Fish oil and omega3 in the prevention of metabolic disorders linked to obesity: role of mitochondrial function
pignalosa, angelica (2011) Fish oil and omega3 in the prevention of metabolic disorders linked to obesity: role of mitochondrial function. [Tesi di dottorato] (Inedito)
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Obesity is one of the most serious public health problems of the 21st century. Along with the increase in obesity, there is a parallel increase in the prevalence of type 2 diabetes, impaired glucose tolerance, atherogenic dyslipidemia (high plasma triglyceride and low HDL cholesterol) and hepatic disease (non alcoholic fatty liver disease, NAFLD). Several epidemiological studies and clinical trials have revealed that fish oil and n-3 polyunsaturated fatty acids (PUFAs) reduce the risk of coronary heart disease. Ecosapentaenoic acid (EPA), one of the major n-3 PUFAs contained in fish oil, has a variety of pharmacologycal effects such as lipid-lowering, anti-platelet, anti-inflammatory and anti-atherogenic effect. The aim of my thesis was mainly to evaluate the effect of high fat diets rich in fish oil (polyunsaturated fatty acids) or in lard (saturated fatty acids) on the obesity and on associated pathologies such as hepatic steatosis and insulin resistance, focusing on the role play by the mitochondrial compartment in the development of such pathologies. In particular, the PhD thesis project included the following experimental series. EXPERIMENTAL SERIES 1: The attention focused on the effect of diet rich in fish oil compared to diet rich in lard on obesity development, lipidemic parameters alterations and hepatic steatosis development. Taking into account, that accumulating evidence indicates that impaired mitochondrial function plays a central role in the fatty liver disease, I analysed liver mitochondrial functionality in terms of oxidative capacities, basal and fatty acid-induced proton leak as well as in terms of oxidative stress induction. Furthermore, since growing evidences suggest that there is an association between mitochondrial functionality and fusion/fission process, I also analysed the effect of both high fat diets on protein involved in the mitochondrial dynamic processes. EXPERIMENTAL SERIES 2: The attention focused on the effect of diet rich in fish oil compared to diet rich in lard on insulin resistance development at both whole-body and skeletal muscle level. It is well known that skeletal muscle is the primary site of insulin action and is thus inherently linked to the development of whole-body insulin resistance. In condition of chronic overfeeding, when the capacity of cells to store fats in the form of triglycerides within lipid droplets is exceeded, endoplasmatic reticulum stress (ER-stress) is induced. ER-stressed cells activate apoptotic and inflammatory pathways, which trigger insulin-resistance and the release of chemokines and cytokines. Whereas ER stress has been widely studied in pancreatic islets, liver and adipose tissue, where it has been proposed to be involved in the pathogenesis of diabetes, much less information exists about ER stress in skeletal muscle. Thus, aim of this experimental series was to evaluate in skeletal muscle the onset of ER-stress, inflammatory pathway and insulin resistance induced by high fat diet rich in lard or in fish oil. Furthermore, taking into account that it has been postulated that defects in mitochondrial performance could contribute to the development of insulin resistance, I found of interest to analyze mitochondrial functionality in terms of respiration rates, energetic efficiency and oxidative stress in both subpopulation of skeletal muscle mitochondria (subsarcolemmal, SS and intermyofibrillar, IMF). EXPERIMENTAL SERIES 3: In the light of the previous results of my PhD project showing a beneficial effect of fish oil on obesity, hepatic steatosis and insulin resistance development, in the last year of my PhD I found of interest to evaluate the effect of polyunsaturated fatty acids (such as EPA of which fish oil is rich) on a new adipokine linked to obesity and insulin resistance, apelin. Apelin appears as a beneficial adipokine with anti-obesity and anti-diabetic properties and thus as a promising therapeutic target in metabolic disorders. The aim of the last year of my PhD project was to evaluate the interaction between EPA and apelin in both in vitro and in vivo experimental model. To this end, I carried out my research in collaboration with Isabelle Castan-Laurelle in the laboratory of the team of Pr. P. Valet in the institute of Molecular and Cardiovascular Medecine (I2MC) in Toulouse.in France (INSERM U1048, team 3). RESULTS. The results of the first experimental series showed that high fat diet rich in lard compared to high fat diet rich in fish oil induced : 1) at the total body level, an higher obesity development and alteration in lipidemic parameters and 2) at the hepatic level, an higher tendency to the mitochondrial fission associated with alteration in mitochondrial function as well as with an higher degree of oxidative stress and hepatic lipid accumulation. The results obtained in this thesis suggested that mitochondria from L rats did not maintain a correct balance between mitochondrial fusion and fission. Indeed, mitochondria isolated from L rats exhibited a higher DRP-1 content and lower Mfn2 content, suggesting a shift of dynamic processes towards fission events. Interestingly, if high fat diet is rich in fish oil instead of lard, this shift toward mitochondrial fission is not induced, mitochondrial function is not impaired and oxidative stress is not induced. The results of the experimental series 2 underlined that high fat diet rich in lard compared to high fat diet rich in fish oil induced: 1) at the total body level, a higher degree of inflammation and insulin resistance and 2) at the skeletal muscle level, a higher degree of lipid accumulation and inflammation associated to ER stress, oxidative stress and SS mitochondrial impairment. Thus, high fat diet rich in lard induced a higher impairment in mitochondrial function and oxidative stress, mainly in SS mitochondria, that due to their localization are the first to be affected by the substrate pressure caused by chronic overfeeding. High fat diet rich in fish oil, through the increase in SS fatty acid induced proton leak, elicited an increased substrates utilization preventing skeletal muscle fat accumulation. The lower fat accumulation found in rats fed high fat diet rich in fish oil, differently from rats fed high fat diet rich in lard, did not induce ER stress and thus inflammatory and insulin resistance pathways. The results of the experimental series 3 showed that, also in muscle cells such as in adipocyte, EPA induces an increase in the expression of the apelin. Moreover, the EPA anti-inflammatory effect is demonstrated by a decrease in TNFα associated with an increase in expression in PPARα. These results obtained in vitro in C2C12 cells as well as the results obtained in the literature on adipose tissue are encouraging. Apelin might be an interesting target of EPA and can mediate its beneficial effects. The results obtained in vivo using male C57Bl6/J mice showed that in mice fed high fat diet rich in EPA there is a lower development of obesity and an improvement in hepatic steatosis and insulin sensitivity, suggesting that EPA could be the component in fish oil responsible for these metabolic effects. CONCLUSIONS The results of my thesis suggest a role for omega 3 in fish oil in preventing not only obesity and hepatic steatosis development but also insulin resistance onset. Mitochondria seems to play an important role both in liver and in skeletal muscle, since, by modulating respiratory capacity and proton leak conductance, regulate the balance between the oxidation and the accumulation of fat substrates. The excessive intracellular fat accumulation is then responsible for both ER stress and the following inflammatory process and insulin resistance on-set. Fish oil, differently from lard, by acting on mitochondrial energetic efficiency in liver and in skeletal muscle, is able to prevent steatosis and insulin resistance development. In addition, the results of my thesis suggest that the effect of fish oil on obesity and insulin resistance development may be due to its composition in omega 3, in particular to EPA. Indeed the treatment with high fat diet rich in EPA showed a similar effect in body weight gain and insulin resistance development compared to high fat diet rich in fish oil. These results suggest that it could be excluded the hypothesis that the effect of fish oil on these parameters may be due to other components of fish oil such as furanic acids. Finally, the results of my thesis give new insight on the interaction between omega 3 (EPA) and the anti-obesity and anti-diabetic hormone apelin, suggesting new perspective in this research field.
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