Olive oil and oleate affect hepatic proteome and mitochondrial dynamics: in vivo and in vitro approaches

• Ano de defesa: 2020
• Autor(a) principal: Sousa, Isy Faria de [UNIFESP]
• Tipo de documento: Tese
• Tipo de acesso: Acesso aberto
• Idioma: eng
• Instituição de defesa: Universidade Federal de São Paulo (UNIFESP)
• Palavras-chave em Português: Extra virgin olive oil; High fat diets; Proteome; Liver; Mitochondria; Azeite extra virgem; Dietas com alto teor de gordura; Fiva; Mitocôndria
• Link de acesso: https://sucupira.capes.gov.br/sucupira/public/consultas/coleta/trabalhoConclusao/viewTrabalhoConclusao.jsf?popup=true&id_trabalho=9383048; https://hdl.handle.net/11600/64584

The Mediterranean diet (MD) has been pointed out as a nutritional approach to prevent metabolic disturbances leading to obesity, diabetes and liver steatosis. These properties have been largely attributed to oleic acid and polyphenol compounds present in olive oil (OO), the main fat source of MD. However, there are controversies regarding the consequences of excessive extra virgin olive oil (EVOO) intake. Prolonged high-fat feeding may induce obesity, insulin resistance, and non-alcoholic fatty liver disease. Dysfunction of mitochondrial dynamics along with endoplasmic reticulum (ER) stress, have been suggested to play a role in the establishment of these disturbances. The mechanism seems to involve a prevalence of the fission process over the fusion process, and endoplasmic reticulum stress, with activation of the unfolded protein response (UPR) and consequent stimulation of inflammatory pathways. The dietary fats differ in their lipotocixity degree, with saturated fatty acids being considered more harmful, while the monounsaturated oleic acid has been suggested to protect against lipotoxicity. However, data on the mechanisms involved as well as on the existence of a dose-dependency are not conclusive. Given that the liver is a key organ for the maintenance of the metabolic homeostasis and is highly susceptible to dietary manipulations, focusing on the consequences of high-fat feeding on this tissue is of relevance. With the aim of evaluating these aspects, we used both in vivo and in vitro approaches. The animal study examined body, serum, and hepatic parameters after the prolonged intake of diets containing either normolipidic or hyperlipidic amounts of EVOO. To perform a broad analysis of liver metabolism, a proteomic approach was used. Two months-old swiss mice were fed for 12 weeks with either normolipidic diets (9.5% energy from fat) containing soy oil (control diet, C) or EVOO (NO diet) or a high fat EVOO diet (HO, 39% energy from fat). Body weight and food intake were measured weekly and serum parameters were analyzed by enzymatic methods. Liver proteome was analyzed by LC-MS/MS and the proteomic data were analyzed by one-way Anova followed by Tukey post-hoc and Benjamini-Hochberg correction. Pathway enrichment analysis was performed with Fisher´s test and corrected by the Bonferroni approach. The high-fat intake of EVOO diet (HO group) inhibited food and energy intake, decreased serum triglycerides while it preserved normal patterns of body weight gain, body adiposity, and glucose levels . However, it increased total cholesterol levels and liver mass and tended to increase hepatic fat content. The proteomic analysis of the liver identified 2318 proteins and, after application of the inclusion criteria, 487 proteins were quantified. They were allocated in 27 pathways significantly enriched, of which 7 pathways were altered in the HO group, in comparison to both C and NO (lipid metabolism, fatty acids metabolism, gluconeogenesis, metabolism of amino acids and derivatives, citric acid cycle, electron transport chain, and biological oxidations). The examination of the pathway analysis derived from the proteomic data suggested stimulation of both mitochondrial and peroxissomal β-oxidation of fatty acids, and inhibition of lipid synthesis from LCFA and of gluconeogenesis in the HO group. On the other hand, although the NO group failed to show significant alteration of the liver proteome, it presented reduced body fat, body weight gain, and serum triglycerides and glucose levels, with no evident hypercholesterolemia. The results allow the hypothesis that the hepatic metabolic adjustments in the HO indicated by the proteomic analysis, were partially successful in avoiding/counteracting the detrimental outcomes of a long term high fat feeding. Contrastingly, since the beneficial effects of the NO diet could not be attributed to overt effects on hepatic metabolism, it is suggested that other tissues may have had a more relevant participation. Because of the indications of altered mitochondrial metabolism by the high EVOO intake, we performed in vitro experiments in HepG2 cells to analyze the consequences of crescent doses of either oleate or palmitate on aspects of mitochondrial dynamics, cell viability, apoptosis, and ER-UPR response. To this end, HEPG-2 cells were treated for 24 hours with 10 μM, 50 μM, 100 μM, 250 μM or 500 μM of either palmitate or oleate. The effects on apoptosis and cell viability were evaluated by the caspase-3 activity and MTT assay, respectively. Western blotting analysis was performed to evaluate the protein content of: a) mitofusin 2 (MFN2) and optic atrophy 1 (OPA1), markers of mitochondrial fusion process; b) dynamin-related protein 1 (DRP1), marker of mitochondrial fission process; and c) 78-kDa glucose-regulated protein (GRP78), marker of early ER stress. Both fatty acids reduced cell viability at doses of 250μM and 500μM, and the highest dose had more pronounced effects. The two highest doses of either palmitate or oleate caused similar increases of Caspase-3 apoptotic activity. GRP78 levels were increased only by palmitate at the lowest and highest doses while no differences were induced by oleate. The levels of MFN2 were not significantly affected by the treatments, although a trend to increased values was observed with all oleate doses, while only the doses of 100 μM, 250 μM and 500 μM of palmitate promoted such a trend. OPA1 and DRP1 levels were not significantly affected by any treatment or dose.