Regulación de la formación de gotas lipídicas por ácido araquidónico en monocitos humanosimportancia del ácido graso 16:1n-9

Resumen

INTRODUCTION Lipid Droplets (LD) are intra-cytosolic organelles present in virtually all cell types. These organelles are composed of a neutral lipid core surrounded by a phospholipid monolayer, which provides them a hydrophobic environment within the cells. LD surface contains multiple proteins, some of them involved in lipid metabolism. LD are formed in response to multiple stimuli, such as lipid overload, stress or cellular activation. The most accepted theory proposes that LD are synthesized between the two leaflets of the cytosolic face of the smooth endoplasmic reticulum in a tight regulated process which includes fatty acyl-CoA generation, neutral lipid synthesis, phospholipid remodeling and protein and phospholipid biosynthesis. LD are currently regarded as greatly dynamic structures which take part in a wide range of cellular processes and whose dysregulation is involved in several diseases, such as metabolic syndrome-related pathologies, neoplastic processes, neurodegenerative diseases or hepatitis C virus infection. In immune cells LD are produced in response to multiple cell-activating agents and are involved in the control and amplification of the inflammatory response. Some PLA2, key enzymes in the lipid metabolism, are related to LD biosynthesis, either providing fatty acids for the neutral lipid synthesis, modifying lipid-containing particles to allow their internalization by cells, producing metabolites that regulate LD generation, or participating in the own biogenesis of the organelles. Atherosclerosis is the main pathology in cardiovascular disease, the world-leading cause of death. This disorder is included within metabolic syndrome-related diseases and shares the same risk factors with other common diseases such as non-alcoholic steatohepatitis, obesity or type 2 diabetes. Atherosclerosis is a multifactorial pathology that initially consists on fat, cells and matrix deposition in the blood vessels intima. Monocyte-derived macrophages are key cells in the fatty streak development through the accumulation of lipid droplets when are exposed to modified LDL in the sub-endothelial space, becoming foam cells. The long-term accumulation of foam cells can lead to fatal consequences. In the last years, circulating monocytes have also been considered as key cells in the early atherosclerotic plaque development through the formation of lipid droplets prior to their extravasation, becoming foamy monocytes, and increasing their pro-inflammatory activation, extravasation potential and atherogenic properties in several models. In this work, LD synthesis by monocytes exposed to arachidonic acid (AA), a fatty acid present in the atherosclerosis foci, is proposed as a necessary study in the search of mechanisms and early biomarkers of foamy monocytes formation. Palmitoleic acid has been described as a lipid hormone or lipokine produced in adipose tissue and released to the bloodstream, with beneficial effects in metabolic syndrome-related pathologies. However, other studies have shown that palmitoleic acid has detrimental effects in other biological models. Moreover, palmitoleic acid has been described as an anti-inflammatory fatty acid in some mouse models, but the measurement of its high plasma levels is related to several markers of disease and inflammation in epidemiologic studies in humans. The discrepancies found in the study of biological effects of this fatty acid could be given by the different chemical forms in which palmitoleic acid is present in cells, ranging from free fatty acid, esterified in other lipids, forming phosphatidylinositol molecules or oxidized, being part of FAHFA. Other isomers of palmitoleic acid with biological implications, such as sapienic and palmitelaidic acid, have also been detected in cells, and the search and study of other major isomers deserves more attention. OBJECTIVES The recent discovery of lipid droplet deposition in circulating monocytes as a mechanism in the early atherosclerosis development leads to the first main goal: the study of the regulation of lipid droplet formation by arachidonic acid in human monocytes. On the other hand, palmitoleic acid is in the spotlight of lipid research due to its behavior as a lipokine with several beneficial metabolic effects. Hence, the design of an experimental method for palmitoleic acid isomers detection and quantification is required, as well as, a functional and biochemical study of new isomers in immune cells. EXPERIMENTAL METHODS A lipidomic approach was employed for the analysis of neutral lipids, main components of lipid droplets, and for the structural study of palmitoleic acid isomers by gas chromatography/mass spectrometry. Other mass spectrometry techniques were used for the determination of palmitoleic acid-containing