All reagents were purchased from Thermo Fisher unless specified otherwise. Collagenase D (11088858001) and dispase II (04942078001) were purchased from Roche. Ammonium acetate (372331), ammonium hydroxide (338818), MAFP (M2939) and C17:1 FA (H8896) were purchased from Sigma. Enzyme inhibitors were as follows: atglistatin (ATGL inhibitor), A 922500 (DGAT1 inhibitor), TSI-01 (lysophosphatidylcholine acyltransferase inhibitor), ML-348 (LYPLA1 inhibitor), ML-349 (LYPLA2 inhibitor) and etomoxir (carnitine palmitoyltransferase 1 inhibitor) were purchased from Cayman. PF-06424439 (DGAT2 inhibitor) was purchased from Sigma. Acyl donors were as follows: TG(18:1) (95%, Cayman 26871), TG(18:1) (99%, Sigma T7140), TG(16:1) (98%, Sigma T2630), TG(18:2) (98%, Sigma T9517), TG(16:0/18:1/18:1) (98%, Cayman 28559), TG(17:1) (≥98%, Cayman, 26996), DG(18:1/18:1/0:0) (≥97%, Sigma D0138), PC(18:1/18:1) (Cayman 15098) and oleoyl-CoA (FA-CoA, Avanti 870719) were purchased from the indicated companies. 9-has (ref. 43), D20-9-HSA, FP-alkyne, [13C16]9-PAHSA (ref. 1) and 9-C9-HSA were synthesized by the laboratory of D.S. Chemical synthesis is described in the Supplementary Methods. D31-palmitic acid was purchased from Cayman (16497). HEK293T, HPEG2 and 3T3L1 cell lines were purchased from ATCC and tested for mycoplasma contamination. WT and catalytically dead mouse ATGL plasmids37 and immunopurified CGI-58 enzyme were described before44,45. Full-length ATGL was expressed with an N-terminal 6xHis-tag in Expi293F cells (Thermo Fisher Scientific) and purified by affinity chromatography using an Akta pure chromatography system (Cytiva). Mouse ATGL-288 (amino acids 1–288) and ATGL-288(S47A) were immunopurified using the following method: mouse ATGL-288 and mouse ATGL-288(S47A) with an N-terminal 6×His-tag and a C-terminal Strep-tag II were expressed in Escherichia coli Arctic Express cells at 10 °C and purified by two-step affinity chromatography using an Äkta avant 25 chromatography system (Cytiva)38. We obtained highly purified WT ATGL (His–mAT288–Strep) and catalytically dead mutant (His–mAT288(S47A)–Strep) ATGL as assessed by Coomassie staining of SDS–polyacrylamide gel electrophoresis gels (Extended Data Fig. 4a, b). Mouse DGAT1/2 plasmid46, DGAT1 and DGAT2 antibodies47 and Dgat1-KO48 mice were a gift from R. V. Farese’s laboratory. Solvents for LC–MS were purchased from Honeywell Burdick & Jackson. Human SVF pre-adipocytes and SQ tissue biopsies were provided by the Adipose Tissue Biology and Nutrient Metabolism Core (Boston Nutrition Obesity Research Center). The samples were completely de-identified and were provided in a manner that does not link the samples to any private protected information. All adipose tissue donors signed an informed consent form approved by the Boston Medical Center and Boston University Medical Campus Institutional Review Board. The use of de-identified human samples for this study was approved by the Institutional Review Board at Beth Israel Deaconess Medical Center.

Cell culture and differentiation

3T3-L1 and SVF cell differentiation

3T3-L1 fibroblasts were cultured in high-glucose DMEM, supplemented with 10% FBS and antibiotic–antimycotic, at 37 °C and 5% CO2. Pre-adipocytes from the SVF of the SQ WAT were isolated from 9–10-week-old mice. Briefly, both fat pads were minced using scissors and digested in 5 mg ml−1 collagenase D, 2 U ml−1 dispase II and 10 mM CaCl2. SVF cells were then separated from adipocytes using 40-µm cell strainers and seeded on a 10-cm plate in DMEM F12 GlutaMAX supplemented with 15% FBS and 1% antibiotic–antimycotic. After 24 h, unattached cells were removed with PBS washing. SVF pre-adipocytes were cultured until 80–90% confluency. Cells from multiple mice were pooled before differentiation, so each treatment condition was performed on replicate wells from 6–9 mice.

