanced vascular function and cardioprotective effects. However, little is known about the effects of EPI on myocardial arginase activity. The aim of the present study was to determine if EPI is able to interact and modulate myocardial arginase and NOS expression and activity. For this purpose, in silico modeling, in vitro activity assays and a rat model of ischemia/reperfusion injury were used. In silico and in vitro results demonstrate that EPI can interact with arginase and significantly decrease its activity. In vivo , 10 days of EPI pretreatment reduces ischemic myocardium arginase expression while increasing NOS expression and phosphorylation levels. Altogether, these results may partially account for the cardioprotective effects of EPI. [ABSTRACT FROM AUTHOR]

Introduction Multiple components of the dystrophin-associated protein complex (DAPC) are expressed in numerous tissues including the brain. Members of the DAPC and dysbindin are abnormally expressed in the brain of Duchenne Muscular Dystrophy (DMD) patients, which has been associated with cognitive impairments. However, little is known about the expression pattern of individual members of the DAPC in animal models of DMD and their relationship with dysbindin. Methods Ten mdx mice were randomly allocated into a control and intervention group [(−)-epicatechin (Epi) 1 mg/kg/day for four weeks] and results compared to a wild-type mice. After sacrifice, brain pre-frontal cortices were collected for Western blotting and immunoprecipitation assays, and sagittal sections processed for immunohistochemistry. Results Epi promotes a partial recovery of DAPC members [α1-Syntrophin, sarcoglycans (SG), dystrophin 71 (Dp71)], dysbindin, and utrophin protein levels. Epi also appears to restore the association of DAPC between dysbindin, and utrophin with Dp71 and ε-SG. Co-immunostaining evidence increased protein levels of dysbindin, dystrophin, and ε-SG and their colocalization. Conclusions Altogether, results suggest that Epi is capable of restoring pre-frontal cortex DAPC and dysbindin levels of mdx mice towards that of healthy brains. The functional implications of such studies warrant further investigation. [ABSTRACT FROM AUTHOR]

In this study, we demonstrate that (-)-epicatechin (Epi), a cacao flavanol, induces the browning of fat by promoting mitochondrial biogenesis, enhancing indicators of mitochondrial structure and function, increasing fatty acid metabolism and upregulating the expression of brown adipose tissue-specific proteins in a high-fat diet mouse model of obesity and in cultured human adipocytes. Epi treatment significantly improved mitochondrial function, as measured by citrate synthase activity, and also reduced protein acetylation of total and specific regulators in both adipose tissue and human adipocytes. Browning of fat via Epi was evidenced by the increased expression of key thermogenic genes, phosphorylation of upstream regulators of fatty acid oxidation, and reduced triglyceride levels. Properly designed clinical trials are needed to explore the potential of Epi as an agent that promotes the browning of fat. [ABSTRACT FROM AUTHOR]

Background Cardiometabolic disruptions such as insulin resistance, obesity, high blood pressure, hyperglycemia, and dyslipidemias, are known to increase the risk for cardiovascular and metabolic diseases such as type 2 diabetes mellitus and atherosclerosis. Several screening tools for assessing cardiometabolic risk have been developed including the TG/HDLc ratio, which has been, demonstrated to possess a strong association with insulin resistance and coronary disease. Dietary modifications, together with regular moderate exercise have proven to be effective in attenuating cardiometabolic disruptions. However, they often exhibit poor long-term patient compliance. Nutraceutics, including (−)-epicatechin (EPI), have gained increasing interest as coadjuvant effective and safe therapies that are able to attenuate hypertension, hyperglycemia, hyperinsulinemia, hypertriglyceridemia and hypoalphalipoproteinemia. Methods The aims of this study were: 1) to compare the in vitro effect of EPI vs. (+)-catechin on fructose induced triglyceride accumulation and mitochondrial function in Hep2 cells in culture, 2) to evaluate the efficacy of EPI treatment in reducing fasting blood triglycerides and improving the TG/HDLc ratio in hypertriglyceridemic patients with a total daily dose of 100 mg of EPI. Secondary clinical variables included total cholesterol, LDLc, fructosamine, glucose, insulin, and high sensitivity C-reactive protein blood levels. Results and conclusion Our results provide preliminary evidence as to favorable effects of EPI on glycemia homeostasis, lipid profile and systemic inflammation such bioactive actions are not class-effects (i.e. limited to their antioxidant potential) but instead, may result from the specific activation of associated downstream signaling pathways since catechin has no effects. [ABSTRACT FROM AUTHOR]

