Distinct roles of adipose triglyceride lipase and hormone-sensitive lipase in the catabolism of triacylglycerol estolides

Branched esters of palmitic acid and hydroxy stearic acid are antiinflammatory and antidiabetic lipokines that belong to a family of fatty acid (FA) esters of hydroxy fatty acids (HFAs) called FAHFAs. FAHFAs themselves belong to oligomeric FA esters, known as estolides. Glycerol-bound FAHFAs in triacylglycerols (TAGs), named TAG estolides, serve as metabolite reservoir of FAHFAs mobilized by lipases upon demand. Here, we characterized the involvement of two major metabolic lipases, adipose triglyceride lipase (ATGL) and hormone-sensitive lipase (HSL), in TAG estolide and FAHFA degradation. We synthesized a library of 20 TAG estolide isomers with FAHFAs varying in branching position, chain length, saturation grade, and position on the glycerol backbone and developed an in silico mass spectra library of all predicted catabolic intermediates. We found that ATGL alone or coactivated by comparative gene identification-58 efficiently liberated FAHFAs from TAG estolides with a preference for more compact substrates where the estolide branching point is located near the glycerol ester bond. ATGL was further involved in transesterification and remodeling reactions leading to the formation of TAG estolides with alternative acyl compositions. HSL represented a much more potent estolide bond hydrolase for both TAG estolides and free FAHFAs. FAHFA and TAG estolide accumulation in white adipose tissue of mice lacking HSL argued for a functional role of HSL in estolide catabolism in vivo. Our data show that ATGL and HSL participate in the metabolism of estolides and TAG estolides in distinct manners and are likely to affect the lipokine function of FAHFAs.

Branched esters of palmitic acid and hydroxy stearic acid (PAHSAs) are antiinflammatory and antidiabetic lipokines (1–3). PAHSA serum and adipose tissue levels correlate with insulin sensitivity and are decreased in insulin-resistant humans (2, 4). PAHSAs increase glucose-stimulated insulin secretion by enhancing the production of the gut-derived incretin glucagon-like peptide-1 (5, 6). The antiinflammatory effects of PAHSA isomers (2, 7, 8) are mediated via free fatty acid receptor 4 (FFAR4, GPR120) and modulate both innate and adaptive immune responses in a mouse colitis model (1) and type-1 diabetes (6). Therefore, PAHSAs have beneficial effects on both metabolism and the immune system (9).PAHSAs belong to the family of fatty acid (FA) esters of hydroxy FAs (HFAs) called FAHFAs, which are part of a much larger family of mono- or oligomeric FAHFA esters named estolides. Since FAHFAs contain only a single ester bond of one FA with one HFA (the estolide bond), they represent monoestolides (10). The position of the branching carbon atom defines the regioisomer (e.g., 5-PAHSA or 9-PAHSA). PAHSAs and other less-well-studied FAHFAs such as the oleic acid esters of hydroxy palmitic acid (OAHPAs) or the docosahexaenoic acid ester of 13-hydroxy linoleic acid (13-DHAHLA) derive from either dietary sources or de novo synthesis in adipose tissue and other organs (2, 11, 12). Nonesterified, free FAHFAs (free mono-estolides) can be esterified to glycerol to form FAHFA acylglycerols, which in combination with other FAs result in the formation of triacylglycerol (TAG) estolides, diacylglycerol (DAG) estolides, or monoacylglycerol (MAG) estolides. TAG estolides represent a major storage form of bioactive free FAHFAs and are present in plant oils (e.g., castor oil) (13, 14) and adipose tissue of mice (3, 15) and humans (16).Both the synthetic and catabolic pathways of FAHFAs and TAG estolides are insufficiently understood. The hydrolytic catabolism of FAHFAs and TAG estolides results in the generation of highly bioactive and physiologically relevant FAHFAs, HFAs, FAs, and DAGs. Given the structural and metabolic similarity between TAGs and TAG estolides, it seemed reasonable to suspect that canonical TAG lipases will be involved in FAHFA and TAG estolide degradation. Generally, the catabolism of TAGs in cells occurs in the cytosol (neutral lipolysis) or in lysosomes (acidic lipolysis). Neutral lipolysis represents the predominant pathway for the hydrolysis of lipid droplet-associated TAGs in adipocytes involving three major enzymes, adipose triglyceride lipase (ATGL), hormone-sensitive lipase (HSL), and monoglyceride lipase (MGL). ATGL catalyzes the initial step of TAG hydrolysis, generating DAG and one FA (3, 17, 18). The enzyme belongs to the patatin-like phospholipase domain-containing (PNPLA) family of proteins comprising a number of lipid hydrolases (3, 15). ATGL is the most potent TAG hydrolase within this family but also exhibits some phospholipase, retinylesterase, and transacylase activities of undefined physiological relevance (17, 19, 20). For full TAG hydrolase activity, ATGL requires a coactivator named comparative gene identification-58 (CGI-58; also called α/β-hydrolase domain containing 5, ABHD5) (21–23). CGI-58 features α/β-hydrolase folds and also exerts some acyltransferase and protease activities (23–25). Yet, the physiological role of these activities remains elusive. ATGL exhibits unique regioselectivity for TAG substrates and preferentially hydrolyzes the sn-2 position of the glycerol chain of TAGs (26). Upon stimulation of ATGL by CGI-58 this regioselectivity broadens to the sn-1 but not the sn-3 position (26).HSL is rate-limiting for the second step of TAG lipolysis converting DAG to one FA and MAG (27). The enzyme preferentially catalyzes DAGs at the sn-3 position and cholesteryl esters (28, 29) but also cleaves TAGs (sn-1 and sn-3 position), retinylesters (30), or medium- and short-chain carboxylic acid glycerol esters (29). The enzyme is structurally unrelated to ATGL and does not require enzyme coactivators. Hormonal stimulation of neutral lipolysis by β-adrenoreceptor agonists such as catecholamines activates both ATGL and HSL by promoting the molecular interaction of ATGL with CGI-58 on the surface of TAG-containing lipid droplets (21, 31) and the translocation of HSL from the cytoplasm to lipid droplets. These processes involve the protein kinase A-dependent phosphorylation of perilipin-1, CGI-58, and HSL (31–33).Previous studies by Tan et al. (15) and our laboratory (3) demonstrated that ATGL and HSL are both able to hydrolyze FAHFA–glycerol ester bonds of TAG estolides. However, enzyme preferences for this reaction, the substrate requirements, or the contribution of these enzymes to hydrolyze the FA–HFA ester bond (estolide bond) in TAG estolides as well as in free FAHFAs remained unaddressed. Using a newly generated library of TAG estolides, we now show that ATGL and HSL play distinct roles in the formation of TAG estolides by transesterification reactions and the degradation of (TAG) estolides by hydrolysis reactions.