Molecular mechanisms of protection by dietary polyphenols against free fatty acid-induced mitochondrial dysfunction and endoplasmic reticulum stress in an in vitro model of non-alcoholic fatty liver disease

Abstract

Non-alcoholic fatty liver disease (NAFLD) is a public health burden. Steatosis as the “first hit”, and oxidative stress, inflammation, mitochondrial dysfunction, and endoplasmic reticulum stress as the “second hits” are the main contributors of the progression of fatty liver to non-alcoholic steatohepatitis (NASH). Dietary polyphenols have shown promise in protecting the liver against NAFLD. The relative effectiveness and mechanisms of different polyphenols however is mostly unknown. In this thesis HepG2 hepatocytes exposed to oleic or palmitic acid were used as a model system to explore the ability of selected polyphenols (resveratrol, quercetin, catechin, cyanidin, cyanidin-3-glucoside, berberine) from different classes to protect against molecular aspects of NAFLD and NASH. In an investigation of the “first hit” (Chapter 3), different polyphenols protected similarly against oleic acid-induced intracellular lipid accumulation, but differed in their effects on the expression of genes and proteins involved in lipogenesis, fatty acid oxidation, mitochondrial biogenesis, and bioenergetics. In an investigation of “second hits” (Chapter 4), most of the polyphenols decreased reactive oxygen species (ROS), prevented the decrease in uncoupling protein 2 (UCP2) mRNA, prevented the increase in tumor necrosis factor alpha (TNFα) mRNA, reversed decreases in mitochondrial biogenesis and increased expression of mitochondrial respiratory complexes and manganese superoxide dismutase (MnSOD). The anthocyanins were unique in decreasing ROS without inducing mitochondrial biogenesis or Mn-SOD mRNA expression. In investigations with palmitic acid (Chapter 5), exposure of HepG2 cells to palmitic acid induced endoplasmic reticulum (ER) stress evidenced by upregulated mRNA for the ER chaperones glucose-regulated protein 94 and 78 (GRP94, GRP78) and oxygen-regulated protein 150 (ORP150), cochaperone endoplasmic reticulum-localized DnaJ homologue 4 (ERdj4), and proapoptotic CCAAT-enhancer-binding protein homologous protein (CHOP). A few of the polyphenols (quercetin, catechin, cyanidin) protected against these changes. In a comparison of flavonoids with their phenolic breakdown/digestion products (Chapter 6), the polyphenols 2,4,6-trihydroxybenzaldehyde, protocatechuic acid, and caffeic acid protected similarly to quercetin and cyanidin against oleic and palmitic acid-induced steatosis and ROS generation. Moreover, in a short-term 1 h exposure (to limit spontaneous degradation in the medium), only breakdown/digestion products prevented an oleic acid-induced decrease of mitochondrial biogenesis. In conclusion, different classes of dietary polyphenols were all able to protect against steatosis and ROS generation in this in vitro model of NAFLD. Part of the mechanism for some polyphenols was through effects on mitochondrial biogenesis and function, bioenergetics, and ER stress. Phenolic breakdown/digestion products of flavonoids were shown to contribute to the protective effects of parent polyphenols.