Materials and Reagents All chemical reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated. Hydroxystearic acid was purchased from Indofine Chemical Company, Inc. (Hillsborough, NJ). [13C 16 ]-Palmitic acid was purchased from Cambridge Isotopes Laboratories, (Andover, MA). Organic solvents for chemical synthesis were purchased from EMD Millipore (Billerica, MA). Solvents for HPLC were purchased from EMD Millipore and solvents for LC-MS were from Honeywell Burdick & Jackson.

Culture and Differentiation of Cells 3T3-L1 fibroblasts were maintained in DMEM supplemented with 10% bovine calf serum, pen/strep and maintained at 37°C and 5% CO 2 . For adipocyte differentiation, 3T3-L1 fibroblasts were grown to confluency in 48-well plates. Cells were maintained at confluency for 2 days after which differentiation was initiated by culturing cells in DMEM containing 10% fetal calf serum (FCS), 4 μg/ml bovine insulin, 0.25 μM dexamethasone, 0.5 mM IBMX and 2 μM Rosiglitazone. 3 days after differentiation initiation, the media was removed and replaced with DMEM supplemented with 10% FCS. Media was changed every 2 days until cells were used for experimental studies at 7–14 days postdifferentiation initiation. STC-1 enteroendocrine cells were maintained in DMEM supplemented with 10% FCS, pen/strep and maintained at 37°C and 5% CO 2 .

GPR120 siRNA Knockdown in Adipocytes 3T3-L1 adipocytes grown in 10cm plates were trypsinized 8 days postdifferentiation initiation and transfected with control siRNA or three different GPR120 targeting siRNA’s (TriFecta siRNA Kit, IDT) individually or in combination by nucleofection following the manufacturers’ instructions (Lonza). Knockdown efficiencies of GPR120 targeting siRNA’s were evaluated by q-PCR 48 hr posttransfection ( Figure S4 B). siRNA #1 showed >93% inhibition of GPR120 mRNA expression compared to control siRNA ( Figure S4 B) and, therefore, GPR120 siRNA #1 was used for the entire study. siRNA sequences and qPCR primers are listed in Table S4

Quantitative PCR RNA was extracted using Tri-Reagent (MRC) and cDNA was generated with random hexamers (Clontech). Quantitative real-time PCR was performed with the ABI Prism sequence detection system. Each sample was run in duplicate, and the quantity of GPR120 mRNA in each sample was normalized to mouse TATA-box binding protein (mTBP) mRNA levels. All primers were obtained from IDT DNA. qPCR primer sequences are listed in Table S4

GLP1 Secretion from Enteroendocrine Cells Hudson et al., 2013 Hudson B.D.

Shimpukade B.

Mackenzie A.E.

Butcher A.J.

Pediani J.D.

Christiansen E.

Heathcote H.

Tobin A.B.

Ulven T.

Milligan G. The pharmacology of TUG-891, a potent and selective agonist of the free fatty acid receptor 4 (FFA4/GPR120), demonstrates both potential opportunity and possible challenges to therapeutic agonism. GLP1 secretion studies were performed as described previously (). Briefly, STC-1 cells were plated in poly-d-lysine coated 24-well plates 72 hr before assay initiation. Cells were washed three times with incubation buffer B (Hanks’ balanced salt solution supplemented with 20 mM HEPES and 2.5 μM KR-62436 (DPP-IV inhibitor, to prevent hydrolysis of GLP1)). Cells were incubated in incubation buffer containing test compounds or DMSO (≤0.1%) control at 37°C for 1 hr. Cell supernatants were collected in microcentrifuge tubes and centrifuged to remove cellular debris and assayed for active-GLP1 concentration using an active-GLP1 ELISA kit (Millipore, Billerica, MA).