For adipocyte differentiation, SVFs and 3T3-L1 cells were seeded in 12-well plates and grown to confluency. Differentiation medium (DMEM F12 GlutaMAX, 10% FBS, 1% antibiotic–antimycotic, 4 µg ml−1 bovine insulin, 1 µM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine and 2 µM rosiglitazone) was added to SVFs the day after confluency, and to 3T3-L1 cells 3 days after confluency. SVFs and 3T3-L1 were cultured for 3 days in differentiation medium. Cells were then cultured in a post-differentiation medium containing 10% FBS, 1% antibiotic–antimycotic and 4 µg ml−1 bovine insulin for 7–8 days. Human SVF pre-adipocytes were cultured and differentiated as described previously49. Uniform lipid droplet formation among cells was confirmed before FAHFA biosynthesis experiments through visual inspection under a microscope and/or oil red O staining.

Transfections in HEK293T cells

Cells were cultured in high-glucose DMEM, supplemented with 10% FBS and 1% antibiotic–antimycotic, at 37 °C and 5% CO2. For FAHFA biosynthesis experiments, HEK293T cells were seeded as 200,000 cells per well on 6-well plates (Primaria). After 18–20 h, cells were transiently transfected with 1.5 µg per well WT ATGL, ATGL(S47A) mutant, DGAT1, DGAT2, GFP or control vector plasmid using Lipofectamine 2000. FAHFA biosynthesis experiments were performed in intact cells 48 h after transfections.

Intact cell FAHFA biosynthesis assays

Transiently transfected HEK293T cells, differentiated mouse and human adipocytes or HEPG2 cells (human hepatoma) were incubated with 9-HSA or D20-9-HSA and C17:1 FA in phenol-free medium containing 1% FBS for 2 h (Fig. 1 and Extended Data Fig. 1) or 4 h (Figs. 2 and 4 and Extended Data Figs. 2, 3 and 6) at 37 °C. The specific concentration used for each experiment is indicated in the figure legends. For MAFP, FP-alkyne and enzyme inhibitor studies, adipocytes were pre-incubated with these compounds for 1 h. Adipocytes were preincubated for 1 h with the following inhibitors: atglistatin (ATGL inhibitor, 10 µM), TSI-01 (lysophosphatidylcholine acyltransferase inhibitor, 25 µM), ML-348 (LYPLA1 inhibitor, 10 µM), ML-349 (LYPLA2 inhibitor, 10 µM) and etomoxir (carnitine palmitoyltransferase 1 inhibitor, 10 µM) for experiments in Fig. 2a, b. Adipocytes were co-incubated with FA and HFA substrates, atglistatin (10 µM), A 922500 (DGAT1 inhibitor, 20 µM) and PF-06424439 (DGAT2 inhibitor, 20 µM) for experiments in Figs. 2c–l and 4 and Extended Data Fig. 2 and 3. After incubation with FAs and HFAs, cells were washed with ice-cold PBS once and collected in sterile 550 µl PBS. Cells were stored at −80 °C until lipid extractions. Protein concentrations were determined using the bicinchoninic acid assay at the time of lipid extraction.

Activity-based proteomics sample preparation

WT and AG4OX SVF adipocytes were pretreated with 5 µM FP-alkyne for 1 h and then co-incubated with 9-HSA for 2 h. Cells were then washed with ice-cold PBS twice, scraped, sonicated to obtain cell lysates and stored at −80 °C.

