Edited by Dennis R. VoelkerFatty acid esters of hydroxy fatty acids (FAHFAs) are a recently discovered class of biologically active lipids. Here we identify the linoleic acid ester of 13-hydroxy linoleic acid (13-LAHLA) as an anti-inflammatory lipid. An oat oil fraction and FAHFA-enriched extract from this fraction showed anti-inflammatory activity in a lipopolysaccharide-induced cytokine secretion assay. Structural studies identified three LAHLA isomers (15-, 13-, and 9-LAHLA) as being the most abundant FAHFAs in the oat oil fraction. Of these LAHLAs, 13-LAHLA is the most abundant LAHLA isomer in human serum after ingestion of liposomes made of fractionated oat oil, and it is also the most abundant endogenous LAHLA in mouse and human adipose tissue. As a result, we chemically synthesized 13-LAHLA for biological assays. 13-LAHLA suppresses lipopolysaccharide-stimulated secretion of cytokines and expression of pro-inflammatory genes. These studies identify LAHLAs as an evolutionarily conserved lipid with anti-inflammatory activity in mammalian cells.Fatty acid esters of hydroxy fatty acids (FAHFAs) 4 are a recently discovered class of lipids with anti-diabetic and anti-inflammatory activity (1). Because there are numerous FAHFAs, they are classified into families based on the composition of fatty acid and hydroxy fatty acid. For example, palmitic acid esters of hydroxy stearic acids (PAHSAs) and oleic acid esters of hydroxy stearic acids (OAHSAs) are two FAHFA families. Furthermore, within a FAHFA family, there are multiple regioisomers that differ in the position of the ester linkage (e.g. 5-PAHSA and 9-PAHSA) (1).Biological testing of 5-and 9-PAHSA revealed potent antidiabetic and anti-inflammatory activity (1-3). Mechanistic studies revealed that FAHFAs regulate several cellular and physiological pathways, with at least some of the biology being attributable to agonism of GPR120 and GPR40, two G proteincoupled receptors (1, 3). Other ligands for these G proteincoupled receptors include saturated and polyunsaturated fatty acids (4, 5). GPR120 is the endogenous receptor for omega-3 fatty acids, and it mediates the anti-inflammatory effects of these lipids (5).The anti-inflammatory activity of FAHFAs has been reported in vitro and in vivo (1, 2). Initially, cellular experiments with bone marrow-derived dendritic cells showed that treatment of cells with 9-PAHSA reduced the amplitude of cytokine secretion and expression of cellular inflammation markers. In addition, administration of 9-PAHSA to mice on a high-fat diet reduced inflammation in adipose tissue of treated mice (1). 9-PAHSA also showed robust anti-inflammatory activity in a mouse colitis model. Administration of 9-PAHSA to mice undergoing chemically induced colitis improved clinical and molecular inflammation (2). Moreover, an analysis of the impact of 9-PAHSA on the immune system revealed effects on the innate and adaptive immune system (2). Most recently, Kuda et al. (6) demonstrated that docosahexaenoic acid of 13-hydroxy linoleic acid (13-DHAHLA), a...

FAHFAs are a class of bioactive lipids which show great promise for treating diabetes and inflammatory diseases. Deciphering the metabolic pathways that regulate endogenous FAHFA levels is critical for developing diagnostic and therapeutic strategies. However, it remains unclear how FAHFAs are metabolized in cells or tissues. Here, we investigate whether FAHFAs can be incorporated into other lipid classes and identify a novel class of endogenous lipids, FAHFAcontaining triacylglycerols (FAHFA-TGs), which contain a FAHFA group esterified to the glycerol backbone. Isotope-labeled FAHFAs are incorporated into FAHFA-TGs when added to differentiated adipocytes which implies the existence of enzymes and metabolic pathways capable of synthesizing these lipids. Induction of lipolysis (i.e., triacylglycerol hydrolysis) in adipocytes is associated with marked increases in nonesterified FAHFA levels demonstrating that FAHFA-TGs breakdown is a regulator of cellular FAHFA levels. To quantify FAHFA levels in FAHFA-TGs and determine their regioisomeric distributions, we developed a mild alkaline hydrolysis method that liberates FAHFAs from triacylglycerols for easier detection. FAHFA-TG concentrations are greater than 1oo-fold than that of nonesterified FAHFAs, indicating that FAHFA-TGs are a major reservoir of FAHFAs in cells and tissues. The discovery of FAHFA-TGs reveals a new branch of TG and FAHFA metabolism with potential roles in metabolic health and regulation of inflammation.

Branched fatty acid (FA) esters of hydroxy FAs (HFAs; FAHFAs) are recently discovered lipids that are conserved from yeast to mammals1,2. A subfamily, palmitic acid esters of hydroxy stearic acids (PAHSAs), are anti-inflammatory and anti-diabetic1,3. Humans and mice with insulin resistance have lower PAHSA levels in subcutaneous adipose tissue and serum1. PAHSA administration improves glucose tolerance and insulin sensitivity and reduces inflammation in obesity, diabetes and immune-mediated diseases1,4–7. The enzyme(s) responsible for FAHFA biosynthesis in vivo remains unknown. Here we identified adipose triglyceride lipase (ATGL, also known as patatin-like phospholipase domain containing 2 (PNPLA2)) as a candidate biosynthetic enzyme for FAHFAs using chemical biology and proteomics. We discovered that recombinant ATGL uses a transacylation reaction that esterifies an HFA with a FA from triglyceride (TG) or diglyceride to produce FAHFAs. Overexpression of wild-type, but not catalytically dead, ATGL increases FAHFA biosynthesis. Chemical inhibition of ATGL or genetic deletion of Atgl inhibits FAHFA biosynthesis and reduces the levels of FAHFA and FAHFA-TG. Levels of endogenous and nascent FAHFAs and FAHFA-TGs are 80–90 per cent lower in adipose tissue of mice in which Atgl is knocked out specifically in the adipose tissue. Increasing TG levels by upregulating diacylglycerol acyltransferase (DGAT) activity promotes FAHFA biosynthesis, and decreasing DGAT activity inhibits it, reinforcing TGs as FAHFA precursors. ATGL biosynthetic transacylase activity is present in human adipose tissue underscoring its potential clinical relevance. In summary, we discovered the first, to our knowledge, biosynthetic enzyme that catalyses the formation of the FAHFA ester bond in mammals. Whereas ATGL lipase activity is well known, our data establish a paradigm shift demonstrating that ATGL transacylase activity is biologically important.