Effects of (−)-epicatechin on molecular modulators of skeletal muscle growth and differentiation

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.     

Synthesis,nitric oxide release,and α-glucosidase inhibition of nitric oxide donating apigenin and chrysin derivatives

α-Glucosidase (AG) play crucial roles in the digestion of carbohydrates. Inhibitors of α-glucosidase (AGIs) are promising candidates for the development of anti-diabetic drugs. Here, five series of apigenin and chrysin nitric oxide (NO)-donating derivatives were synthesised and evaluated for their AG inhibitory activity and NO releasing capacity in vitro. Except for 9a–c, twelve compounds showed remarkable inhibitory activity against α-glucosidase, with potency being better than that of acarbose and 1-deoxynojirimycin. All organic nitrate derivatives released low concentrations of NO in the presence of l-cysteine. Structure activity relationship studies indicated that 5-OH, hydrophobic coupling chain, and carbonyl groups of the coupling chain could enhance the inhibitory activity. Apigenin and chrysin derivatives therefore represents a new class of promising compounds that can inhibit α-glucosidase activity and supply moderate NO for preventing the development of diabetic complications.     

Biological activities of (−)-epicatechin and (−)-epicatechin-containing foods: Focus on cardiovascular and neuropsychological health

Recent studies have suggested that certain (?)-epicatechin-containing foods have a blood pressure-lowering capacity. The mechanisms underlying (?)-epicatechin action may help prevent oxidative damage and endothelial dysfunction, which have both been associated with hypertension and certain brain disorders. Moreover, (?)-epicatechin has been shown to modify metabolic profile, blood's rheological properties, and to cross the blood-brain barrier. Thus, (?)-epicatechin causes multiple actions that may provide unique synergy beneficial for cardiovascular and neuropsychological health. This review summarises the current knowledge on the biological actions of (?)-epicatechin, related to cardiovascular and brain functions, which may play a remarkable role in human health and longevity.     

Killing of Bacillus spores is mediated by nitric oxide and nitric oxide synthase during glycoconjugate–enhanced phagocytosis

Nitric oxide (NO) is a signaling and defense molecule of major importance. NO endows macrophages with bactericidal, cytostatic as well as cytotoxic activity against various pathogens. Bacillus spores can produce serious diseases, which might be attenuated if macrophages were able to kill the spores on contact. Present research was carried out to study whether glycoconjugates stimulated NO and nitric oxide synthase (NOS2) production during phagocytosis killing of Bacillus spores. Murine macrophages exposed to glycoconjugate-treated spores induced NOS2 and NO production that was correlated with high viability of macrophages and killing rate of bacterial spores. Increased levels of inducible NOS2 and NO production by macrophages in presence of glycoconjugates suggested that the latter provide an activation signal directed to macrophages. Glycoconjugates were shown to exert a protective influence, sparing macrophages from spore-induced cell death. In presence of glycoconjugates, macrophages efficiently kill the organisms. Without glycoconjugate activation, murine macrophages were ineffective at killing Bacillus spores. These results suggest that glycoconjugates promote killing of Bacillus spores by blocking spore-induced macrophage cell death, while increasing their activation level and NO and NOS2 production. Glycoconjugates suggest novel antimicrobial approaches to prevention and treatment of infection caused by bacterial spores.     

Does nitric oxide modulate mitochondrial energy generation and apoptosis?

The physiological role of nitric oxide (NO) in the maintenance of vascular tone, in synaptic transmission and in cellular defence is now firmly established. Recent evidence indicates that NO can also affect mitochondrial function. Here, we review findings indicating that NO through its interaction with components of the electron-transport chain might function not only as a physiological regulator of cell respiration, but also to augment the generation of reactive oxygen species by mitochondria, and thereby trigger mechanisms of cell survival or death.     

Effects of (−)-epicatechin on the time course of the expression of perilipins in a diet-induced model of nonalcoholic steatohepatitis

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.     

Synthesis of novel (−)-epicatechin derivatives as potential endothelial GPER agonists: Evaluation of biological effects

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.     

Activation of macrophage nuclear factor-κB and induction of inducible nitric oxide synthase by LPS

Background  

Chronic lung disease (CLD) of prematurity is a major problem of neonatal care. Bacterial infection and inflammatory response have been thought to play an important role in the development of CLD and steroids have been given, with some benefit, to neonates with this disease. In the present study, we assessed the ability of lipopolysaccharide (LPS) to stimulate rat alveolar macrophages to produce nitric oxide (NO), express inducible nitric oxide synthase (iNOS) and activate nuclear factor-κB (NF-κB) in vitro. In addition, we investigated the impact of dexamethasone and budesonide on these processes.     

