Adipose triglyceride lipase: the first transacylase for FAHFAs

**Background:** Fatty acid esters of hydroxy fatty acids (FAHFAs) are a diverse class of bioactive lipids with over 50 families and 600 regioisomers identified to date. Since their discovery in mammals in 2014, FAHFAs have been recognized for their anti-inflammatory and anti-diabetic properties. However, progress in understanding their physiological roles has been limited by two major challenges: technical difficulties in detecting low (nanomolar) levels of specific FAHFA species without artifact interference, and an incomplete understanding of their in vivo biosynthesis and degradation. While four putative FAHFA hydrolases (carboxyl ester lipase, androgen-dependent tissue factor pathway inhibitor regulating protein, androgen-induced gene 1, and hormone-sensitive lipase) had been identified, no biosynthetic enzyme for FAHFAs was known prior to this work.

**Methods:** The study by Patel et al., published in Nature, built on previous observations that adipose-specific Glut4 overexpression (AG4OX) mice showed 8- to 16-fold elevation of FAHFA levels in white adipose tissue. The authors established an FAHFA biosynthetic assay in adipocytes by adding exogenous 9-hydroxy stearic acid (9-HSA) and Cis-10-heptadecenoic acid (C17:1) and measuring 9-C17:1-HSA formation. C17:1 was selected due to its low endogenous abundance to minimize background interference. AG4OX adipocytes showed a 2-fold increase in 9-C17:1-HSA compared to wild-type adipocytes. To block FAHFA degradation, cells were treated with methyl arachidonyl fluorophosphonate (MAFP), a covalent inhibitor of serine/threonine lipases that inhibits all four known FAHFA lipases. Unexpectedly, MAFP caused a dose-dependent decrease in FAHFA, suggesting the biosynthetic enzyme(s) were also MAFP-sensitive. Using this finding, the authors performed activity-based protein profiling with a fluorophosphonate (FP)-alkyne probe. Candidate proteins were biotinylated via click chemistry, enriched by streptavidin pull-down, and analyzed by proteomics.

**Key Results:** Among several candidates identified by proteomics, only ATGL (adipose triglyceride lipase, also known as PNPLA2) passed all subsequent in vitro and in vivo validation as a FAHFA biosynthetic enzyme. Notably, ATGL does not synthesize FAHFA directly from free fatty acids and hydroxy fatty acids. Instead, ATGL utilizes triglyceride (TG) as the primary acyl donor, and to a lesser extent diglyceride (DG), transferring the fatty acid to hydroxy fatty acid to generate FAHFA. Both the transacylase and lipase activities of ATGL require the co-activator CGI-58 and show higher substrate specificity for TG than DG. The transacylase activity is abolished when ATGL lipase activity is blocked by the chemical inhibitor atglistatin or by inactivating point mutations, indicating the two activities are intimately linked. The authors propose a model where both activities proceed through the same acyl-enzyme intermediate, with the key difference being whether the acyl group is transferred to water (lipase activity) or to hydroxy fatty acid (transacylase activity).

**Clinical Implications:** This identification of ATGL as the first FAHFA biosynthetic enzyme represents a major breakthrough in bioactive lipid research. It provides a critical missing piece for understanding FAHFA dynamics in vivo and opens the door to studying how FAHFA levels are regulated in metabolic diseases such as diabetes and obesity. The discovery that ATGL possesses transacylase activity beyond its well-characterized lipase function may represent a paradigm shift in the field. Important questions remain, including how hydroxy fatty acids are generated and delivered to ATGL, what factors regulate the switch between ATGL's lipase and transacylase activities, and whether these activities can be pharmacologically dissociated. Structural studies of ATGL in complex with its substrates would provide valuable insights for targeting this enzyme therapeutically.