(-)-Epicatechin (EPI) is cardioprotective in the setting of ischemia/reperfusion (IR) injury and doxycycline (DOX) is known to preserve cardiac structure/function after myocardial infarction (MI). The main objective of this study was to examine the effects of EPI and DOX co-administration on MI size after IR injury and to determine if cardioprotection may involve the mitigation of mitochondrial swelling. For this purpose, a rat model of IR was used. Animals were subjected to a temporary 45 min occlusion of the left anterior descending coronary artery. Treatment consisted of a single or double dose of EPI (10 mg/kg) combined with DOX (5 mg/kg). The first dose was given 15 min prior to reperfusion and the second 12 h post-MI. The effects of EPI +/− DOX on mitochondrial swelling (i.e. mPTP opening) were determined using isolated mitochondria exposed to calcium overload and data examined using isobolographic analysis. To ascertain for the specificity of EPI effects on mitochondrial swelling other flavonoids were also evaluated. Single dose treatment reduced MI size by ~46% at 48 h and 44% at three weeks. Double dosing evidenced a synergistic, 82% reduction at 3 weeks. EPI plus DOX also inhibited mitochondrial swelling in a synergic manner thus, possibly accounting for the cardioprotective effects whereas limited efficacy was observed with the other flavonoids. Given the apparent lack of toxicity in humans, the combination of EPI and DOX may have clinical potential for the treatment of myocardial IR injury. [ABSTRACT FROM AUTHOR]

Background Targeting the mitochondria during ischemia/reperfusion (IR) can confer cardioprotection leading to improved clinical outcomes. The cardioprotective potential of (-)-epicatechin (EPI) during IR via modulation of mitochondrial function was evaluated. Methods and results Ischemia was induced in rats via a 45min occlusion of the left anterior descending coronary artery followed by 1h, 48h, or 3week reperfusion. EPI (10mg/kg) was administered IV 15min prior to reperfusion for the single dose group and again 12h later for the double dose group. Controls received water. Experiments also utilized cultured neonatal rat ventricular myocytes (NRVM) and myoblasts. A single dose of EPI reduced infarct size by 27% at 48h and 28% at 3week. Double dose treatment further decreased infarct size by 80% at 48h, and 52% by 3weeks. The protective effect of EPI on mitochondrial function was evident after 1h of reperfusion when mitochondria demonstrated less respiratory inhibition, lower mitochondrial Ca2+ load, and a preserved pool of NADH that correlated with higher tissue ATP levels. Mechanistic studies in NRVM revealed that EPI acutely stimulated maximal rates of respiration, an effect that was blocked by inhibitors of the mitochondrial pyruvate carrier, nitric oxide synthase, or soluble guanylyl cyclase. In myoblasts, knockdown of components of the mitochondrial pyruvate carrier blocked EPI-induced respiratory stimulation. Conclusions IV EPI confers cardioprotection via preservation of mitochondrial function potentially through enhanced substrate provision. These provocative results document a novel mechanism of a natural product with potential clinical utility. [ABSTRACT FROM AUTHOR]

