Discovery of FAHFA-Containing Triacylglycerols and Their Metabolic Regulation

Abstract

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.

AIG1 and ADTRP are endogenous hydrolases of fatty acid esters of hydroxy fatty acids (FAHFAs) in mice

“…Indeed, the absolute levels of TG-esterified FAHFAs are higher in BAT compared to WAT depots (Table S3). TG-esterified FAHFAs are depots for release of FAHFAs (28). TG-esterified FAHFAs are unlikely to be direct substrates of AIG1 and ADTRP because TG-esterified FAHFAs and FAHFAs are markedly different substrates.…”

“…Instead, the regulation of FAHFAs by AIG1 and ADTRP likely requires the release of FAHFAs from the TGs. Adipose triglyceride lipase (ATGL) has been identified as a hydrolase that can release FAHFAs from the TG pool (28,36). Hence the activity of ATGL, and AIG1 and ADTRP might coordinate FAHFA hydrolysis and signaling.…”

“…We recently described a novel pathway for FAHFA metabolism in which FAHFAs are esterified into triglycerides (TGs) to generate FAHFA-containing TGs (FAHFA-TG or TGesterified FAHFAs) (28). FAHFA-TGs are potentially a storage form of FAHFAs; they are >100-fold more abundant in tissues than nonesterified FAHFAs.…”

“…Tissues from ad lib fed female mice in C57/BL6J background at the age of 12-13 weeks were used for FAHFA analyses. FAHFA measurements and data analysis were performed as explained previously (28). 25-150 mg tissue was homogenized in 1.5:1.5:3 ml PBS:methanol:chloroform.…”

“…The lower phase was transferred into a new glass vial and dried. Dried lipids were fractionated using SPE columns (28). First, columns were equilibrated with 6 ml hexanes, then the samples were resuspended in 200 µl chloroform and loaded on the column.…”