[3H]Deoxy-Glucose Uptake in Adipocytes Differentiated 3T3-L1 adipocytes (7–14 days postdifferentiation) were treated with test compounds 2 days or 6 days (chronic treatment) or for 30 min (acute treatment) with PAHSAs or vehicle (DMSO, ≤ 0.1%) control. For transport assays, cells were washed twice with serum-free DMEM and incubated in serum-free DMEM for 2–3 hr at 37°C. Cells were then washed 3 times with Krebs-Ringer-HEPES (KRH) buffer (50 mM HEPES pH 7.4, 137 mM NaCl, 1.25 mM CaCl 2 , 4.7 mM KCl, 5.0 mM, 1.25 mM MgSO 4 ). Cells were incubated in KRH for 30 min then incubated in KRH containing test compounds or DMSO (≤0.1%) control ± insulin for 25 min before the addition of a [3H]deoxy-glucose/2-deoxyglucose solution, yielding final assay concentrations of 1 μCi of [3H]deoxy-glucose and 100 μM 2-deoxy-glucose. After 5 min the KRH buffer was removed immediately by aspiration. Cells were washed 3 times in ice-cold PBS to inhibit further glucose uptake and wash away unincorporated radiolabeled [3H]deoxy-glucose. Cells were solubilized with 1% Triton X-100. [3H] levels were measured by liquid scintillation counting and normalized to protein concentration.

Human Pancreatic Islets Human islets from a nondiabetic, 48-year-old female donor (80% pure and 95% viable) and a nondiabetic 44-year-old male donor (85% pure & 95% viable) were obtained from Prodo Laboratories, Inc. (Irvine, CA). Islets were incubated for 1 day at 37°C in human islet media provided by Prodo laboratories before insulin secretion experiments were performed. See Table S3 for metabolic characteristics of human donors.

Quantitation of Glucose-Stimulated Insulin Secretion in Human Islets Kowluru et al., 2010 Kowluru A.

Veluthakal R.

Rhodes C.J.

Kamath V.

Syed I.

Koch B.J. Protein farnesylation-dependent Raf/extracellular signal-related kinase signaling links to cytoskeletal remodeling to facilitate glucose-induced insulin secretion in pancreatic beta-cells. GSIS was assessed as outlined previously (). Briefly, human islets were cultured overnight in low serum (2.5%)—and low glucose (2.5 mM) human islet media. Following a 2 hr incubation in Krebs-Ringer Bicarbonate buffer (KRB) with 0.5% BSA (pH 7.4), islets were stimulated with either low (2.5 mmol/l) or high (20 mmol/l) glucose in KRB buffer in the presence or absence of vehicle for 5-PAHSA (Methanol, 0.25%) or 5-PAHSA (20 μM) for 45 min at 37°C. At the end of the incubation, media was collected and insulin released into the medium was quantitated by ELISA (Alpco).

GPR120 Activity Assay GPR120 activity assays were performed using DiscoverRx assay kit (PathHunter eXpress GPR120L (Long Isoform) CHO-K1 β-Arrestin GPCR Assay (Part # 93-0366E2CP0M)) as per manufacturer's instructions.

Generation of Bone-Marrow-Derived Dendritic Cells and Treatment with PAHSAs Mouse bone marrow cells were flushed from the femurs and tibiae of 8- to 12-week-old C57BL6/J male mice on a chow diet. Red blood cells were lysed with RBC lysis buffer (Biolegend), and the cells were plated in 6 wells flat bottom plate at a density of 1.0x106 cells/ml in RPMI1640 (GIBCO) containing 10% FBS and supplemented with essential amino acids, pyruvate, glutamine, vitamins, β-mercaptoethanol and antibiotics (GIBCO). GM-CSF (R&D) at a concentration of 20 ng/ml was used for BMDC differentiation. The medium was replaced on day 4, and the cells were harvested on day 6 to obtain immature DC (nonactivated iDC). To obtain activated DC (mDC), LPS was added and the cells were cultured for an additional 24 hr. LPS was used at a final concentration of 100 ng/ml. 9-PAHSA was added 10 min prior to LPS treatment at a final concentration from 2 to 80 μM. Palmitic acid and oleic acid or vehicle (ethanol) control were added 10 min prior to LPS treatment at a final concentration of 40 μM. DMSO (≤0.2%) was added as a vehicle control for 9-PAHSA in experiments where indicated. Cytokines (Il-12p70, IL-1β, IL-6, and TNF-α) secreted into the media were measured by ELISA according to the manufacturers' instructions (Biolegend).

Animal Studies and Measurement of Metabolic Parameters Herman et al., 2012 Herman M.A.

Peroni O.D.

Villoria J.

Schön M.R.

Abumrad N.A.

Blüher M.

Klein S.

