Effects of dietary catechins and proanthocyanidins on zinc homeostasis in hepatic cells

Resumen

Catechins and their polymers procyanidins are health-promoting flavonoids found in ediblevegetables and fruits. They act as antioxidants by scavenging reactive oxygen species andby chelating the redox-active metals iron and copper. They also behave as signalingmolecules, modulating multiple cell signaling and metabolic pathways and gene expression,including that of antioxidant enzymes. Previous results of the Nutrigenomics Reseach Groupshowed that an oral acute dose of a grape-seed procyanidin extract (GSPE) represses theexpression of the zinc-binding protein metallothionein (MT) genes in rat liver, and enhancesthe expression of the orfan nuclear receptor small heterodimer partner (SHP/Nr0b2) (Del Baset al., 2005). In addition, it was shown that procyanidins act as transcriptional coactivators ofthe nuclear bile acid receptor Farnesoid X Receptor (FXR), which in turns upregulates SHPexpression, thereby exerting an hypotrygliceridemic effect (Del Bas et al., 2008; Del Bas etal., 2009).The objectives of this Ph.D. Thesis were to determine whether catechins and procyanidinsinteract with the redox-inactive metal zinc, to evaluate their effect on zinc homeostasis inhepatic cells -including the expression of MT genes, used here as a biomarkers ofprocyanidin activity in hepatic cells-, and to disect the mechanisms by which procyanidinsaffect cellular zinc homeostasis, in particular to asses whether MT genes are targets of SHPand FXR.Our results show that GSPE, as well as individual catechins and procyanidins tested,including the green tea flavonoid (-)-epigallocatechin-3-gallate (EGCG), bind zinc cations insolution with higher affinity than the zinc-specific chelator Zinquin. In humanhepatocarcinoma HepG2 cells, GSPE inhibits intracellular zinc accumulation and counteractsthe toxic effects of excess zinc on cell viability. At the mRNA expression level, GSPEdownregulates MTs and zinc-efflux transporters while upregulating zinc-influx transporters.Zinc importers of the Trans-Golgi network are upregulated by GSPE. In addition, GSPEblocks the induction of MTs expression by the proinflammatory cytokine IL-6, the ROSgenerator tBOOH, the glucocorticoid receptor agonist dexamethasone, and the metalscopper and zinc.EGCG reproduces the major effects of GSPE on zinc homeostasis in HepG2, downregulingthe expression of MTs and zinc-efflux transporters, while upregulating the expression of zincinfluxtransporters, concomitantly inhibiting intracellular zinc accumulation and the toxicity of high zinc doses. Procyanidin dimer B1 and trimer C1 behave opposite to GSPE and EGCGwith regard to MT expression and intracellular zinc accumulation in HepG2 cells.Concerning cytoplasmic labile zinc, the tiny fraction of total cellular zinc that modulatessignaling and metabolic pathways, we found that GSPE, EGCG and trimeric procyanidin C1greatly elevate Zinquin-detectable labile zinc in HepG2 cells.Experiments with SHP-null and FXR-null mice demonstrate that GSPE downregulatespostprandial expression of MT genes in the liver, in a SHP-independent but FXR-dependentmanner. In addition, chenodeoxycholic acid, a physiological ligand and activator of FXR,represses the expression of MT genes in HepG2 cells. Thus, MT genes are targets of FXRand, consequently, FXR is revealed as a modulator of zinc homeostasis.To explain these results, we postulate that catechins and procyanidis may act both assequestrants of zinc -thereby impeding the entrance of zinc cations to the cell throughplasma membrane zinc transporters-, and as zinc ionophores -thereby cotransporting zinccations through the lipid bilayer and increasing the levels of cytoplasmic labile zinc.Repression of MT expression by procyanidin-activated FXR might also contribute to theincrement of the labile pool of zinc, by hindering the sequestration of zinc-cations by de novosynthesized apo-thionein.Given the role of labile zinc as modulator of multiple intracellular signaling and metabolicpathways, we forward the hypothesis that extracellular complexation of zinc cations andsubsequent elevation of cytoplasmic labile zinc may be relevant mechanisms underlying thehealth-promoting activity of catechins and procyanidins and, therefore, that the signaling andmetabolic pathways modulated by labile zinc will be aslo a target of these flavonoids.