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. 2021 Jan 12;118(2):e2020999118.
doi: 10.1073/pnas.2020999118.

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

Kristyna Brejchova  1 , Franz Peter Walter Radner  2 , Laurence Balas  3 , Veronika Paluchova  1 , Tomas Cajka  1 , Hana Chodounska  4 , Eva Kudova  4 , Margarita Schratter  2 , Renate Schreiber  2 , Thierry Durand  3 , Rudolf Zechner  5   6 , Ondrej Kuda  7
Affiliations

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

Kristyna Brejchova et al. Proc Natl Acad Sci U S A. .

Abstract

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.

Keywords: ATGL; FAHFA; HSL; lipokine.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
Synthesis of TAG estolide 70:4 isomers. (A) Scheme of the organic synthesis of TAG estolide (TAG EST) 70:4 isomers. 1,2- and 1,3-dilinoleoyl glycerol (1,2/1,3-DAG18:2/18:2) were esterified with palmitic acid ester of hydroxy stearic acid with branching positions at carbon atom 5, 7, or 9 (5-, 7-, or 9-PAHSA). Both sn-1 and sn-3 products were synthesized, as the starting glycerol (red structure) was a racemic mixture. The internal ester linkage within the PAHSA (blue structure) is the estolide bond (branching position). (B) Illustrative chromatograms of the TAG EST 70:4 and 66:1 isomers. The FAHFA (PAHSA or oleic acid ester of hydroxy palmitic acid [OAHPA]) is bound at the sn-1/3 position of the glycerol.
Fig. 2.
Fig. 2.
The FAHFA–glycerol ester bond in TAG estolides is preferentially hydrolyzed by ATGL. (A) Release of PAHSAs from TAG estolide 66:0 with 5-, 7-, or 9-PAHSA bound at the sn-1/3 position. (B) Release of 5-, 7-, or 9-OAHPAs from TAG estolide 66:1 series. (C) Release of 16-OAHPA from the TAG estolide 66:1 with 16-OAHPA bound at the sn-1/3 position. Data are presented as means ± SEM (n = 3). Statistical differences were determined by one-way ANOVA with multiple comparison test (Tukey). *Significantly different at P < 0.05 from the β-Gal group. Additional statistics are presented in SI Appendix.
Fig. 3.
Fig. 3.
Regioselectivity of ATGL-, ATGL+CGI-58–, and HSL-mediated glycerol ester bond hydrolysis. (A and B) Lipolytic products formed by the hydrolysis of TAG estolide (TAG EST) 70:4 with 5-PAHSA bound at sn-1/3 (marked sn-1 due to ATGL position specificity) or sn-2 position are shown. Data are presented as means ± SEM (n = 3). Statistical differences were determined by one-way ANOVA with multiple comparison test (Tukey). *Significantly different at P < 0.05 from the β-Gal group. Additional statistics are presented in SI Appendix. DAG 52:2 stands for DAG 5-PAHSA_18:2.
Fig. 4.
Fig. 4.
TAG estolide remodeling yields alternative TAG estolide combinations. (A) Schematic representation of the main biochemical reactions running simultaneously or sequentially within the TAG estolide (TAG EST) 70:4 (5-PAHSA at sn-1/3) hydrolysis assay. The substrate as well as other substrates present in the cell lysate are metabolized by ATGL and other enzymes in the lysate. These pools of intermediates serve as both acyl donors and acceptors in reactions catalyzed by ATGL (acyltransferase, transacylase reaction). TAG estolides with one, two, or three PAHSAs and alternative estolides originating from FAHFA transfer or estolide remodeling were detected mainly in the samples where ATGL was coactivated by CGI-58. All structures with acyl chain composition were annotated based on MS/MS spectra. (B–F) Levels of selected estolides from A including MS/MS spectra. Data are presented as means ± SEM (n = 3). Fold change over ATGL+CGI-58 group.
Fig. 5.
Fig. 5.
The estolide bond in TAG estolides is preferentially hydrolyzed by HSL. Hydrolysis of TAG estolide 66:1 series with OAHPA bound at the sn-1/3 position and with OAHPA branching at the carbon atom 5, 7, 9 (A), and 16 (B), respectively. Data are presented as means ± SEM (n = 3). Statistical differences were determined by one-way ANOVA with multiple comparison test (Tukey). *Significantly different at P < 0.05 from the β-Gal group. Additional statistics are presented in SI Appendix. TAG 48:0(OH), monohydroxylated TAG intermediate; HPA, hydroxy palmitic acid. The 5-, 7-, and 9-HPA and the corresponding TAGs 48:0 (OH) were not found in A reactions.
Fig. 6.
Fig. 6.
Hydrolysis of free OAHPAs with branching at the carbon atom 5, 7, 9, or 16. Production of FA 18:1 (A) and the corresponding HPA (B). Data are presented as means ± SEM (n = 3). Statistical differences were determined by one-way ANOVA with multiple comparison test (Tukey). *Significantly different at P < 0.05 from the β-Gal group. Additional statistics are presented in SI Appendix.
Fig. 7.
Fig. 7.
Concentrations of TAG estolides and free FAHFAs in epididymal white adipose tissue of ATGL and HSL KO mice. Epididymal white adipose tissue from WT, ATGL KO, and HSL KO mice was collected in the ad libitum-fed and 14-h-fasted state. (A and B) TAG estolide species were quantified and data sorted according to the number of carbon atoms and the number of double bonds. The color scale represents the mean concentration (n = 5 to 7). When multiple isomers were detected, the square was divided into sections. (C) Concentrations of total TAG estolide and (D) various free FAHFAs in HSL KO white adipose tissue compared to WT control samples. Data are presented as means ± SEM (n = 5). Statistical differences were determined by two-way ANOVA with multiple comparison test (Tukey). *Significantly different at P < 0.05 for genotype. Additional statistics are presented in SI Appendix.
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