Kahn B.B. A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism. Shepherd et al., 1993 Shepherd P.R.

Gnudi L.

Tozzo E.

Yang H.

Leach F.

Kahn B.B. Adipose cell hyperplasia and enhanced glucose disposal in transgenic mice overexpressing GLUT4 selectively in adipose tissue. Herman et al., 2012 Herman M.A.

Peroni O.D.

Villoria J.

Schön M.R.

Abumrad N.A.

Blüher M.

Klein S.

Kahn B.B. A novel ChREBP isoform in adipose tissue regulates systemic glucose metabolism. 2 O). Mice received 1 g/Kg glucose by gavage 30 min after PAHSA or vehicle administration and glycemia was monitored over a 2 hr period. In OGTT studies with 5-PAHSA in chow-fed 45 week old male mice ( 2 O). Glycemia was measured before the gavage (time 0) and 150 min (5-PAHSA) or 180 min (9-PAHSA) postgavage. Mice were bled from the tail vein using heparin coated capillary tubes and serum insulin was measured by ELISA (Crystal Chem Inc.). All animals were kept on a 14 hr light, 10 hr dark schedule (no changes for daylight savings) at an ambient temperature between 72 and 74°F and fed ad libitum unless specified otherwise. All mice were housed in a barrier facility in individually ventilated cages. Male mice were housed individually. Female mice were housed 2–3 per cage. All animal care and use procedures were in strict accordance with, and approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center, Boston, MA. Female AG4OX mice and WT littermate controls (FVB background) were described previously (). ChREBP-KO, ChREBP-KO/AG4OX and control females, described previously () were used at 16- to 18-week-old. Wild-type female FVB mice were also used for studies of FAHFA tissue distribution and the effects of fasting and HFD on FAHFA levels. For measurement of FAHFAs in serum and tissues of chow- and HFD-fed mice, C57BL6/J male mice were maintained on chow (Lab Diet, 5008) or HFD (Harlan Teklad, 93075) for 9 weeks. Animals were euthanized by decapitation and tissues were dissected, flash frozen in liquid nitrogen, and stored at −80°C. For metabolic studies (OGTT and glycemia with food removal), male C57BL6/J mice were fed on either chow (Lab Diet, 5008) or HFD (Harlan Teklad, 93075) for 42–52 weeks. Oral glucose tolerance tests (OGTT) were performed in awake mice after 5 hr food removal. At 4.5 hr after food removal (0.5 hr before initiation of the OGTT) mice were gavaged with 30 mg/kg (chow) or 45 mg/kg (HFD) 9-PAHSA, 5-PAHSA or an equivalent volume of vehicle (50% PEG400, 0.5% Tween-80, 49.5% HO). Mice received 1 g/Kg glucose by gavage 30 min after PAHSA or vehicle administration and glycemia was monitored over a 2 hr period. In OGTT studies with 5-PAHSA in chow-fed 45 week old male mice ( Figure 5 C), mice were bled 5 min postglucose gavage from the tail vein using heparin coated capillary tubes and blood was transferred into tubes containing KR-62436 (DPP-IV inhibitor, 2.5 μM final conc.) and serum insulin and total GLP-1 were measured by ELISA (insulin; Crystal Chem Inc., GLP-1; Millipore). In studies elucidating 9- and 5-PAHSA effects on ambient glycemia, HFD-fed mice were fasted for 2.5 hr and gavaged with either 5- or 9-PAHSA (45 mg/kg) or vehicle (50% PEG400, 0.5% Tween 80, 49.5% HO). Glycemia was measured before the gavage (time 0) and 150 min (5-PAHSA) or 180 min (9-PAHSA) postgavage. Mice were bled from the tail vein using heparin coated capillary tubes and serum insulin was measured by ELISA (Crystal Chem Inc.). All animals were kept on a 14 hr light, 10 hr dark schedule (no changes for daylight savings) at an ambient temperature between 72 and 74°F and fed ad libitum unless specified otherwise. All mice were housed in a barrier facility in individually ventilated cages. Male mice were housed individually. Female mice were housed 2–3 per cage. All animal care and use procedures were in strict accordance with, and approved by the Institutional Animal Care and Use Committee at Beth Israel Deaconess Medical Center, Boston, MA.

