Background: The incidence of metabolic syndrome (MetS) in the world’s adult population is increasing and is becoming an important health problem given that it can lead to insulin-resistance and type 2 diabetes. MetS-associated insulin-resistance can be in part triggered by the associated increased plasma levels of free fat acid (FFAA). The activation by FFAA of NADPH oxidase and the consequent production of superoxide anion and derived oxidants can in part lead to hepatic insulin resistance. Oxidants can activate kinases (JNK, IKK) which negatively phosphorylate the insulin receptor and IRS1 leading to an impaired insulin signaling. Epidemiological studies show that the consumption of fruit and vegetables in humans decreases the risk for MetS. The flavan-3-ol (-)-epicatechin (EC) is present in large amounts in fruit and vegetables. Furthermore, we have shown that EC improves parameters of inflammation and insulin sensitivity in adipocytes. Objective: The aim of this study was to evaluate the effects of EC and its metabolites on the expression and activity of NADPH oxidase, and the consequent production of oxidants, in human hepatocytes (HepG2 cells) treated with the FFAA palmitate (Pal). Material and Methods: HepG2 cells were incubated with Pal (0.25-0.75 mM), in the absence and the presence of EC and EC metabolites (ECM) (0.25-1 µM) for 24 h. The protein levels of NADPH oxidase subunits (NOX3, p22, p47) were assessed by Western Blot. NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence assay and cell oxidants by fluorescence with the oxidant-sensitive 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF) probe. Results: Pal caused a dose-dependent increase in the HepG2 cell expression of NOX3 and p22. EC and its metabolites acted preventing Pal-induced NOX3 and p22 expression. The incubation with Pal also caused a significant increase in NADPH oxidase activity, measured by both enzyme activity, and p47 translocation from the cytosol to the membrane. EC and ECM (1 µM) prevented Pal-mediated NADPH oxidase activation. Pal also caused an increase in cellular oxidants (DCF fluorescence) which was significantly decreased by EC and ECM. Conclusion: Results show that PAL increases the production of oxidants in HepG2 through an up regulation of NOX3/p22 expression. EC and its metabolites, at concentrations found in plasma after consumption of EC-rich foods, prevented Pal-induced activation of NADPH oxidase and increased oxidant production. This EC effects could be relevant in the improvement of insulin sensitivity
(-)-Epicatechin prevents palmitate-induced increase in NADPH oxidase expression and activation in the human hepatocyte cell line HepG2
CREMONINI, Eleonora;CERVELLATI, Carlo;
2013
Abstract
Background: The incidence of metabolic syndrome (MetS) in the world’s adult population is increasing and is becoming an important health problem given that it can lead to insulin-resistance and type 2 diabetes. MetS-associated insulin-resistance can be in part triggered by the associated increased plasma levels of free fat acid (FFAA). The activation by FFAA of NADPH oxidase and the consequent production of superoxide anion and derived oxidants can in part lead to hepatic insulin resistance. Oxidants can activate kinases (JNK, IKK) which negatively phosphorylate the insulin receptor and IRS1 leading to an impaired insulin signaling. Epidemiological studies show that the consumption of fruit and vegetables in humans decreases the risk for MetS. The flavan-3-ol (-)-epicatechin (EC) is present in large amounts in fruit and vegetables. Furthermore, we have shown that EC improves parameters of inflammation and insulin sensitivity in adipocytes. Objective: The aim of this study was to evaluate the effects of EC and its metabolites on the expression and activity of NADPH oxidase, and the consequent production of oxidants, in human hepatocytes (HepG2 cells) treated with the FFAA palmitate (Pal). Material and Methods: HepG2 cells were incubated with Pal (0.25-0.75 mM), in the absence and the presence of EC and EC metabolites (ECM) (0.25-1 µM) for 24 h. The protein levels of NADPH oxidase subunits (NOX3, p22, p47) were assessed by Western Blot. NADPH oxidase activity was measured by lucigenin-enhanced chemiluminescence assay and cell oxidants by fluorescence with the oxidant-sensitive 5-(and-6)-carboxy-2′,7′-dichlorodihydrofluorescein diacetate (DCF) probe. Results: Pal caused a dose-dependent increase in the HepG2 cell expression of NOX3 and p22. EC and its metabolites acted preventing Pal-induced NOX3 and p22 expression. The incubation with Pal also caused a significant increase in NADPH oxidase activity, measured by both enzyme activity, and p47 translocation from the cytosol to the membrane. EC and ECM (1 µM) prevented Pal-mediated NADPH oxidase activation. Pal also caused an increase in cellular oxidants (DCF fluorescence) which was significantly decreased by EC and ECM. Conclusion: Results show that PAL increases the production of oxidants in HepG2 through an up regulation of NOX3/p22 expression. EC and its metabolites, at concentrations found in plasma after consumption of EC-rich foods, prevented Pal-induced activation of NADPH oxidase and increased oxidant production. This EC effects could be relevant in the improvement of insulin sensitivityI documenti in SFERA sono protetti da copyright e tutti i diritti sono riservati, salvo diversa indicazione.