Click chemistry and biotinylated protein enrichment was performed as described previously50. Alkyne-labelled proteomes (1.5 mg ml−1 in 1 ml PBS) were incubated with biotin-N3 (100 μM), TCEP (1 mM), TBTA (100 μM) and CuSO4 (1 mM) for 1 h at room temperature while rotating. After labelling, the proteomes were denatured and precipitated using 2 ml cold methanol, and protein pellets were washed with 1 ml of 1:1 cold methanol/CHCl3 (twice), sonicated in 2.5 ml 4:1 methanol/CHCl3 and resuspended in 1 ml PBS with 0.5% SDS. The biotinylated proteins were enriched with PBS-washed avidin–agarose beads (80 µl; Thermo Scientific, catalogue number 20357) by rotating at room temperature for 2 h. The beads were then washed sequentially with 0.5% SDS in PBS (four times). To elute proteins, beads were incubated in 4% SDS, 5% glycerol, 200 mM 2-mercaptoethanol and 100 mM Tris pH 6.8 (160 µl) at 95 °C for 10 min. The supernatant sample was collected.

Proteins were precipitated using methanol–chloroform. Dried pellets were dissolved in 8 M urea/100 mM TEAB, pH 8.5, reduced with 5 mM tris(2-carboxyethyl) phosphine hydrochloride (TCEP), and alkylated with 50 mM chloroacetamide. Samples were diluted to 2 M urea/100 mM TEAB, and proteins were then trypsin digested overnight at 37 °C.

Proteomics LC–MS and data analysis

The digested samples were analysed on an Orbitrap Fusion mass spectrometer (Thermo). Samples were injected directly onto a 25-cm, 100-μm ID column packed with BEH 1.7-μm C18 resin (Waters). Samples were separated at a flow rate of 300 nl min−1 on an nLC 1200 (Thermo). The LC solvents were as follows: buffer A, 0.1% formic acid in water; buffer B, 90% acetonitrile/0.1% formic acid. A typical LC run was 240 min long with a binary gradient consisting of the following steps: 1–25% buffer B over 180 min, 25–40% B over 40 min, 40–90% B over 10 min, 90% B for 10 min. The column was re-equilibrated with buffer A before the injection of sample. Peptides were eluted directly from the tip of the column and nanosprayed directly into the mass spectrometer by application of 2.5 kV voltage at the back of the column. The mass spectrometer was operated in a data-dependent mode. Full MS1 scans were collected in the Orbitrap at 120,000 resolution. The cycle time was set to 3 s, and within this 3 s the most abundant ions per scan were selected for collision-induced dissociation MS/MS in the ion trap. Monoisotopic precursor selection was enabled and dynamic exclusion was used with an exclusion duration of 5 s.

Protein and peptide identification were performed with Integrated Proteomics Pipeline (IP2, Integrated Proteomics Applications). Tandem mass spectra were extracted from raw files using RawConverter51 and searched with ProLuCID52 against the UniProt mouse database. The search space included all fully tryptic and half-tryptic peptide candidates. Carbamidomethylation on cysteine was considered as a static modification. Data were searched with a 50-ppm precursor ion tolerance and a 600-ppm fragment ion tolerance. Identified proteins were filtered to a 10-ppm precursor ion tolerance using DTASelect53 utilizing a target–decoy database search strategy to control the false discovery rate to 1% at the protein level54.

Western blotting

Frozen adipose tissue (human or mouse) and HEK293T cells were dounce homogenized on ice in buffer A (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 µg ml−1 leupeptin, 2 µg ml−1 antipain and 1 µg ml−1 pepstatin, pH 7.0). Lysates were passed through a 26-G or 30-G needle attached to a 1-cc syringe 30 times to break open the cells. Samples were sonicated in a 550 Sonic Dismembrator Cup Horn (Fisher Scientific) filled with ice-cold water for 5 min. Homogenates were centrifuged to remove nuclei and unbroken cells (1,000g, 2 °C, 15 min). Protein concentrations were determined using the reducing reagent bicinchoninic acid assay (Thermo Fisher). Fifty micrograms of proteins were separated by SDS– polyacrylamide gel electrophoresis (7.5% Bio-Rad TGX gels) and transferred to nitrocellulose membranes. Membranes were stained using Revert 700 (Licor) to visualize and quantify total proteins on the blots. Blots were blocked with 5% milk in TBS for 30 min. Membranes were probed with antibodies to ATGL (Cell Signaling number 2439, 30A4, 1:2,000), DGAT1 and DGAT2 (gift from R. V. Farese’s laboratory, 1:1,000)47 and GAPDH (Cell Signaling number 2118, 14C10, 1:5,000). IR Dye 800CW anti-rabbit and anti-mouse secondary antibodies (LI-COR,1:15,000) were used as secondary antibodies for visualization. The uncropped and unprocessed scans are shown in Supplementary Figs. 13. Band intensities were quantified using Image Studio Lite software.