Human Samples 2 per min, essentially as described ( DeFronzo et al., 1979 DeFronzo R.

Tobin J.

Andres R. Glucose clamp technique: a method for quantifying insulin secretion and resistance. Laakso et al., 2008 Laakso M.

Zilinskaite J.

Hansen T.

Boesgaard T.W.

Vänttinen M.

Stancáková A.

Jansson P.A.

Pellmé F.

Holst J.J.

Kuulasmaa T.

et al. EUGENE2 Consortium

Insulin sensitivity, insulin release and glucagon-like peptide-1 levels in persons with impaired fasting glucose and/or impaired glucose tolerance in the EUGENE2 study. Laakso et al., 2008 Laakso M.

Zilinskaite J.

Hansen T.

Boesgaard T.W.

Vänttinen M.

Stancáková A.

Jansson P.A.

Pellmé F.

Holst J.J.

Kuulasmaa T.

et al. EUGENE2 Consortium

Insulin sensitivity, insulin release and glucagon-like peptide-1 levels in persons with impaired fasting glucose and/or impaired glucose tolerance in the EUGENE2 study. Thirteen nondiabetic subjects (9F/4M) with varying degrees of insulin sensitivity and BMI were recruited through advertisements in local media. Individuals diagnosed with diabetes or taking any chronic medication were excluded from participation. Height, weight and waist circumference were measured with conventional methods, BMI was calculated as kg body weight divided by height (m) squared. Fasting blood samples were drawn after an overnight fast. Circulating free fatty acid and triglyceride levels were determined by standard methods in the accredited central hospital laboratory using Wako kits (Nordic Biolabs, Täby, Sweden). To evaluate insulin sensitivity a hyperinsulinaemic-euglycaemic clamp was performed for 120 min with an insulin infusion rate of 40 mU/mper min, essentially as described () Blood glucose was clamped at 5 mmol/l by infusion of 20% glucose at various rates according to the blood glucose measurements performed at 5 min intervals. The mean amount of glucose infused during the last hour was used to calculate the rate of whole-body glucose uptake and expressed per Kg lean body mass (LBM). Fat mass and LBM were calculated from bioimpedance analysis (). SQ WAT biopsies were obtained from the peri-umbilical, abdominal region after an overnight fast and snap frozen in liquid nitrogen. The study was approved by the local Ethical Committees at the Sahlgrenska Academy at the University of Gothenburg and performed in agreement with the Declaration of Helsinki. All subjects received written information and gave written consent to participate. See Table S2 for metabolic parameters of human participants.

9-PAHSA Biosynthetic Activity Assay Fresh Liver and PG-WAT tissue was Dounce homogenized in buffer A (10 mM Tris-HCL pH 7.4, 250 mM Sucrose containing protease inhibiters [Roche]). Lysates were centrifuged at 1,200 g to remove incompletely lysed cells and debris. Lysates were then adjusted to 1 mg/ml protein and 100 μl was incubated with 100 μM palmitoyl-CoA and 100 μM 9-hydroxy stearic acid (PAHSA substrates) for 2 hr at 37°C. Control samples were heat denatured by boiling for 10 min prior to incubation with PAHSA substrates. After 2 hr the reaction was stopped by the addition of 300 μl cold buffer A followed by 400 μl of methanol and 800 μl of chloroform. Samples were vortexed and centrifuged at 1,200 g for 5 min. The bottom organic layer was transferred to a new glass vial and dried under a stream of nitrogen. 9-PAHSA levels were measured by LC-MS.

FAHFA Synthesis In Vivo Two hours postfood removal C57BL6/J mice were gavaged with 25 mg/Kg of 9-hydroxy heptadecanoic acid or vehicle (50% PEG-400, 0.5% Tween-80, 49.5% H 2 0) control. Three hours later mice were sacrificed by decapitation and serum was collected. Serum lipids were extracted and palmitic-acid-9-hydroxy-heptadecanoic-acid (9-PAHHA) levels were measured by LC-MS.

Anti-Inflammatory Effects of 9-PAHSA In Vivo 2 O). On the fourth day mice were euthanized by decapitation and perigonadal stromal vascular (SVF) fraction obtained as described ( Moraes-Vieira et al., 2014 Moraes-Vieira P.M.

Yore M.M.

Dwyer P.M.

