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.

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.

In vitro measurements and interpretation of total antioxidant capacity.

BACKGROUND: One of the strategies most commonly used to assess a free radical-antioxidant balance in chemical and biological systems is the determination of the total antioxidant capacity (TAC). A large amount of research has been published using TAC. However, it remains unclear which is the significance of these investigations for understanding the biological importance of free radical reactions. SCOPE OF REVIEW: This review discusses the relevance and limitations of TAC for the assessment of the antioxidant activities present in food and food derivatives, and in body tissues and fluids. MAJOR CONCLUSIONS: TAC determinations are simple, inexpensive, and able to evaluate the capacity of known and unknown antioxidants and their additive, synergistic and/or antagonistic actions, in chemical and biological systems. However, different TAC assays correlate poorly with each other, since each TAC assay is sensitive to a particular combination of compounds, but exclude many others. The TAC values for foods cannot be translated to the in vivo (human) antioxidant defenses, and furthermore, to health effects provided by that food. GENERAL SIGNIFICANCE: Up to date, conclusions that can be drawn from the extensive amount of research done using TAC of foods or populations should not be considered when used for making decisions affecting population health. This article is part of a Special Issue entitled Current methods to study reactive oxygen species - pros and cons and biophysics of membrane proteins. Guest Editor: Christine Winterbourn.

Amaranth peptides from simulated gastrointestinal digestion: antioxidant activity against reactive species.

We evaluated the capacity of simulated gastrointestinal digests or alcalase hydrolysates of protein isolates from amaranth to scavenge diverse physiologically relevant reactive species. The more active hydrolysate was obtained with the former method. Moreover, a prior alcalase treatment of the isolate followed by the same simulated gastrointestinal digestion did not improve the antioxidant capacity in any of the assays performed and even produced a negative effect under some conditions. Gastrointestinal digestion produced a strong increment in the scavenging capacity against peroxyl radicals (ORAC assay), hydroxyl radicals (ESR-OH assay), and peroxynitrites; thus decreasing the IC50 values to approximately 20, 25, and 20%, respectively, of the levels attained with the nonhydrolyzed proteins. Metal chelation (HORAC assay) also enhanced respect to isolate levels, but to a lesser extent (decreasing IC50 values to only 50%). The nitric-oxide- and superoxide-scavenging capacities of the digests were not relevant with respect to the methodologies used. The gastrointestinal digests from amaranth proteins acted against reactive species by different mechanisms, thus indicating the protein isolate to be a potential polyfunctional antioxidant ingredient.

Coenzyme Q in pregnant women and rats with intrahepatic cholestasis.

BACKGROUND & AIMS: Intrahepatic cholestasis of pregnancy is a high-risk liver disease given the eventual deleterious consequences that may occur in the foetus. It is accepted that the abnormal accumulation of hydrophobic bile acids in maternal serum are responsible for the disease development. Hydrophobic bile acids induce oxidative stress and apoptosis leading to the damage of the hepatic parenchyma and eventually extrahepatic tissues. As coenzyme Q (CoQ) is considered an early marker of oxidative stress in this study, we sought to assess CoQ levels, bile acid profile and oxidative stress status in intrahepatic cholestasis. METHODS: CoQ, vitamin E and malondialdehyde were measured in plasma and/or tissues by HPLC-UV method whereas serum bile acids by capillary electrophoresis in rats with ethinyl estradiol-induced cholestasis and women with pregnancy cholestasis. RESULTS: CoQ and vitamin E plasma levels were diminished in both rats and women with intrahepatic cholestasis. Furthermore, reduced CoQ was also found in muscle and brain of cholestatic rats but no changes were observed in heart or liver. In addition, a positive correlation between CoQ and ursodeoxycholic/lithocholic acid ratio was found in intrahepatic cholestasis suggesting that increased plasma lithocholic acid may be intimately related to CoQ depletion in blood and tissues. CONCLUSION: Significant CoQ and vitamin E depletion occur in both animals and humans with intrahepatic cholestasis likely as the result of increased hydrophobic bile acids known to produce significant oxidative stress. Present findings further suggest that antioxidant supplementation complementary to traditional treatment may improve cholestasis outcome.