Biotransformation of (−)-epicatechin, (+)-epicatechin, (−)-catechin,and (+)-catechin by intestinal bacteria involved in isoflavone metabolism

Isoflavone-metabolizing bacteria, Adlercreutzia equolifaciens, Asaccharobacter celatus, Slackia equolifaciens, and Slackia isoflavoniconvertens catalyzed C-ring cleavage of (–)-epicatechin and (+)-catechin, (+)-epicatechin, and (–)-catechin in varying degrees. The cleaving abilities of (–)-epicatechin and (+)-catechin were enhanced by hydrogen, except (+)-catechin cleavage by S. equolifaciens, which was not accelerated. (?)-Catechin cleavage by Ad. equolifaciens was remarkably accelerated by hydrogen.     

Differential effect of nitric oxide on glutathione metabolism and mitochondrial function in astrocytes and neurones: implications for neuroprotection/neurodegeneration?

Primary culture rat astrocytes exposed to the long acting nitric oxide donor (Z)-1-[2-aminoethyl)-N-(2-ammonioethyl)amino]diazen-1-ium-1,2-diolate (DETA-NO) for 24 h approximately double their concentration of glutathione (GSH) and show no sign of cell death. In contrast, GSH was depleted by 48%, and significant loss of mitochondrial respiratory chain complex activity and cell death were observed in primary culture rat neurones subjected to DETA-NO for 18 h. Northern blot analysis suggested that mRNA amounts of both subunits of glutamate-cysteine ligase (GCL), the rate-limiting enzyme in GSH synthesis, were elevated in astrocytes following nitric oxide (NO) exposure. This correlated with an increase in astrocytic GCL activity. Neurones on the other hand did not exhibit increased GCL activity when exposed to NO. In addition, the rate of GSH efflux was doubled and gamma-glutamyltranspeptidase (gamma-GT) activity was increased by 42% in astrocytes treated with NO for 24 h. These results suggest that astrocytes, but not neurones, up-regulate GSH synthesis as a defence mechanism against excess NO. It is possible that the increased rate of GSH release and activity of gamma-GT in astrocytes may have important implications for neuroprotection in vivo by optimizing the supply of GSH precursors to neurones in close proximity.     

Cell membrane mediated (−)-epicatechin effects on upstream endothelial cell signaling: Evidence for a surface receptor

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 10 min with EPI or EPI–Dx at equimolar concentrations (100 nM). 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.     

Effects of α-mangostin on mitochondrial energetic metabolism

Although α-mangostin prevents from toxicity associated to oxidative stress, it also promotes apoptotic cell death in cancer cells. Such effects have been associated with mitochondrial membrane depolarization and cytochrome c release. Therefore, the aim of this work was to analyze the potentially harmful effect of this natural compound on relevant parameters of mitochondrial function from normal tissue. Our results showed that α-mangostin protected mitochondria from peroxidative damage, but at high concentration, it acted as an uncoupler, reduced dramatically ADP-stimulated respiration and inhibited the activity of respiratory complex IV, making mitochondria prone to permeability transition, which is a mitochondrial player on cell fate.     

In vivo and in silico anti-inflammatory mechanism of action of the semisynthetic (−)-cubebin derivatives (−)-hinokinin and (−)-O-benzylcubebin

(?)-Cubebin (CUB), isolated from seeds of Piper cubeba, was used as starting material to obtain the derivatives (?)-hinokinin (HK) and (?)-O-benzyl cubebin (OBZ). Using paw edema as the experimental model and different chemical mediators (prostaglandin and dextran), it was observed that both derivatives were active in comparison with both negative (5% Tween® 80 in saline) and positive (indomethacin) controls. The highest reduction in the prostaglandin-induced edema was achieved by OBZ (66.0%), while HK caused a 59.2% reduction. Nonetheless, the dextran-induced paw edema was not significantly reduced by either of the derivatives (HK or OBZ), which inhibited edema formation by 18.3% and 3.5%, respectively, in contrast with the positive control, cyproheptadine, which reduced the edema by 56.0%. The docking analysis showed that OBZ presented the most stable ligand-receptor (COX-2 – cyclooxygenase-2) interaction in comparison with CUB and HK.     