FAHFA biosynthesis assay with affinity-purified ATGL and CGI-58

The in vitro FAHFA biosynthesis assay was adapted from a previously described ATGL activity assay55. For the preparation of the substrate for each reaction, 15 µg PC/phosphatidylinositol 3:1 w/w) was added to the substrates in a glass tube. Organic solvent was evaporated off under a N2 stream. The mixture was emulsified by sonication (550 Sonic Dismembrator, Fisher Scientific) in 100 µl of 0.1 M potassium phosphate, 5% BSA pH 7.0. For the acyl donor screen experiment, PC/phosphatidylinositol was omitted from the substrate preparation. Final acyl donor concentrations were 850 µM for TG(18:1), TG(17:1), TG(16:1), TG(18:2), TG(16:0/18:1/18:1), C17:1 FFA, DG(18:1/18:1/0:0), PC(18:1/18:1) and 100 µM for oleoyl-CoA (18:1-CoA). An 800 µM concentration (final) of 9-HSA or D20-9-HSA was the acyl acceptor. (See purity of each substrate above.) For the assay, 10 µg immunopurified mouse enzymes WT full-length ATGL, WT ATGL-288 or ATGL-288(S47A) alone or with the cofactor CGI-58 were constituted in 100 µl reaction buffer (0.25 M sucrose, 1 mM EDTA, 1 mM dithiothreitol, 20 µg ml−1 leupeptin, 2 µg ml−1 antipain and 1 µg ml−1 pepstatin, pH 7). For atglistatin inhibition experiments (Fig. 3a, right), full-length ATGL enzyme was preincubated with the indicated doses of atglistatin or 0.5% dimethylsulphoxide (DMSO) for 15 min on a shaking thermo mixer at 37 °C. To initiate the FAHFA biosynthesis reaction, substrate emulsion was added to the enzymes. For each assay, 100 µl substrate with 100 µl reaction buffer with or without enzymes was incubated for 60 min at 37 °C while shaking. The reaction was stopped by addition of 500 µl methanol. Samples were stored at −80 °C for FAHFA extraction.

Using 99% pure TG(18:1) substrate from Sigma, compared to 95% pure TG(18:1), from Cayman resulted in a higher rate of 9-OAHSA synthesis in our in vitro biosynthesis experiments. As all comparisons were made within the same experiment, this did not affect the relative values within an experiment. For the IC50 calculation in Fig. 3a (right), we used a nonlinear regression log[inhibitor] versus response (four parameters) model and used the mean value of the biosynthesis assay with no enzyme as the bottom constraint.

Human SQ WAT lysate FAHFA biosynthesis assay

The assay was performed as described above, except 400 µg quantities of human SQ WAT lysates from three different donors were incubated with substrates TG and D20-9-HSA for 60 min at 37 °C while shaking.

Mouse studies

All experimental procedures were approved by the Institutional Animal Care and Use Committee of Beth Israel Deaconess Medical Center and performed following its policies. Mice were housed at the Beth Israel Deaconess Medical Center at 23.3 °C temperature and 40–60% humidity, on ventilated racks (25 ACH) under a 12 h light/12 h dark cycle and fed on chow diet (Lab Diet, 5008). AG4OX mice were generated in our laboratory as previously described39,56. AG4OX and control WT female and male mice (FVB background) of 9–10 weeks of age were used for SVF adipocyte preparations. AT-Atglfl/fl (C57BL/6J) mice were purchased from Jackson Laboratory (stock number 024278) and crossed to adiponectin-Cre mice (C57BL/6J), a gift from E. D. Rosen57 (Beth Israel Deaconess Medical Center) to generate AT-Atgl-KO mice. The metabolic phenotype of AT-Atgl-KO mice was previously described39 and was confirmed in our colony. AT-Atgl-KO and littermate Atglfl/fl control female mice of 10–11 weeks of age were used for studies of endogenous FAHFA levels in tissues and in vivo measurement of FAHFA biosynthesis. Animals were euthanized by CO2 inhalation, blood was collected by cardiac puncture to obtain serum, and tissues were rapidly dissected, flash-frozen in liquid nitrogen and stored at −80 °C.