phospholipids and a phospholipidome profile. Several complementary techniques such as western blot, fluorescence microscopy, qPCR, radioactivity assays or ELISA were used for the measurement of protein and gene expression levels and the synthesis of lipid droplets by human monocytes. RESULTS AND DISCUSSION REGULATION OF LIPID DROPLET FORMATION BY ARACHIDONIC ACID IN HUMAN MONOCYTES AA (10 M, 2 hours), induced neutral lipid synthesis and LD formation in human monocytes. On one hand, the neutral lipid synthesis was due to the direct AA incorporation into TAG and, on the other hand, to the mobilization of other fatty acids such as 16:0, 16:1, 18:0 or 18:1n-9 towards TAG and CE molecules. Both mechanisms can be dissociated by the arachidonoyl-CoA synthetases inhibitor triacsin C, which inhibited the former but conserved the latter. Other unsaturated fatty acids (18:1n-9, 18:2n-6, 18:3n-6) induced LD to a lesser extent, but unlike AA, the LD synthesis was inhibited by the preincubation of cells with TC. Therefore, in all subsequent experiments, the study of the signaling pathway leading to LD formation by AA was carried out in the presence of TC. AA triggered de novo fatty acid synthesis by human monocytes. AA did not alter the phospholipid levels in human monocytes, ruling out the transfer of fatty acids from PL to TAG and CE during the biosynthesis of LD. However, the total amount of cellular fatty acids was increased after AA treatment, showing a higher rate of acetyl-CoA use for fatty acid synthesis without changes in the -oxidation rate and suggesting that de novo fatty acid synthesis was activated by AA. This fatty acid increased the gene expression of several proteins involved in lipogenesis, such as ACC, FAS, ELOVL6, SCD-1, SREBP-1c and SREBP-2. The chemical inhibition of the de novo fatty acid synthesis pathway in different steps resulted in a decrease of TAG synthesis, suggesting that fatty acids recruited for neutral lipid and lipid droplet formation in the presence of TC, came from the de novo fatty acid synthesis pathway. Triacsin C inhibited exogenous AA and 16:0 incorporation into cellular lipids, but did not affect the incorporation of fatty acids coming from this pathway. It has been hypothesized that acyl-CoA pools might be compartmentalized, and different ACSL, affected or not by TC inhibition, could catalyze fatty acid thioesterification to coenzyme A. Lipid droplet formation by AA in human monocytes required the cPLA2 phosphorylation at Ser505 by the simultaneous effect of p38 and JNK MAPK. cPLA2 inhibition by its selective inhibitor pyrrophenone resulted in LD formation abrogation, suggesting a key role of this enzyme in LD biosynthesis by AA. Exogenous AA promoted the cPLA2 phosphorylation at Ser505 residue alongside with an increase in its catalytic activity and the activation of the signaling pathways of p38 and JNK, but not ERK. MAPK are well-known activators of cPLA2 by phosphorylation in response to different stimuli. In order to identify which kinases were involved in cPLA2 phosphorylation, some experiments with inhibitors of MAPK were performed, identifying p38 and JNK kinases as regulators of cPLA2 phosphorylation in response to exogenous AA. The total phosphorylation of cPLA2 was achieved when both kinases were acting at the same time. Since cPLA2 is a key enzyme in LD formation in response to AA, the inhibition of both, p38 and JNK pathways, resulted in LD formation by AA suppression, parallel to the inhibition of cPLA2 phosphorylation. The inhibition of only one of MAPK proteins resulted in neutral lipid synthesis and LD formation, since cPLA2 phosphorylation levels were higher than controls. In summary, AA activated p38 and JNK pathways, whose simultaneous effect phosphorylated cPLA2 at Ser505, a key event in lipid droplet formation in human monocytes. JNK, but not p38, was previously described as a key cPLA2 activating pathway in LD formation. Since two parallel MAPK pathways were involved in the activation by phosphorylation of cPLA2 on the same residue, it is reasonable to consider the hypothesis that an intermediate kinase was being phosphorylated by p38 and JNK and then it was phosphorylating cPLA2 at Ser505, as it has been described in other models of cPLA2activation. The mechanism by which cPLA2 directly regulates LD biosynthesis could be due to its catalytic action on the smooth endoplasmic reticulum phospholipids during organelle budding, catalyzing the conversion of cylindrical-shape phospholipids into wedge-shape lysophospholipids, which induces a necessary local positive curvature during organelle synthesis. The same studies made on mature macrophages, other immune cells that are involved in atherosclerosis development through the accumulation of lipid droplets, gave rise to similar qualitative results. AA promoted neutral lipid synthesis in the presence of TC, but to a lesser extent than in human