M. Josepa Salvadó

Oligomers of grape-seed procyanidin extract activate the insulin receptor and key targets of the insulin signaling pathway differently from insulin

Procyanidins are bioactive flavonoid compounds from fruits and vegetables that possess insulinomimetic properties, decreasing hyperglycaemia in streptozotocin-diabetic rats and stimulating glucose uptake in insulin-sensitive cell lines. Here we show that the oligomeric structures of a grape-seed procyanidin extract (GSPE) interact and induce the autophosphorylation of the insulin receptor in order to stimulate the uptake of glucose. However, their activation differs from insulin activation and results in differences in the downstream signaling. Oligomers of GSPE phosphorylate protein kinase B at Thr308 lower than insulin does, according to the lower insulin receptor activation by procyanidins. On the other hand, they phosphorylate Akt at Ser473 to the same extent as insulin. Moreover, we found that procyanidins phosphorylate p44/p42 and p38 MAPKs much more than insulin does. These results provide further insight into the molecular signaling mechanisms used by procyanidins, pointing to Akt and MAPK proteins as key points for GSPE-activated signaling pathways. Moreover, the differences between GSPE and insulin might help us to understand the wide range of biological effects that procyanidins have.

Dietary procyanidins enhance transcriptional activity of bile acid-activated FXR in vitro and reduce triglyceridemia in vivo in a FXR-dependent manner

Consumption of dietary flavonoids has been associated with reduced mortality and risk of cardiovascular disease, partially by reducing triglyceridemia. We have previously reported that a grape seed procyanidin extract (GSPE) reduces postprandial triglyceridemia in normolipidemic animals signaling through the orphan nuclear receptor small heterodimer partner (SHP) a target of the bile acid receptor farnesoid X receptor (FXR). Our aim was to elucidate whether FXR mediates the hypotriglyceridemic effect of procyanidins. In FXR-driven luciferase expression assays GSPE dose-dependently enhanced FXR activity in the presence of chenodeoxycholic acid. GSPE gavage reduced triglyceridemia in wild type mice but not in FXR-null mice, revealing FXR as an essential mediator of the hypotriglyceridemic actions of procyanidins in vivo. In the liver, GSPE downregulated, in an FXR-dependent manner, the expression of the transcription factor steroid response element binding protein 1 (SREBP1) and several SREBP1 target genes involved in lipogenesis, and upregulated ApoA5 expression. Altogether, our results indicate that procyanidins lower triglyceridemia following the same pathway as bile acids: activation of FXR, transient upregulation of SHP expression and subsequent downregulation of SREBP1 expression. This study adds dietary procyanidins to the arsenal of FXR ligands with potential therapeutic use to combat hypertriglyceridemia, type 2 diabetes and metabolic syndrome.

Grape seed proanthocyanidins repress the hepatic lipid regulators miR-33 and miR-122 in rats

SCOPE One major health problem in westernized countries is dysregulated fatty acid and cholesterol metabolism that causes pathologies such as metabolic syndrome. Previous studies from our group have shown that proanthocyanidins, which are the most abundant polyphenols in the human diet, regulate lipid metabolism and are potent hypolipidemic agents. The noncoding RNAs, miR-33 and miR-122, regulate genes that are involved in lipid metabolism. METHODS AND RESULTS Here, we show that grape seed proanthocyanidins rapidly and transiently repressed the expression of miR-33 and miR-122 in rat hepatocytes in vivo and in vitro. Furthermore, the miR-33 target gene ATP-binding cassette A1 and the miR-122 target gene fatty acid synthase were also modulated by proanthocyanidins. Specifically, ATP-binding cassette A1 mRNA and protein levels were increased, and fatty acid synthase mRNA and protein levels were reduced after the miRNA levels were altered. CONCLUSION These results suggest that proanthocyanidin treatment increased hepatic cholesterol efflux to produce new HDL particles by repressing miR-33, and it reduced lipogenesis by repressing miR-122. These results highlight a new mechanism by which grape seed proanthocyanidins produce hypolipidemia through their effects on miRNA modulators of lipid metabolism.