FAHFA biosynthesis in vivo

For in vivo experiments, mice were randomly assigned to different groups. The experiment was performed between 08:00 and 13:15. Ad-libitum-fed AT-Atgl-KO and Atglfl/fl control mice were intraperitoneally injected with 5 mg kg−1 D20-9-HSA in 49.5% H2O/ 0.5% Tween 20, PEG 400/50% vehicle. The volume of the injection was adjusted on the basis of body weight (5 µl g−1 body weight; that is, 100 µl for a 20-g mouse). Food was immediately removed. In our experience with this strain of mice, 5-h food removal in the morning does not increase serum non-esterified FA levels (ad libitum fed 0.68 ± 0.03; after 5-h food removal 0.75 ± 0.04 mmol l−1, n = 7 per group), which is consistent with no significant increase in lipolysis. Injections were staggered by 7 min, so each mouse was euthanized exactly 4 h after substrate injection for tissue and serum collection. Serum, liver and PG WAT lipids were extracted and D20-9-HSA incorporation into FAHFAs and TG-esterified FAHFAs was measured by LC–MS. A further biosynthesis study with a D20-9-HSA dose of 25 mg kg−1 (Extended Data Fig. 6a) was also performed in the same manner in WT mice.

Lipid extraction

For the measurement of FAHFAs from cultured cells, total lipids were extracted using the modified Bligh–Dyer method58. In brief, 1.5 ml of 2:1 chloroform/methanol with the internal standard [13C16]9-PAHSA (5 pmol) was added to 500 µl of cell suspension in PBS. Samples were vortexed and centrifuged at 2,000g for 7 min. The bottom organic phase was transferred into a new vial, and dried under a N2 stream.

For tissues (50–75 mg) and serum (100 µl), total lipids were extracted as described above. FAHFAs were enriched using a solid-phase extraction (SPE) column (Hypersep silica 500 mg) as described previously59. Briefly, columns were equilibrated with 15 ml hexane, and the samples were then resuspended in 200 µl chloroform and loaded on the SPE column. The neutral lipid fraction containing TGs was eluted with 16 ml of 95:5% hexane/ethyl acetate, and the polar lipid fraction containing non-esterified FAHFAs was eluted using 15 ml ethyl acetate. The FAHFA fractions were dried under a stream of N2 and stored at −40 °C until LC–MS/MS analysis. Total TG levels were quantified from the same piece of tissue as FAHFAs. Neutral lipid fractions of SPE eluents were used to quantify total tissue TG levels using the Infinity triglyceride colorimetric assay kit (Thermo, TR22421) as previously described60.

TG-esterified FAHFAs were extracted, hydrolysed and enriched by multi-step lipid extractions and SPE fractionations with a mild LiOH hydrolysis method as described elsewhere27. Internal standard TG ([13C16]PAHSA/16:0/16:0) was added at the beginning of sample processing and D31-PAHSA internal standard was added after 24-h mild hydrolysis of neutral fractions. The amount of internal standard was adjusted on the basis of the type of tissue or cell. Samples were stored at −40 °C until FAHFAs released by mild hydrolysis (TG-esterified FAHFAs) were measured by LC–MS analysis.

TG-esterified C17:1 and D20-9-HSA were extracted in the same manner as TG-esterified FAHFAs for the experiment in Fig. 2l. An internal standard TG(D9-16:0/16:0/16:0) (Cayman 30181) was added at the beginning of sample processing and D31-16:0 (Cayman 16497) internal standard was added to the samples after mild hydrolysis of neutral fractions to quantify TG-esterified C17:1 and D20-9HSA.