Therapeutic Effect of Alpha Lipoic Acid in a Rat Preclinical Model of Preeclampsia: Focus on Maternal Signs, Fetal Growth and Placental Function.

Chronic hypertension is a major risk factor for preeclampsia (PE), associated with significant maternal and neonatal morbidity. We previously demonstrated that pregnant stroke-prone spontaneously hypertensive rats (SHRSP) display a spontaneous PE-like phenotype with distinct placental, fetal, and maternal features. Here, we hypothesized that supplementation with alpha lipoic acid (ALA), a potent antioxidant, during early pregnancy could ameliorate the PE phenotype in this model. To test this hypothesis, timed pregnancies were established using 10 to 12-week-old SHRSP females (n = 19-16/group), which were assigned to two treatment groups: ALA (injected intraperitoneally with 25 mg/kg body weight ALA on gestation day (GD1, GD8, and GD12) or control, receiving saline following the same protocol. Our analysis of maternal signs showed that ALA prevented the pregnancy-dependent maternal blood pressure rise (GD14 blood pressure control 169.3 ± 19.4 mmHg vs. 146.1 ± 13.4 mmHg, p = 0.0001) and ameliorated renal function, as noted by the increased creatinine clearance and improved glomerular histology in treated dams. Treatment also improved the fetal growth restriction (FGR) phenotype, leading to increased fetal weights (ALA 2.19 ± 0.5 g vs. control 1.98 ± 0.3 g, p = 0.0074) and decreased cephalization indexes, indicating a more symmetric fetal growth pattern. This was associated with improved placental efficiency, decreased oxidative stress marker expression on GD14, and serum soluble fms-like tyrosine kinase 1 (sFlt1) levels on GD20. In conclusion, ALA supplementation mitigated maternal signs and improved placental function and fetal growth in SHRSP pregnancies, emerging as a promising therapy in pregnancies at high risk for PE.

(-)-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.

(Poly)phenols and the regulation of NADPH oxidases.

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidases (NOXs) are enzymes that generate superoxide anion (O2•-) and hydrogen peroxide (H2O2), and that are widely distributed in mammalian tissues. Many bioactives, especially plant (poly)phenols are being studied for their capacity to regulate NOXs. The modulation of these enzymes are of central relevance to maintain redox homeostasis and regulate cell signaling. In in vitro and ex vivo assays, and in experimental animal models, different (poly)phenols are able to modulate NOX-dependent generation of O2•- and H2O2. Mechanistically, most of the known effects of (poly)phenols and of their metabolites on NOX1, NOX2, and NOX4, include the modulation of: i) the expression of the different constituent subunits, and/or ii) posttranslational modifications involved in the assembly and translocation of the protein complexes. Very limited evidence is available on a direct action of (poly)phenols on NOX active site (electron-transferring protein). Moreover, it is suggested that the regulation by (poly)phenols of systemic events, e.g. inflammation, is frequently associated with their capacity to regulate NOX activation. Although of physiological significance, more studies are needed to understand the specific targets/mechanisms of NOX regulation by (poly)phenols, and the (poly)phenol chemical structures and moieties directly involved in the observed effects. It should be kept in mind the difficulties of NOX's studies associated with the complexity of NOXs biochemistry and the methodological limitations of O2•- and H2O2 the determinations. Studies relating human ingestion of specific (poly)phenols, with NOX activity and disease conditions, are guaranteed to better understand the health importance of (poly)phenol consumption and the involvement of NOXs as biological targets.

(Poly)phenols and nitrolipids: Relevant participants in nitric oxide metabolism.