monocytes. Oxidized LDL, a classical atherosclerosis stimulus, also induced neutral lipid synthesis in circulating monocytes, but to a lesser extent than AA, showing the great potential of this fatty acid as a LD promoter in monocytes. It is tempting to speculate that prior to perform its biological effect, cells convert AA into some eicosanoids that could be the bioactive compounds in this model. The enzymatic synthesis of eicosanoids by monocytes is unlikely to happen according to preliminary experiments and bibliography, but some of the products formed by the non-enzymatic oxidation of AA need a further research to determine if some of them are directly involved in LD formation by exogenous AA in these cells. A central role of cPLA2 in lipid droplet formation in human monocytes has been identified. Hence, this enzyme might be an interesting target in the prevention of foamy monocytes formation in human models. Interestingly, the cPLA2 deficient mice present less atherosclerotic injuries in aorta under a high-fat diet than their wild type littermates. Preventing foamy monocytes formation by the loss of this enzyme could be one of the mechanisms by which these mice would be protected against this disease. Moreover, this enzyme has also been related to other metabolic diseases, and its deficiency protects mice from abdominal fat accumulation, TAG deposition in liver and hepatic steatosis induced by high-fat diet. 16:1n-9, A FATTY ACID FORMED FROM OLEIC ACID BY -OXIDATION, ACCUMULATES IN NEUTRAL LIPIDS Among all fatty acids increased by AA-exposed monocytes, one of them, preliminarily identified as a 16:1 isomer, was especially augmented by the treatment. After the experimental design of a method that includes a double FAME/DMOX derivatization, the fatty acid was identified as 16:1n-9, a rare fatty acid with barely references in the bibliography, which had not been previously studied in-depth. Monocytes were more than 3-fold enriched in 16:1n-9 than in palmitoleic acid, the main 16:1 isomer in almost all cells and tissues. Regarding the distribution among cellular lipids, 16:1n-9 showed a different location than palmitoleic and the rest of fatty acids. 75 % of basal 16:1n-9 was esterified in neutral lipids (DAG, TAG and CE) and only 21 % in phospholipids, conversely to the rest of fatty acids, primarily incorporated into PL, which is the main fatty acid reservoir in cells (fatty acids esterified in PL constitute the 80 % of total cellular fatty acids). Palmitoleic acid was only esterified in PL. The incorporation of both 16:1 isomers into phospholipid classes was also altered. 16:1n-7 was incorporated into PC species to a greater extent, whereas 16:1n-9 was preferentially incorporated into PA species. All these differences between both isomers suggest possible different roles in cells. 16:1n-9 was formed from oleic acid by partial -oxidation in mitochondria in human monocytes. The enrichment of cells with oleic acid at physiological concentrations resulted in the increase of total 16:1n-9 levels. To confirm the biochemical origin of 16:1n-9, monocytes were labeled with deuterated oleic acid. Labeled 16:1n-9 was detected by GC/MS esterified essentially in TAG, followed by phospholipids. In order to determine whether 16:1n-9 was produced from oleic acid by -oxidation, monocytes were preincubated with the CPT1 inhibitor etomoxir, which inhibits the fatty acid importation into mitochondria. Etomoxir decreased the total 16:1n-9 formed from oleic acid in human monocytes, suggesting that this fatty acid was formed by one cycle of partial -oxidation from oleic acid in the mitochondria, since peroxisome, the other organelle in which -oxidation could be carried out, does not use the CPT system to import fatty acids. Finally, the retroconversion of oleic acid into 16:1n-9 was increased when cells were exposed to exogenous AA. This result is in agreement with the high amount of 16:1n-9 produced in response to AA, which could not be only explained in terms of the increase of its substrate, the oleic acid. A direct functional and physical contact between mitochondria and LD has been described, and some fatty acids from TAG lipolysis in LD are used by mitochondria for -oxidation, suggesting that this process would be bidirectional and the shortened fatty acids could reverse to LD. 16:1n-9, A POTENTIAL ANTI-INFLAMMATORY FATTY ACID IN MOUSE Using the experimental approach previously designed, the fatty acid 16:1n-9 was also found in mouse resident peritoneal macrophages (mRPM), as well as in multiple organs and tissues such as liver, brain, heart, lung, kidney, white and brown adipose tissue, muscle and spleen. mRPM presented 4-fold more 16:1n-9 than 16:1n-7, conversely to the rest of tissues and organs, including mouse serum, which exhibited a higher mass of palmitoleic acid than 16:1n-9. In terms of the distribution of both isomers among all tissues and organs, either in