Syed I.

Aryal P.

Kahn B.B. RBP4 activates antigen-presenting cells, leading to adipose tissue inflammation and systemic insulin resistance. Moraes-Vieira et al., 2014 Moraes-Vieira P.M.

Yore M.M.

Dwyer P.M.

Syed I.

Aryal P.

Kahn B.B. RBP4 activates antigen-presenting cells, leading to adipose tissue inflammation and systemic insulin resistance. Male C57BL6/J mice were fed on either chow (Lab Diet, 5008) or HFD (Harlan Teklad, 93075) for 42–52 weeks. Mice were gavaged once a day for 3 days with 30 mg/kg (chow) or 45 mg/kg (HFD) of 9-PAHSA or an equivalent volume of vehicle (50% PEG400, 0.5% Tween-80, 49.5% HO). On the fourth day mice were euthanized by decapitation and perigonadal stromal vascular (SVF) fraction obtained as described (). SVF cells were cultured for 5 hr with ionomycin, PMA and brefendin at 37°C and the intracellular cytokine content measured as previously described ().

Data Analysis All values are given as mean ± SEM. Differences between groups were assessed using unpaired two-tailed Student’s t tests and/or ANOVA with Fisher’s LSD multiple comparisons as specified in figure legends. Correlations were determined by linear regression analysis yielding “r” and “p” values. All statistical analyses were performed with GraphPad Prism 5.0.

Lipid Extraction from Serum and Tissues Bligh and Dyer, 1959 Bligh E.G.

Dyer W.J. A rapid method of total lipid extraction and purification. Saghatelian et al., 2004 Saghatelian A.

Trauger S.A.

Want E.J.

Hawkins E.G.

Siuzdak G.

Cravatt B.F. Assignment of endogenous substrates to enzymes by global metabolite profiling. 13C-9-PAHSA standard (0.5–5 pmol per sample depending on tissue type) was added to chloroform prior to extraction. The resulting mixture was centrifuged at 2,200 g, 6 min, 4°C to separate organic and aqueous phases, and the organic phase containing extracted lipids was removed with a Pasteur pipette, dried under a gentle stream of Nitrogen and stored at −80°C prior to solid phase extraction (SPE). Lipids were extracted from mouse serum (100 to 200 μl) and human serum (300 μl) using similar protocol, Citric acid buffer or PBS was added to serum such that the final volume was 1 ml for mouse serum and 1.5 ml for human serum, followed by addition of methanol and chloroform to maintain an aqueous: methanol: chloroform ratio of 1:1:2. The resulting mixture was shaken for 30 s by hand, vortexed for 15 s and then centrifuged to separate organic and aqueous phases. Organic phase containing lipids was removed, dried and stored following same method as tissues. Lipid extraction was performed based on known protocol (). Murine tissues (60–150 mg), human fat biopsy (50–70 mg) were Dounce homogenized on ice for 40 strokes in a mixture of 1.5 ml: 1.5 ml: 3 ml citric acid buffer (100 mM trisodium citrate, 1 M NaCl, pH 3.6): methanol: chloroform.C-9-PAHSA standard (0.5–5 pmol per sample depending on tissue type) was added to chloroform prior to extraction. The resulting mixture was centrifuged at 2,200 g, 6 min, 4°C to separate organic and aqueous phases, and the organic phase containing extracted lipids was removed with a Pasteur pipette, dried under a gentle stream of Nitrogen and stored at −80°C prior to solid phase extraction (SPE). Lipids were extracted from mouse serum (100 to 200 μl) and human serum (300 μl) using similar protocol, Citric acid buffer or PBS was added to serum such that the final volume was 1 ml for mouse serum and 1.5 ml for human serum, followed by addition of methanol and chloroform to maintain an aqueous: methanol: chloroform ratio of 1:1:2. The resulting mixture was shaken for 30 s by hand, vortexed for 15 s and then centrifuged to separate organic and aqueous phases. Organic phase containing lipids was removed, dried and stored following same method as tissues.