Abstract: The consumption of cacao-derived products, particularly in the form of dark chocolate is known to provide beneficial cardiovascular effects in normal individuals and in those with vascular dysfunction (reduced nitric oxide [NO] bioavailability and/or synthesis). Upstream mechanisms by which flavonoids exert these effects are poorly understood and may involve the participation of cell membrane receptors. We previously demonstrated that the flavanol (−)-epicatechin (EPI) stimulates NO production via Ca+2-independent eNOS activation/phosphorylation. We wished to investigate the plausible participation of a cell surface receptor using a novel cell-membrane impermeable EPI–Dextran conjugate (EPI–Dx). Under Ca2+-free conditions, human coronary artery endothelial cells (HCAEC) were treated for 10min with EPI or EPI–Dx at equimolar concentrations (100nM). Results demonstrate that both EPI and EPI–Dx induced the phosphorylation/activation of PI3K, PDK-1, AKT and eNOS. Interestingly, EPI–Dx effects were significantly higher in magnitude than those of EPI alone. The capacity of EPI–Dx to stimulate cell responses supports the existence of an EPI cell membrane receptor mediating eNOS activation. [Copyright &y& Elsevier]

Abstract: Overweight and obesity have been associated with increase in cardiometabolic risk. Therapeutics include lifestyle changes and/or pharmacologic agents. However, such interventions are often limited by poor compliance and/or significant side effects. The consumption of certain dietary products, such as cocoa, exerts positive effects on cardiometabolic risk factors. (−)-Epicatechin (EPI), the most abundant flavonoid in cacao has been reported to replicate such effects. However its mechanisms of action have not been fully elucidated.In a rat model of high-fat diet-induced obesity and its associated crdiometabolic risk factors, we administered 1mg/kg of EPI, by gavage, for 2 weeks. Endpoints included weight-gain, glycemia, triglyceridemia, and systolic blood pressure. We also assessed food intake and fecal excretion. Mitochondrial function and structure related proteins were measured by Westerns.Obesity, hyperglycemia, hypertriglyceridemia, and systolic hypertension were developed after the administration of the high-fat diet for five weeks. EPI significantly decreased the rate of weight gain, glycemia and hypertriglyceridemia. The ratio between energy intake and excretion was not significantly modified by treatment. EPI restored the obesity-induced decreases in the levels of skeletal muscle and abdominal tissue sirtuins (SIRTs), peroxisome proliferator-activated receptor coactivator (PGC-1α), mitofilin, transcription factor A mitochondrial (TFAM), uncoupling protein 1 (UCP1), and deiodinase.EPI treatment yielded beneficial effects on high fat diet-induced endpoints thus may be considered as a potential agent for the treatment of obesity and its cardiometabolic associated abnormalities. Mechanism of action may be attributed to the modulation of cellular/mitochondrial function, thus improving overall metabolism. [Copyright &y& Elsevier]

Sarcopenia is a notable and debilitating age-associated condition. Flavonoids are known for their healthy effects and limited toxicity. The flavanol (-)-epicatechin (Epi) enhances exercise capacity in mice, and Epi-rich cocoa improves skeletal muscle structure in heart failure patients. (-)-Epicatechin may thus hold promise as treatment for sarcopenia. We examined changes in protein levels of molecular modulators of growth and differentiation in young vs. old, human and mouse skeletal muscle. We report the effects of Epi in mice and the results of an initial proof-of-concept trial in humans, where muscle strength and levels of modulators of muscle growth were measured. In mice, myostatin and senescence-associated β-galactosidase levels increase with aging, while those of follistatin and Myf5 decrease. (-)-Epicatechin decreases myostatin and β-galactosidase and increases levels of markers of muscle growth. In humans, myostatin and β-galactosidase increase with aging while follistatin, MyoD and myogenin decrease. Treatment for 7 days with (-)-epicatechin increases hand grip strength and the ratio of plasma follistatin/myostatin. In conclusion, aging has deleterious effects on modulators of muscle growth/differentiation, and the consumption of modest amounts of the flavanol (-)-epicatechin can partially reverse these changes. This flavanol warrants its comprehensive evaluation for the treatment of sarcopenia. [ABSTRACT FROM AUTHOR]