Sample analysis using LC–MS/MS

FAHFA isomers were quantified using an Agilent 6470 Triple Quad LC–MS/MS instrument through multiple reaction monitoring (MRM) in the negative ionization mode as described previously27. In brief, cell culture lipid extracts were reconstituted in 50 µl methanol, and tissue extracts were reconstituted in 100 µl methanol. A 7 µl volume of sample was injected onto a UPLC BEH C18 Column (Waters Acquity, 186002352). FAHFA regioisomers from different FAHFA families were resolved using a 93:7 methanol/water with 5 mM ammonium acetate and 0.01% ammonium hydroxide solvent through an isocratic gradient at 0.15 ml min−1 flow rate for 45 min. Transitions for targeted FAHFAs are listed in Supplementary Table 1. MS acquisition parameters for tandem MS were: gas temperature = 250 °C, gas flow = 12 l min−1, nebulizer = 20 psi, sheath gas temperature = 250 °C, sheath gas flow = 11 l min−1. Spray voltage was −1.0 kV.

Each FAHFA regioisomer as well as newly synthesized 9-FA-D20HSA levels were quantified by normalizing their peak area (extracted using MassHunter 10.0) to the internal standard [13C16]9-PAHSA peak area and total protein amount or tissue weight. For the in vivo biosynthesis experiment, enrichment of newly synthesized PAHSA is calculated as:

$$frac{9-{rm{PA}}-{{rm{D}}}_{20}{rm{HSA}}times 100}{(9-{rm{PAHSA}}+9-{rm{PA}}-{{rm{D}}}_{20}{rm{HSA}})}$$

For TG-esterified FAHFA quantifications, levels of FAHFAs hydrolysed from TGs were normalized to levels of [13C16]PAHSA hydrolysed from the internal standard TG ([13C16]PAHSA/16:0/16:0). Percentage TG hydrolysis was corrected by the internal standard D31-PAHSA as described previously27.

Quantification of FA/HSA and D20-9-HSA for in vivo/intact cells and in vitro biosynthesis studies

A 25 mg quantity of PG WAT was homogenized in 1.5:1.5:3 ml PBS/methanol/chloroform with the internal standard 20 pmol 13C9-9-HSA. Samples were vortexed and centrifuged at 2,000g for 7 min. Bottom organic phase was transferred into a new vial, dried under a stream of N2.

FFA and HFAs were quantified using an Agilent 6470 Triple Quad LC–MS/MS instrument through MRM in the negative ionization mode. Solvent A was water and solvent B was acetonitrile; solvents contained 5 mM ammonium acetate and 0.01% ammonium hydroxide. A UHPLC BEH C18 column (Waters Acquity, 186002352) was used. FA and HFA were chromatographically separated using a 40-min stepwise gradient. The gradient was held at 20% B at 0 min, increased linearly from 20 to 70% B between 0 and 5 min, increased linearly from 70 to 95% B between 5 and 20 min, held at 95% B between 20 and 25 min, raised to 100% B at 25.1 min, held at 95% B between 25.1 and 30 min, returned to 20% B at 31 min, and held at 20% B. The flow rate was 0.15 ml min−1. Samples were resuspended in 500 µl 1:1 chloroform/methanol and 7 µl of the sample was used for the quantifications. Transitions for targeted HFA pseudo MRM are: HSA, 299.3→299.3 (collision energy (CE) = 0); D20-9-HSA, 319.4→319.4 (CE = 0); 13C9-9-HSA, 308.3→308.3 (CE = 0). The parameters for MS were set as follows: gas temperature = 275 °C, gas flow = 12 l min−1, nebulizer = 20 psi, sheath gas temperature = 250 °C, sheath gas flow = 11 l min−1. Spray voltage was −1.0 kV. Levels of HSA were quantified by normalizing with levels of 13C9-9-HSA and grams of tissue. The percentage enrichment of D20-9-HSA was calculated as:

$$frac{{{rm{D}}}_{20}-9-{rm{HSA}}times 100}{({rm{HSA}}+{{rm{D}}}_{20}-9-{rm{HSA}})}$$