Resveratrol and EGCG bind directly and distinctively to miR-33a and miR-122 and modulate divergently their levels in hepatic cells

Modulation of miR-33 and miR-122 has been proposed to be a promising strategy to treat dyslipidemia and insulin resistance associated with obesity and metabolic syndrome. Interestingly, specific polyphenols reduce the levels of these mi(cro)RNAs. The aim of this study was to elucidate the effect of polyphenols of different chemical structure on miR-33a and miR-122 expression and to determine whether direct binding of the polyphenol to the mature microRNAs (miRNAs) is a plausible mechanism of modulation. The effect of two grape proanthocyanidin extracts, their fractions and pure polyphenol compounds on miRNA expression was evaluated using hepatic cell lines. Results demonstrated that the effect on miRNA expression depended on the polyphenol chemical structure. Moreover, miR-33a was repressed independently of its host-gene SREBP2. Therefore, the ability of resveratrol and epigallocatechin gallate to bind miR-33a and miR-122 was measured using 1H NMR spectroscopy. Both compounds bound miR-33a and miR-122 and differently. Interestingly, the nature of the binding of these compounds to the miRNAs was consistent with their effects on cell miRNA levels. Therefore, the specific and direct binding of polyphenols to miRNAs emerges as a new posttranscriptional mechanism by which polyphenols could modulate metabolism.

Dietary catechins and procyanidins modulate zinc homeostasis in human HepG2 cells

Catechins and their polymers procyanidins are health-promoting flavonoids found in edible vegetables and fruits. They act as antioxidants by scavenging reactive oxygen species and by chelating the redox-active metals iron and copper. They also behave as signaling molecules, modulating multiple cell signalling pathways and gene expression, including that of antioxidant enzymes. This study aimed at determining whether catechins and procyanidins interact with the redox-inactive metal zinc and at assessing their effect on cellular zinc homeostasis. We found that a grape-seed procyanidin extract (GSPE) and the green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG) bind zinc cations in solution with higher affinity than the zinc-specific chelator Zinquin, and dose-dependently prevent zinc-induced toxicity in the human hepatocarcinoma cell line HepG2, evaluated by the lactate dehydrogenase test. GSPE and EGCG hinder intracellular accumulation of total zinc, measured by atomic flame absorption spectrometry, concomitantly increasing the level of cytoplasmic labile zinc detectable by Zinquin fluorescence. Concurrently, GSPE and EGCG inhibit the expression, evaluated at the mRNA level by quantitative reverse transcriptase-polymerase chain reaction, of zinc-binding metallothioneins and of plasma membrane zinc exporter ZnT1 (SLC30A1), while enhancing the expression of cellular zinc importers ZIP1 (SLC39A1) and ZIP4 (SLC39A4). GSPE and EGCG also produce all these effects when HepG2 cells are stimulated to import zinc by treatment with supplemental zinc or the proinflammatory cytokine interleukin-6. We suggest that extracellular complexation of zinc cations and the elevation of cytoplasmic labile zinc may be relevant mechanisms underlying the modulation of diverse cell signaling and metabolic pathways by catechins and procyanidins.

The lipid-lowering effect of dietary proanthocyanidins in rats involves both chylomicron-rich and VLDL-rich fractions

Proanthocyanidins have been shown to improve postprandial hypertriacylglycerolaemia. The present study aims to determine the actual contribution of chylomicrons (CM) and VLDL in the hypotriacylglycerolaemic action of grape seed proanthocyanidin extract (GSPE) in the postprandial state and to characterise the mechanisms by which the GSPE treatment reduces TAG-rich lipoproteins in vivo. A plasma lipid tolerance test was performed on rats fasted for 14 h and orally loaded with lard containing either GSPE or not. GSPE (250 mg/kg body weight) markedly blocked the increase in plasma TAG induced by lard, with a statistically significant reduction of 22 % in the area under the curve. The VLDL-rich fraction was the major contributor (72 %) after 1 h, whereas the CM-rich fraction was the major contributor (85 %) after 3 h. At 5 and 7 h after treatment, CM-rich and VLDL-rich fractions showed a similar influence. Plasma post-heparin lipoprotein lipase (LPL) activity and LPL mRNA levels in white adipose tissue and muscle were not affected by GSPE. On the contrary, GSPE treatment significantly repressed (30 %) the secretion of VLDL-TAG. In the liver, GSPE treatment induced different effects on the expression of acyl-coenzyme A synthetase long-chain family member 1, Apoc3 and 3-hydroxy-3-methylglutaryl-coenzyme A reductase at 1 h and Cd36 at 5 h, compared to those induced by lard. Furthermore, GSPE treatment significantly increased the activity of carnitine palmitoyltransferase 1a at 1 h. In conclusion, both CM-rich and VLDL-rich fractions contributed to the hypotriacylglycerolaemic action of GSPE, but their influence depended on time. GSPE induces hypotriacylglycerolaemic actions by repressing lipoprotein secretion and not by increasing LPL activity.

