Mapping the diverse functions of dietary fatty acids via target gene regulation

Dietary fat is a strong predictor of chronic diseases, such as cardiovascular diseases, obesity, diabetes, dyslipidemia and metabolic syndrome. A great number of epidemiological and observational studies clearly show that in addition to the amount of fat consumed in a diet, fat composition is an equally important factor in the development of chronic diseases. Evidence abounds indicating that adherence to a diet with high content of polyunsaturated (PUFAs) and monounsaturated fatty acids (MUFAs) such as the Mediterranean diet has substantial health benefits, while diets with high content of saturated fatty acids (SFAs) such as the Western type diet increase the risk for the development of several chronic diseases. Nutritional genomics or nutrigenomics investigates the interaction between nutrients and genes at the molecular level, by using genomic tools. Within the field of nutrigenomics, dietary fatty acids and their metabolites are seen as signaling molecules that target specific cellular response systems. Dietary fatty acids have been reported to bind physically to PPARs, a family of ligand activated transcription factors, that play a major role in metabolic homeostasis. Three PPAR isotypes have been identified, PPARα, PPARβ/δ and PPARγ. Their expression and target genes vary among different tissues and cell types. After a meal triglycerides are packed into chylomicrons in the small intestine and via the lymph system, they reach the blood and the peripheral tissues. Triglyceride chylomicrons deliver free fatty acids to the organs after being lipolylised by lipoprotein lipase (LPL), which is anchored to the capillary endothelium. Among different organs, heart and liver show the highest uptake of dietary triglycerides, postprandialy.  However, opposite to the liver, heart is a constant working muscle, which covers its demands on energy mainly by fatty acids, delivered to the heart via hydrolysis of circulating triglyceride-rich lipoproteins. Unbalanced fatty acid uptake and fatty acid oxidation is common in cardiac diseases, such as cardiac failure, myocardial ischemia and diabetes. Heart is characterized by decreased lipid storage capacity, therefore chronic elevated levels of lipids uptake and intracellular storage is considered harmful and may lead to lipotoxic cardiomyopathy. Our first aim was to explore the whole genome effects of individual dietary fatty acids in the intact heart via transcriptional profiling. By conducting these experiments in wild-type and PPARα−/− mice, we aimed to determine the specific contribution of PPARα, which has been previously described as a master regulator of lipid homeostasis in the heart. We took advantage of a unique experimental model, where mice were given a single oral bolus of synthetic triglycerides composed of a single fatty acid. We sacrificed the mice 6hours after the oral gavage and we compared the effects of different fatty acids on gene expression by microarray analysis in the total heart. Many genes were regulated by one particular treatment only and among those most of them showed large functional divergence. Although, the majority of genes responding to fatty acid treatment were regulated in a PPARα-dependent manner, emphasizing the importance of PPARα in mediating transcriptional regulation by fatty acids in the heart, we observed a substantial number of genes regulated in a PPARα- independent manner. Finally, we observed that deletion and activation of PPARα had a major effect on expression of numerous genes involved in metabolism and immunity. We identified response to oxidative stress as the top upregulated process activated by all  administered  fatty  acids  in  the  heart.  High  rates  of  mitochondria  oxidation,  due  to increased supply of substrate after the oral gavage are coupled with enzymatic and non- enzymatic mechanisms aiming to counterbalance the production of highly reactive secondary products of the respiratory chain, the reactive oxygen species (ROS) in the heart. Under conditions such as chronic high fat diet or insulin resistance, increased lipid influx in combination with uncontrolled production of ROS and lipid intermediates may result in mitochondrial malfunctioning and lipid accumulation. Myocardial lipotoxicity refers to the accumulation of intramyocardial lipids and is associated with contractile dysfunction and even myocytes death. We found Angptl4 to be the top upregulated gene, in all groups that received the fatty acids oral gavage. Angptl4 has been described as a target gene of PPARs and an endogenous inhibitor of the triglyceride hydrolyzing enzyme lipoprotein lipase (LPL), which catalyzes uptake of circulating lipids into tissues.  We were able to show that the strong upregulation of Angptl4 by dietary fatty acids is mediated by PPARβ/δ and is part of a feedback mechanism aimed at protecting the heart against lipid overload and consequently fatty acid–induced oxidative stress, one of the hallmarks of lipotoxic cardiomyopathy. Angptl4 has been shown to have a potent inhibitory effect in LPL activity and subsequent reduction in uptake of lipids by several tissues and cell types, including macrophages. Furthermore, Angptl4 was shown to prevent the formation of foam cells in mesenteric lymph nodes upon high fat feeding. Accordingly, we hypothesized that Angptl4 may affect atherosclerosis development by reducing foam cell formation. Thus, our second aim  was  to  investigate  the  role  of  Angptl4  on  atherosclerosis  development.  We  studied Angptl4 expression in atherosclerotic lesions and macrophages and determined the effect of Angptl4 transgenic overexpression in atherosclerosis prone ApoE3Leiden (E3L) mice fed a Western diet containing 0.4% cholesterol. We observed a decrease in atherosclerosis in Angptl4 overexpressing mice on an ApoE3L background. This effect was independent of the plasma cholesterol and triglyceride levels. Importantly, Angptl4Tg.E3L exhibited a less pro- inflammatory phenotype with decreased accumulation of monocytes/macrophages in the atherosclerotic plaque, suggesting an anti-inflammatory role of Angptl4 in atherosclerosis development. Finally, we set out to identify transcriptional targets of fatty acids in macrophages, as part of a general goal to elucidate mechanisms through which fatty acids exhibit a direct role in modulating inflammatory processes in macrophages. We identified Hig-2 to be strongly upregulated by all treatments. We found expression of Hig-2 to be the highest in peritoneal macrophages and white adipose tissue. Chronic high fat feeding increased Hig-2 expression levels in adipose tissue but not in liver. Immunohistochemistry indicated colocalization of Hig-2 with Cd68 in infiltrating macrophages as part of crown-like structures. Based on these findings we propose that Hig-2 has a specific role in macrophages and may function as an interesting target in the study of obese adipose tissue. In conclusion, this thesis contributes new information on gene regulation by dietary PUFA in the mammalian heart and provides mechanistic insight on their previous reported beneficial effects. Furthermore, we reveal a novel protective role of Angptl4 in atherosclerosis development. We propose that this effect is mediated by a mechanism, which is independent of inhibition of LPL-mediated systemic lipid clearance and it is probably related to the effect of Angptl4 on macrophage oxLDL uptake and chemotaxis. Finally, in the present thesis we start up an effort to identify fatty acid target genes in macrophages, which open new future research paths