PL or TAG, 16:1n-7 was more abundant than 16:1n-9 with the exception of mRPM. In CE, 16:1n-9 esterified in mRPM was 10-fold higher than the rest of biological samples. Comparing the distribution of both isomers in mRPM, 16:1n-9 was esterified preferentially in CE, similarly to monocytes, whereas palmitoleic acid was only incorporated into cellular phospholipids and was not present in other lipid classes. Finally, the modulation of the inflammatory response by both fatty acid isomers was studied. Mouse resident peritoneal macrophages were preincubated for 2 hours with 16:1n-9, 16:1n-7 or DHA as a positive control, and cells were challenged with bacterial lipopolysaccharide. As previously described, palmitoleic acid-enriched macrophages resulted in the inhibition of Tnf and Nos2 gene expression. 16:1n-9 cell preincubation significantly inhibited those two markers and Il6, Il12a, Il23a and Il1 as well, to a higher extent than palmitoleic acid. To amplify these results, several in vivo experiments were carried out. Fatty acids and endotoxin were injected intraperitoneally to mice. Serum circulating levels of Il6 were measured by ELISA, finding that 16:1n-9 strongly inhibited Il6 production in response to LPS. This effect was not observed with palmitoleic acid or DHA. This suggests that the novel described fatty acid 16:1n-9 possesses anti-inflammatory properties in local and systemic models of inflammation in mouse. There are only a few studies describing the biological effects of 16:1n-9 and all of them are epidemiological. High levels of this fatty acid in plasma and erythrocytes are related to an elevated risk of sudden cardiac arrest and more incidence of metabolic syndrome and hepatic steatosis. In that way, 16:1n-9 behaves as its isomer palmitoleic acid, which presents an anti-inflammatory component in vitro, but whose increased circulating levels are related to the development of damaging effects. These discrepancies could be due to the different biological forms in which this fatty acid is measured and the wide range of biological metabolites through which might be acting. The limited literature about this fatty acid, the interest generated by its isomer palmitoleic acid, the recent discovery of its biochemical pathways of synthesis and its behavior as a potent anti-inflammatory fatty acid in vitro and in vivo invite to a more in-depth study of 16:1n-9 properties. CONCLUSIONS  Regarding the first general goal, the study of the regulation of lipid droplet formation by arachidonic acid in human monocytes in the context of their conversion into foamy monocytes in a model of atherosclerosis, the obtained results led to the following conclusions: 1. AA induced neutral lipid synthesis and lipid droplet formation in human monocytes, to a greater extent than other tested fatty acids. On one hand, neutral lipid synthesis was due to the direct AA incorporation into cellular TAG, and on the other hand, to the incorporation of other fatty acids towards TAG and CE molecules. Both pathways can be dissociated by using triacsin C, an acyl-CoA synthetases inhibitor. 2. AA stimulated the de novo fatty acid synthesis in human monocytes. Fatty acids synthesized by such pathway were used to neutral lipid synthesis by cells. 3. AA promoted cPLA2 phosphorylation at Ser505 by a simultaneous effect of p38 and JNK. This is a key event prior to lipid droplet biosynthesis by human monocytes. 4. AA also induced neutral lipid synthesis in human monocyte-differentiated macrophages. Similarly, oxidized LDL promoted neutral lipid accumulation in human monocytes, although to a lesser extent than AA.  Regarding the second general goal, which was the study of palmitoleic acid isomers in innate immune cells, the experimental evidences generated the following results: 5. The most increased fatty acid in neutral lipids of monocytes and macrophages challenged to atherosclerosis stimuli was a palmitoleic acid isomer. The design and application of an experimental method for its analysis allowed the determination of the fatty acid 16:1n-9. 6. Unlike palmitoleic acid and the rest of cellular fatty acids, 16:1n-9 was esterified in neutral lipids in non-treated human monocytes. These cellular lipids were more enriched in 16:1n-9 than in palmitoleic acid, showing differences in their incorporation patterns into phospholipid classes. 7. The fatty acid 16:1n-9 was formed from oleic acid by partial-oxidation in mitochondria in human monocytes. The conversion rate was increased when cells were exposed to AA. 8. The fatty acid 16:1n-9 was detected in mouse cells, tissues and organs, and it was especially abundant in peritoneal macrophages, where it was esterified to a greater proportion than palmitoleic acid. 9. Both in vitro and in vivo preincubation of peritoneal cells with the fatty acid 16:1n-9 reduced the expression of molecules involved in the local and systemic inflammatory response to LPS.