Myristic acid: in vivo evaluation of connection with cardiovascular risk factors and in vitro proteomic investigations of its biochemical effects

Fatty acids (FAs) are fundamental constituents of cell structure, but they can also influence cellular functions and molecular mechanisms with different effects according to their chain length and degree of saturation. Different pathological conditions have been linked to FAs profile, including dyslipidemia and hypertriglyceridemia. However, data concerning the effects of FAs on lipid metabolism and cardiovascular disease are still scarce and controversial. Therefore, the aim of the first part of the present PhD thesis (Section 1) has been to investigate the presence of possible significant correlations between plasma FAs profile and lipid parameters (including levels of the major apolipoproteins) in a population of Coronary Artery Disease (CAD) patients and controls. The analysis, performed on plasma of 1,370 subjects, revealed that Myristic acid (C14:0) positively predicted both Triglycerides (TG) and Apolipoprotein C-III (ApoC-III) plasma levels, confirming the preliminary data obtained in my master’s degree thesis on 57 CAD patients. ApoC-III being an important regulator of plasma TG levels, the influence of C14:0 on the expression of this protein has been investigated in a HepG2 cell model. Mass spectrometry results, together with Real Time PCR findings, suggest a slight but significant increase in ApoC-III protein and mRNA levels in C14:0 treated cells. Therefore, the in vitro investigations supported the positive connection observed in-vivo between C14:0, TG and ApoC-III plasma levels, suggesting a possible important role of this saturated FA in the onset of cardiovascular disease. The effects of FAs on liver metabolism have been studied during the last few years for the influence they have on lipid metabolism, the onset of nonalcoholic fatty liver disease (NAFLD) and cardiovascular disease. However, proteomics investigations in this direction are still scarce and the influence of C14:0 on liver metabolism still needs to be elucidated. Therefore, in the second part of the present PhD thesis (Section 2) the effects of different concentrations of C14:0 on HepG2 cells proteome and secretome have been investigated by means of high–resolution mass spectrometry. The results obtained highlighted the influence of this FA on proteins involved in lipid droplets formation, cytoskeleton organization, endoplasmic reticulum stress, exosome release and cell-cell stress communication. To highlight the proteome changes specifically related to C14:0, a comparative study of the proteomic modulations induced by C14:0, palmitic (C16:0) and oleic acid (C18:1) has been performed. Interestingly, the overlapping of modulated proteins induced by the three FAs treatments was limited to just one protein, highlighting their different mechanisms of action. 40 proteins were found to be deregulated specifically by C14:0. These results suggested a unique influence of this saturated FA on specific proteins involved in different biological processes, mainly protein homeostasis counteracting ER stress (e.g. ENTPD5, VAPB and SGTA) and lipid metabolism (e.g. ApoE). In conclusion, the present PhD thesis highlights for the first time a possible in-vivo fundamental role of C14:0 on lipid metabolism, particularly on ApoC-III and TG plasma levels and represents the first investigation shedding light on the in-vitro C14:0 effects on a human hepatocyte-derived cell line.