Proanthocyanidins in health and disease

Proanthocyanidins (PAs) are the most abundant flavonoids in the human diet. Several epidemiological studies connect PA consumption and health benefits and the designation of PAs as healthy compounds started at the early stages of the 20th century. The beneficial health properties of PAs are attributed to their conjugated and colonic metabolites. Therefore, gut microbial compositions can determine the effectiveness of PAs. Reciprocally, dietary polyphenols can act as prebiotics. Recently, it has also been described that PAs modulate the circadian rhythm. Biochemical and epigenetic mechanisms, including the modulation of microRNAs, allow PAs to modulate cell functionality. PA effects in metabolic diseases are also reviewed.

Epigallocatechin gallate counteracts oxidative stress in docosahexaenoxic acid-treated myocytes

Skeletal muscle is a key organ of mammalian energy metabolism, and its mitochondria are multifunction organelles that are targets of dietary bioactive compounds. The goal of this work was to examine the regulation of mitochondrial dynamics, functionality and cell energy parameters using docosahexaenoic acid (DHA), epigallocatechin gallate (EGCG) and a combination of both in L6 myocytes. Compounds (at 25μM) were incubated for 4h. Cells cultured with DHA displayed less oxygen consumption with higher ADP/ATP ratio levels concomitant with downregulation of Cox and Ant1 gene expression. The disruption of energetic homeostasis by DHA, increases intracellular reactive oxygen species (ROS) levels and decreases mitochondrial membrane potential. The defence mechanism to counteract the excess of ROS production was by the upregulation of Ucp2, Ucp3 and MnSod gene expression. Moreover myocytes cultured with DHA had a higher mitochondrial mass with a higher proportion of large and elongated mitochondria, whereas the fission genes Drp1 and Fiss1 and the fusion gene Mfn2 were downregulated. In myocytes co-incubated with DHA and EGCG, ROS levels and the adenosine diphosphate (ADP)/adenosine triphosphate (ATP) ratio were similar to untreated myocytes and the decrease of oxygen consumption, higher mitochondrial mass and the overexpression of Ucp2 and Ucp3 genes were similar to the DHA-treated cells with also a higher amount of mitochondrial deoxyribonucleic acid (DNA), and reduced Drp1 and Fiss1 gene expression levels. In conclusion the addition of EGCG to DHA returned the cells to the control conditions in terms of mitochondrial morphology, energy and redox status, which were unbalanced in the DHA-treated myocytes.

Combination of grape seed proanthocyanidin extract and docosahexaenoic acid-rich oil increases the hepatic detoxification by GST mediated GSH conjugation in a lipidic postprandial state

The ingestion of dietary lipids leads to oxidative stress. This postprandial oxidative stress may potentiate the adverse effects of postprandial hyperlipidaemia. Proanthocyanidins have been shown to alleviate oxidative stress and hypertriglyceridaemia associated with the postprandial state. Additionally, omega-3 polyunsaturated fatty acids (PUFAs) also have beneficial effects on lipoprotein metabolism and oxidative stress. The present study was designed to investigate the possible additive effects in liver of an acute dose of grape seed proanthocyanidins extract (GSPE) and oil rich in docosahexaenoic acid (DHA-OR) on lipidic postprandial oxidative stress in Wistar rats. GSPE+DHA-OR modifies the hepatic antioxidant enzymatic activities (GST and GPx), clearly showing that this combination increases the detoxification of postprandial xenobiotics via the GST action mediated hepatic GSH conjugation. In conclusion, this study provides evidence that the combination of GSPE and DHA-OR ameliorate the transient imbalance between the lipid hydroperoxide level and antioxidant status related to a lipidic postprandial state.

A novel form of the human manganese superoxide dismutase protects rat and human livers undergoing ischaemia and reperfusion injury

Hepatic microcirculatory dysfunction due to cold storage and warm reperfusion (CS+WR) injury during liver transplantation is partly mediated by oxidative stress and may lead to graft dysfunction. This is especially relevant when steatotic donors are considered. Using primary cultured liver sinusoidal endothelial cells (LSECs), liver grafts from healthy and steatotic rats, and human liver samples, we aimed to characterize the effects of a new recombinant form of human manganese superoxide dismutase (rMnSOD) on hepatic CS+WR injury. After CS+WR, the liver endothelium exhibited accumulation of superoxide anion (O2-) and diminished levels of nitric oxide (NO); these detrimental effects were prevented by rMnSOD. CS+WR control and steatotic rat livers exhibited markedly deteriorated microcirculation and acute endothelial dysfunction, together with liver damage, inflammation, oxidative stress, and low NO. rMnSOD markedly blunted oxidative stress, which was associated with a global improvement in liver damage and microcirculatory derangements. The addition of rMnSOD to CS solution maintained its antioxidant capability, protecting rat and human liver tissues. In conclusion, rMnSOD represents a new and highly effective therapy to significantly upgrade liver procurement for transplantation.