Abstract: Sarcopenia is a notable and debilitating age-associated condition. Flavonoids are known for their healthy effects and limited toxicity. The flavanol (−)-epicatechin (Epi) enhances exercise capacity in mice, and Epi-rich cocoa improves skeletal muscle structure in heart failure patients. (−)-Epicatechin may thus hold promise as treatment for sarcopenia. We examined changes in protein levels of molecular modulators of growth and differentiation in young vs. old, human and mouse skeletal muscle. We report the effects of Epi in mice and the results of an initial proof-of-concept trial in humans, where muscle strength and levels of modulators of muscle growth were measured. In mice, myostatin and senescence-associated β-galactosidase levels increase with aging, while those of follistatin and Myf5 decrease. (−)-Epicatechin decreases myostatin and β-galactosidase and increases levels of markers of muscle growth. In humans, myostatin and β-galactosidase increase with aging while follistatin, MyoD and myogenin decrease. Treatment for 7 days with (−)-epicatechin increases hand grip strength and the ratio of plasma follistatin/myostatin. In conclusion, aging has deleterious effects on modulators of muscle growth/differentiation, and the consumption of modest amounts of the flavanol (−)-epicatechin can partially reverse these changes. This flavanol warrants its comprehensive evaluation for the treatment of sarcopenia. [Copyright &y& Elsevier]

Abstract: Background: Type 2 diabetes (T2D) and heart failure (HF) are associated with high levels of skeletal muscle (SkM) oxidative stress (OS). Health benefits attributed to flavonoids have been ascribed to antioxidation. However, for flavonoids with similar antioxidant potential, end-biological effects vary widely suggesting other mechanistic venues for reducing OS. Decreases in OS may follow the modulation of key regulatory pathways including antioxidant levels (e.g. glutathione) and enzymes such as mitochondrial superoxide dismutase (SOD2) and catalase. Methods: We examined OS-related alterations in SkM in T2D/HF patients (as compared vs. healthy controls) and evaluated the effects of three-month treatment with (−)-epicatechin (Epi) rich cocoa (ERC). To evidence Epi as the mediator of the improved OS profile we examined the effects of pure Epi (vs. water) on SkM OS regulatory systems in a mouse model of insulin resistance and contrasted results vs. normal mice. Results: There were severe alterations in OS regulatory systems in T2D/HF SkM as compared with healthy controls. Treatment with ERC induced recovery in glutathione levels and decreases in the nitrotyrosilation and carbonylation of proteins. With treatment, key transcriptional factors translocate into the nucleus leading to increases in SOD2 and catalase protein expression and activity levels. In insulin resistant mice, there were alterations in muscle OS and pure Epi replicated the beneficial effects of ERC found in humans. Conclusions: Major perturbations in SkM OS can be reversed with ERC in T2D/HF patients. Epi likely mediates such effects and may provide an effective means to treat conditions associated with tissue OS. [Copyright &y& Elsevier]

Abstract: Impaired mitochondrial function represents an early manifestation of endothelial dysfunction and likely contributes to the development of cardiovascular diseases (CVD). The stimulation of mitochondrial function and/or biogenesis is seen as a means to improve the bioenergetic and metabolic status of cells and thus, reduce CVD. In this study we examined the capacity of the flavanol (−)-epicatechin and two novel derivatives to enhance mitochondrial function and protein levels in cultured bovine coronary artery endothelial cells. As nitric oxide production by endothelial cells is suspected in mediating mitochondria effects (including biogenesis), we also examined the dependence of responses on this molecule using an inhibitor of nitric oxide synthase. Results indicate that the flavanol (−)-epicatechin and derivatives are capable of stimulating mitochondrial function as assessed by citrate synthase activity as well as induction of structural (porin, mitofilin) and oxidative phosporylation protein levels (complex I and II). Effects were blocked by the use of the chemical inhibitor of the synthase thus, evidencing a role for nitric oxide in mediating these effects. The results observed indicate that the three agents are effective in enhancing mitochondria function and protein content. The effects noted for (−)-epicatechin may serve to explain the healthy effects on cardiometabolic risk ascribed to the consumption of cocoa products. [Copyright &y& Elsevier]

