Carboxylesterase 1 (Ces1) Releases Oxylipins From Oxidized Triacylglycerols: Examination of Its Substrate Selectivity

Abstract

Triacylglycerols (TAGs) are the most quantitatively important storage form of fat and are found primarily in cytoplasmic lipid droplets within cells. TAGs are broken down to its component free fatty acids by lipolytic enzymes when fuel reserves are required. However, when TAGs possess large quantities of polyunsaturated fatty acids (PUFAs) they are prone to nonenzymatic oxidation reactions, leading to formation of oxylipins (i.e., oxidized forms of fatty acids) that are esterified to the glycerol backbone (termed oxTAGs). Human carboxylesterase (CES1) is a member of the serine hydrolase superfamily and defined by its ability to catalyze the hydrolysis of carboxyl ester bonds in both toxicants and lipids. Although it is known that CES1 is a bona fideTAG hydrolase, it is unclear which specific fatty acids are preferentially released during lipolysis. Moreover, to better understand the biochemical function of CES1 in macrophages, we need to determine its substrate selectivity when it encounters oxidized PUFAs in TAG lipid droplets. Thus, the goal of this study is to systematically identify those oxidized fatty acids that are liberated from oxTAGs by CES1, because their release activates signaling pathways important for the development of lipid‐driven inflammation. Gaining this knowledge will fill data gaps that exist between CES1 and the lipid‐sensing nuclear receptors, PPARγ and LXRα, that are abundantly expressed in macrophages and are important drivers of lipid metabolism and inflammation. Oxidized forms of triarachidonoylglycerol (oxTAG C20:4) and trilinoleoylglycerol (oxTAG C18:2) – containing physiologically relevant levels of oxidized PUFAs (<5 mol%) – were incubated with recombinant CES1, or Pseudomonaslipase (a positive control), to assess the release of oxylipins and non‐oxidized arachidonic acid (AA) and linoleic acid (LA), which were quantified by LC‐MS. CES1 was shown to efficiently metabolize oxTAG C20:4 and oxTAG C18:2, yielding several regioisomers of hydroxyeicosatetraenoic acid (5‐, 12‐, and 15‐HETE) and hydroxyoctadecadienoic acid (9‐ and 13‐HODE), respectively. The CES1‐catalyzed cumulative release of HODEs was faster than that of HETEs from the respective oxTAGs (7 pmol HODEs/min vs. 1 pmol HETEs/min). Thus, for oxTAGs, hydroxy fatty acids derived from LA are preferred by CES1 to those derived from AA. A similar trend was noted for the release of non‐oxidized PUFAs from the respective oxTAGs (470 pmol LA/min vs. 83 pmol AA/min). CES1 also preferentially liberated 5‐HETE over 12‐HETE and 15‐HETE from oxTAG C20:4, whereas no differences were noted between 9‐HODE and 13‐HODE. This study indicates that CES1 can metabolize oxTAG lipids to release oxylipins and PUFAs. It further specifies the substrate selectivity of CES1 in the metabolism of bioactive lipid mediators that can regulate inflammatory activities of immune cells.