For quantification of the FFA in the intact cell culture experiment in Fig. 2m and the in vitro biosynthesis study in Fig. 3e, 20 pmol D31-PA internal standard was also added at the beginning of the lipid extraction as described above in the section describing the method for FAHFA lipid extraction. Transitions for targeted pseudo MRM are: PA, 255.2→255.3 (CE = 0); PO, 253.2→253.2 (CE = 0); OA, 281.2→281.2 (CE = 0); D31-PA, 286.6→286.6 (CE = 0). To quantify ATGL lipase activity in vitro (Fig. 3e), average background (no enzyme) was subtracted from each sample.

Semi-targeted lipidomics in 3T3-L1 adipocyte lipid extraction and LC–MS

Lipids were extracted using a modified version of the Bligh–Dyer method58. In brief, samples were manually shaken in a glass vial (VWR) with 1 ml PBS, 1 ml methanol and 2 ml chloroform containing internal standards ([13C16]palmitic acid and D7-cholesterol) for 30 s. The resulting mixture was vortexed for 15 s and centrifuged at 2,400g for 6 min to induce phase separation. The organic (bottom) layer was retrieved using a Pasteur pipette, dried under a gentle stream of nitrogen, and reconstituted in 2:1 chloroform/methanol for LC–MS analysis.

Lipidomics analysis was performed on a Vanquish HPLC online with a Q-Exactive quadrupole-Orbitrap mass spectrometer equipped with an electrospray ion source (Thermo). Data were acquired in positive and negative ionization modes. Solvent A consisted of 95:5 water/methanol; solvent B was 70:25:5 isopropanol/methanol/water. For positive mode, solvents A and B contained 5 mM ammonium formate with 0.1% formic acid; for negative mode, solvents contained 0.028% ammonium hydroxide. A Bridge (Waters) C8 column (5 μm, 4.6 mm × 50 mm) was used. The gradient was held at 0% B between 0 and 5 min, increased to 20% B at 5.1 min, increased linearly from 20% to 100% B between 5.1 and 55 min, held at 100% B between 55 min and 63 min, returned to 0% B at 63.1 min, and held at 0% B until 70 min. The flow rate was 0.1 ml min−1 from 0 to 5 min, 0.3 ml min−1 between 5.1 min and 55 min, and 0.4 ml min−1 between 55 min and 70 min. The spray voltage was 3.5 kV and 2.5 kV for the positive and negative ionization modes, respectively; the S-lens RF level was 65. The sheath, auxiliary and sweep gases were 50, 10 and 1, respectively. The capillary temperature was 325 °C and the auxiliary gas heater temperature was 200 °C. Data were collected in full MS/dd-MS2 (top 10) mode. The full MS scan was acquired in the range 150–1,500 m/z with a resolution of 70,000, an automatic gain control (AGC) target of 1 × 106 and a maximum injection time of 100 ms. MS2 was acquired with a resolution of 17,500, a fixed first mass of 50 m/z, an AGC target of 1 × 105 and a maximum injection time of 200 ms. Stepped normalized collision energies were 20, 30 and 40%. The inclusion list was on, and the instrument was set to pick other ions when idle.

A lipid target list was generated with LipidCreator. Mass accuracy, chromatography retention time and peak integration of all targeted lipids were verified with Skyline61. Peak areas were used in data reporting, and data were normalized using internal standards.

Statistical analyses

Data are shown as individual data points and mean ± s.e.m. The numbers of participants (human WAT lysate study), mice and wells of cells per group are annotated as n. Each cell culture biosynthesis experiment was replicated with similar results at least twice. Graphs and statistical analyses were generated using GraphPad Prism 6.0 and 8.0 (GraphPad Software). Specific statistical tests and n used for each experiment are listed in the figure legends. Data were analysed using unpaired t-tests, two-tailed for single comparisons, t-test corrected for Holm–Sidak multiple comparison, one-way ANOVA for multiple comparisons (Holm–Sidak multiple comparison) and two-way ANOVAs for comparison of genotype and treatments (Holm–Sidak multiple comparison) as listed in the figure legends.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this paper.

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