Endothelial dysfunction (EnD) occurs with aging and endothelial nitric oxide (NO) production by NO synthase (NOS) can be impaired. Low NO levels have been linked to increased arginase (Ar) activity as Ar competes with NOS for L-arginine. The inhibition of Ar activity can reverse EnD and (−)-epicatechin (Epi) inhibits myocardial Ar activity. In this study, through in silico modeling we demonstrate that Epi interacts with Ar similarly to its inhibitor Norvaline (Norv). Using in vitro and in vivo models of aging, we examined Epi and Norv-inhibition of Ar activity and its endothelium-protective effects. Bovine coronary artery endothelial cells (BCAEC) were treated with Norv (10 μM), Epi (1 μM) or the combination (Epi + Norv) for 48 h. Ar activity increased in aged BCAEC, with decreased NO generation. Treatment decreased Ar activity to levels seen in young cells. Epi and Epi + Norv decreased nitrosylated Ar levels by ~25% in aged cells with lower oxidative stress (~25%) (dihydroethidium) levels. In aged cells, Epi and Epi + Norv restored the eNOS monomer/dimer ratio, protein expression levels and NO production to those of young cells. Furthermore, using 18 month old rats 15 days of treatment with either Epi (1 mg/kg), Norv (10 mg/kg) or combo, decreased hypertension and improved aorta vasorelaxation to acetylcholine, blood NO levels and tetra/dihydribiopterin ratios in cultured rat aortic endothelial cells. In conclusion, results provide evidence that inhibiting Ar with Epi reverses aged-related loss of eNOS function and improves vascular function through the modulation of Ar and eNOS protein levels and activity. • In aged endothelium, arginase (Ar) activity is increased, leading to a reduction in NO production. • (−)-Epicatechin (Epi) inhibits Ar activity and modulates L-arginine availability. • Inhibition of Ar with Epi reverses aged-related loss of eNOS function. • Epi and Epi plus Norvaline improves endothelial and vascular function in aged endothelium. [ABSTRACT FROM AUTHOR]

The existing treatments for nonalcoholic steatohepatitis (NASH) are not completely effective. The need for new alternatives without adverse effects and low cost, such as the flavonoid (-)-epicatechin (EC), which has beneficial effects on lipid metabolism and cardiovascular diseases, arises. The objective of this work was to analyze EC effects in the NASH induced by a Paigen-type diet (PD). Mice were administered with (1) normal chow and water, (2) PD + fructose 30% and (3) PD + fructose 30% + EC (1 mg/kg) per gavage during 9 weeks. At the end of each treatment, serum was collected for analysis of the biochemical profile and liver enzymes. The liver was collected for microscopic analysis and for the evaluation of the relative expression of Plin2, Plin3, CD36, adiponectin and UCP2. Results showed that EC reduced weight gain and decreased triglyceride (TG), low-density lipoprotein cholesterol, TG/high-density lipoprotein and the activity of liver enzymes (alanine aminotransferase and alkaline phosphatase), suggesting lower liver damage. The microscopic analysis showed less "balloonization" of the hepatocyte, small drops of lipids, less accumulation of collagen and infiltration of inflammatory cells as compared to nontreated group. Finally, a decrease in the expression of Plin 2 was observed. While CD36 decreased, adiponectin and UCP2 increased. In conclusion, EC improves the biochemical profile, the microscopic characteristics and protein expression. Therefore, it may be a possible therapeutic approach for NASH since it prevents the progression of the hepatic and metabolic damage induced by high-fat diets. [ABSTRACT FROM AUTHOR]