Nitric oxide (•NO) is an essential molecule able to control and regulate many biological functions. Additionally, •NO bears a potential toxicity or damaging effects under conditions of uncontrolled production, and because of its participation in redox-sensitive pathways and oxidizing reactions. Several plant (poly)phenols present in the diet are able to regulate the enzymes producing •NO (NOSs). In addition, (poly)phenols are implicated in defining •NO bioavailability, especially by regulating NADPH oxidases (NOXs), and the subsequent generation of superoxide and •NO depletion. Nitrolipids are compounds that are present in animal tissues because of dietary consumption, e.g. of olive oil, and/or as result of endogenous production. This endogenous production of nitrolipids is dependent on the nitrate/nitrite presence in the diet. Select nitrolipids, e.g. the nitroalkenes, are able to exert •NO-like signaling actions, and act as •NO reservoirs, becoming relevant for systemic •NO bioavailability. Furthermore, the presence of (poly)phenols in the stomach reduces dietary nitrite to •NO favoring nitrolipids formation. In this review we focus on the capacity of molecules representing these two groups of bioactives, i.e. (poly)phenols and nitrolipids, as relevant participants in •NO metabolism and bioavailability. This participation acquires especial relevance when human homeostasis is lost, for example under inflammatory conditions, in which the protective actions of (poly)phenols and/or nitrolipids have been associated with local and systemic •NO bioavailability.

Fe allocation in liver during early stages of endotoxemia in Fe-overload rats.

The hypothesis of this study was that alterations in Fe distribution triggered by lipopolysaccharide (LPS) administration were affected in vivo by Fe overload. Lipopolysaccharide treatment by itself significantly decreased Fe content in serum and increased the blood NO-hemoglobin (NO-Hb) EPR signal and nitrotyrosine protein content in liver, as compared to values in control animals. Fe overload (produced by Fe-dextran ip administration) caused an increase, as compared to values in control animals, in Fe content in serum, and a significant enhancement in ferritin (Ft) content, Fe content in Ft, the labile Fe pool (LIP), and the protein carbonyl content in the liver. The simultaneous administration of LPS and Fe-dextran lead to a significant increase in the Fe content in serum, blood NO-Hb EPR signal, the content of Fe, Fe in Ft, LIP, protein carbonyl, and nitrotyrosine protein in liver, as compared to values in control animals. The data reported here indicate that the protective strategy against endotoxemia of sequestering serum Fe content is not fully operative under Fe overload conditions. However, the oxidative condition of the liver does not seem to be being affected, since endogenous mechanisms were able to regulate the amount of catalytically active Fe to the same levels observed after Fe-dextran administration, even in the presence of LPS, over the initial six-hour period.

Linking biomarkers of oxidative stress and disease with flavonoid consumption: From experimental models to humans.

Identification of the links among flavonoid consumption, mitigation of oxidative stress and improvement of disease in humans has significantly advanced in the last decades. This review used (-)-epicatechin (EC) as an example of dietary flavonoids, and inflammation, endothelial dysfunction/hypertension and insulin resistance/diabetes as paradigms of human disease. In these pathologies, oxidative stress is part of their development and/or their perpetuation. Evidence from both, rodent studies and characterization of mechanisms in cell cultures are encouraging and mostly support indirect antioxidant actions of EC and EC metabolites in endothelial dysfunction and insulin resistance. Human studies also show beneficial effects of EC on these pathologies based on biomarkers of disease. However, there is limited available information on oxidative stress biomarkers and flavonoid consumption to allow establishing conclusive associations. The evolving discovery of metabolites that could serve as reliable markers of intake of specific flavonoids constitutes a powerful tool to link flavonoid consumption to disease and prevention of oxidative stress in human populations.

Placental Glycoredox Dysregulation Associated with Disease Progression in an Animal Model of Superimposed Preeclampsia.

Pregnancies carried by women with chronic hypertension are at increased risk of superimposed preeclampsia, but the placental pathways involved in disease progression remain poorly understood. In this study, we used the stroke-prone spontaneously hypertensive rat (SHRSP) model to investigate the placental mechanisms promoting superimposed preeclampsia, with focus on cellular stress and its influence on galectin-glycan circuits. Our analysis revealed that SHRSP placentas are characterized by a sustained activation of the cellular stress response, displaying significantly increased levels of markers of lipid peroxidation (i.e., thiobarbituric acid reactive substances (TBARS)) and protein nitration and defective antioxidant enzyme expression as early as gestation day 14 (which marks disease onset). Further, lectin profiling showed that such redox imbalance was associated with marked alterations of the placental glycocode, including a prominent decrease of core 1 O-glycan expression in trophoblasts and increased decidual levels of sialylation in SHRSP placentas. We also observed significant changes in the expression of galectins 1, 3 and 9 with pregnancy progression, highlighting the important role of the galectin signature as dynamic interpreters of placental microenvironmental challenges. Collectively, our findings uncover a new role for the glycoredox balance in the pathogenesis of superimposed preeclampsia representing a promising target for interventions in hypertensive disorders of pregnancy.