Effects of acute and chronic endurance exercise on intracellular nitric oxide and superoxide in circulating CD34⁺ and CD34⁻ cells

We investigated the influence of acute and chronic endurance exercise on levels of intracellular nitric oxide (NO), superoxide (O?·?), and expression of genes regulating the balance between these free radicals in CD34? and CD34? peripheral blood mononuclear cells (PBMCs; isolated by immunomagnetic cell separation). Blood samples were obtained from age- and body mass index (BMI)-matched endurance-trained (n = 10) and sedentary (n = 10) men before and after 30 min of exercise at 75% maximal oxygen uptake (·VO(?max)). Baseline levels of intracellular NO (measured by DAF-FM diacetate) and O?·? (measured by dihydroethidium) were 26% (P < 0.05) and 10% (P < 0.05) higher, respectively, in CD34? PBMCs from the sedentary group compared with the endurance-trained group. CD34? PBMCs from the sedentary group at baseline had twofold greater inducible nitric oxide synthase (iNOS) mRNA and 50% lower endothelial NOS (eNOS) mRNA levels compared with the trained group (P < 0.05). The baseline group difference in O?·? was eliminated by acute exercise. Experiments with apocynin indicated that the training-related difference in O?·? levels was explained by increased NADPH oxidase activity in the sedentary state. mRNA levels of additional angiogenic and antioxidant genes were consistent with a more angiogenic profile in CD34? cells of trained subjects. CD34? PBMCs, examined for exploratory purposes, also displayed a more angiogenic mRNA profile in trained subjects, with vascular endothelial growth factor (VEGF) and eNOS being more highly expressed in trained subjects. Overall, our data suggest an association between the sedentary state and increased nitro-oxidative stress in CD34? cells.     

Effects of inhibition of nitric oxide synthesis on TNFα serum levels in e. coli endotoxemic rats

We investigated the effects of nitric oxide (NO) synthesis inhibition on mortality rate and TNFa serum levels in rats inoculated with E. Coli endotoxin (30 mg/kg i.V.). Pre-treatment of endotoxemic rats with NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NO synthesis by both the constitutive and the inducible isoforms of the NO synthase, did not change the mortality rate but significantly reduced TNFa serum levels. By contrast, administration of aminoguanidine, a more specific inhibitor of the inducible NO synthase, did not modifiy serum TNFα. These results suggest that, in E. Coli endotoxemic rats, NO synthetized by the constitutive isoform of the NO synthase positively modulates TNFa synthesis.     

Competitive, reversible, physiological? Inhibition of mitochondrial cytochrome oxidase by nitric oxide

In the mid 1990s a number of research groups recognized that mitochondrial oxygen consumption could be reversibly inhibited by nitric oxide at the level of the enzyme cytochrome c oxidase. The inhibition was apparently competitive with respect to the oxygen concentration. This review critically assesses the present state of knowledge as regards the hypothesis that nitric oxide is a competitive, reversible, physiological inhibitor of cytochrome oxidase.     

Synthesis and biological evaluation of truncated α-galactosylceramide derivatives focusing on cytokine induction profile

A series of truncated analogs of α-galactosylceramide with altered ceramide moiety was prepared, and evaluated for Th2-biased response in the context of IL-4/IFN-γ ratio. Phytosphingosine-modified analogs including cyclic, aromatic and ethereal compounds as well as the C-glycoside analog of OCH (2) with their cytokine inducing profile are disclosed.     

Structure and evolution of mitochondrial outer membrane proteins of β-barrel topology

Gram-negative bacteria are the ancestors of mitochondrial organelles. Consequently, both entities contain two surrounding lipid bilayers known as the inner and outer membranes. While protein synthesis in bacteria is accomplished in the cytoplasm, mitochondria import 90–99% of their protein ensemble from the cytosol in the opposite direction. Three protein families including Sam50, VDAC and Tom40 together with Mdm10 compose the set of integral β-barrel proteins embedded in the mitochondrial outer membrane in S. cerevisiae (MOM). The 16-stranded Sam50 protein forms part of the sorting and assembly machinery (SAM) and shows a clear evolutionary relationship to members of the bacterial Omp85 family. By contrast, the evolution of VDAC and Tom40, both adopting the same fold cannot be traced to any bacterial precursor. This finding is in agreement with the specific function of Tom40 in the TOM complex not existent in the enslaved bacterial precursor cell. Models of Tom40 and Sam50 have been developed using X-ray structures of related proteins. These models are analyzed with respect to properties such as conservation and charge distribution yielding features related to their individual functions.