Antioxidant actions of flavonoids: thermodynamic and kinetic analysis.

The benefits of flavonoids on human health are very often ascribed to their potential ability to act diminishing free radical steady state concentration in biological systems providing antioxidant protection. This is an assumption based on the chemical structures of flavonoids that support their capacity to scavenge free radicals and chelate redox-active metals. In this paper we will use thermodynamic and kinetic approaches to analyze the interactions of flavonoids with biological material and from there, extrapolate the physiological relevance of their antioxidant actions. Thermodynamic analysis predicts that both, scavenging of oxygen-derived radicals and the sequestration of redox-active metals are energetically favored. Nevertheless, the actual concentrations reached by flavonoids in most animal and human tissues following dietary ingestion are incompatible with the kinetic requirements necessary to reach reaction rates of physiological relevance. This incompatibility becomes evident when compared to other antioxidant compounds, e.g. alpha-tocopherol (vitamin E), ascorbate (vitamin C), and glutathione. Alternatively, lipid-flavonoid and protein-flavonoid interactions can indirectly mediate a decrease in oxidant (free radical) production and/or oxidative damage to both cell and extracellular components. The final mechanisms mediating the antioxidant actions of flavonoid will be determined by their actual concentration in the tissue under consideration.

(-)-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.

(-)-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.

Cocoa, chocolate, and cardiovascular disease.

A significant body of evidence demonstrates that diets rich in fruits and vegetables promote health and attenuate, or delay, the onset of various diseases, including cardiovascular disease, diabetes, certain cancers, and several other age-related degenerative disorders. The concept that moderate chocolate consumption could be part of a healthy diet has gained acceptance in past years based on the health benefits ascribed to selected cocoa components. Specifically, cocoa as a plant and chocolate as food contain a series of chemicals that can interact with cell and tissue components, providing protection against the development and amelioration of pathological conditions. The most relevant effects of cocoa and chocolate have been related to cardiovascular disease. The mechanisms behind these effects are still under investigation. However, the maintenance or restoration of vascular NO production and bioavailability and the antioxidant effects are the mechanisms most consistently supported by experimental data. This review will summarize the most recent research on the cardiovascular effects of cocoa flavanols and related compounds.

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.

Plant bioactives and redox signaling: (-)-Epicatechin as a paradigm.

Polyphenols are bioactives claimed to be responsible for some of the health benefits provided by fruit and vegetables. It is currently accepted that the bioactivities of polyphenols can be mostly ascribed to their interactions with proteins and lipids. Such interactions can affect cell oxidant production and cell signaling, and explain in part the ability of polyphenols to promote health. EC can modulate redox sensitive signaling by: i) defining the extent of oxidant levels that can modify cell signaling, function, and fate, e.g. regulating enzymes that generate superoxide, hydrogen peroxide and nitric oxide; or ii) regulating the activation of transcription factors sensible to oxidants. The latter includes the regulation of the nuclear factor E2-related factor 2 (Nfr2) pathway, which in turn can promote the synthesis of antioxidant defenses, and of nuclear factor kappa-light-chain-enhancer of activated B cells (NF-kB) pathway, which mediates the expression of oxidants generating enzymes, as well as proteins not involved in redox reactions. In summary, a significant amount of data vindicates the participation of EC in redox regulated signaling pathways. Progress in the understanding of the molecular mechanisms involved in EC biological actions will help to define recommendations in terms of which fruit and vegetables are healthier and the amounts necessary to provide health effects.

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.