Solid Phase Extraction for FAHFA Enrichment SPE was performed at room temperature via gravity flow. SPE cartridge (500 mg silica, 6 ml, Thermo Scientific, 60108-411) was conditioned with 15 ml hexane. Extracted lipids (reconstituted in 200 μl chloroform) were loaded onto column. Vial containing lipids was washed with an additional 100 μl chloroform and the wash also loaded onto the column. Neutral lipids were eluted with 16 ml 5% ethyl acetate in hexane, followed by elution of FAHFAs with 16 ml ethyl acetate. FAHFA fraction was dried under nitrogen and stored at −80 °C prior to LC-MS.

Mass Spectrometry 13C-9-PAHSA was m/z 553.5 → m/z 271.3 (CE = 30 V). Fragmentor voltage and dwell time were 205 V and 300 ms, respectively, for each transition. Skimmer voltage was 15 V and ΔEMV was 400 V. MS1 resolution was set to wide and MS2 resolution to unit. Capillary voltage was 4.0 kV, drying gas temperature was 350°C, drying gas flow rate was 8 L min−1 and nebulizer pressure was 35 psi. Identical gradient and instrument parameters were used for detection of all additional FAHFAs with the exception of dwell time, which was reduced to 30 ms to accommodate additional transitions. All FAHFA transitions are listed in FAHFAs were measured on an Agilent 6410 Triple Quad LC/MS instrument via Multiple Reaction Monitoring (MRM) in negative ionization mode. A Luna C18(2) (Phenomenox, 00G-4251-B0) column (3 μm, 100 Å, 250 × 2.0 mm) was used with an in-line filter (Phenomenex, AF0-8497). The solvent was 93:7 methanol:water with 5 mM ammonium acetate (Aldrich, 372331) and 0.01% ammonium hydroxide (Sigma-Aldrich, 338818), and distinct PAHSA species were resolved via isocratic flow at 0.2 ml/min for 120 min. Each extracted and fractionated sample was reconstituted in 25 μl methanol and 10 μl was injected for analysis. Transitions for endogenous PAHSAs were m/z 537.5 → m/z 255.2 (CE = 30 V), m/z 537.5 → m/z 281.2 (CE = 25 V) and m/z 537.5 → m/z 299.3 (CE = 23 V), and transition forC-9-PAHSA was m/z 553.5 → m/z 271.3 (CE = 30 V). Fragmentor voltage and dwell time were 205 V and 300 ms, respectively, for each transition. Skimmer voltage was 15 V and ΔEMV was 400 V. MS1 resolution was set to wide and MS2 resolution to unit. Capillary voltage was 4.0 kV, drying gas temperature was 350°C, drying gas flow rate was 8 L minand nebulizer pressure was 35 psi. Identical gradient and instrument parameters were used for detection of all additional FAHFAs with the exception of dwell time, which was reduced to 30 ms to accommodate additional transitions. All FAHFA transitions are listed in Table S1

Targeted LC-MS Quantitation of PAHSA Biosynthetic Reactions Performed In Vivo and In Vitro Samples for analysis of 9-PAHSA biosynthetic activity were dissolved in 50 μl methanol. The LC-MS method was slightly modified from the targeted FAHFA method. The column was changed to an ACQUITY UPLC BEH C18 column (1.7 μm, 130Å, 2.1 mm X 100 mm) and maintained at 40°C during separation. The elution time was shortened to 40 min. The injection volume is 10 μl. For analysis of 9-PAHHA synthesis in vivo, similar 40 min gradient LC-MS method was employed, but the corresponding MRM transitions used for 9-PAHHA were m/z 523.5 → m/z 255.2, m/z 523.5 → m/z 267.2 and m/z 523.5 → m/z 285.3.

Lipidomics of WT and AG4OX SQ WAT A Gemini C18 reversed phase column (5 μm, 4.6 × 50 mm, Phenomonex) and a C18 reversed phase guard column (3.5 μm, 2 × 20 mm, Western Analytical) were used for LC-MS analysis in negative mode. In positive mode, a Luna C5 reversed phase column (5 μm, 4.6 × 50 mm, Phenomonex) was used together with a C4 reversed phase guard column (3.5 μm, 2 × 20 mm, Western Analytical). 30 μl of each sample was injected using an autosampler. Mobile phase A consisted of a 95:5 water:methanol mixture and mobile phase B consisted of 60:35:5, 2-propanol:methanol:water. In negative mode 0.1% ammonium hydroxide was added to the mobile phases and in positive mode 0.1% formic acid plus 5 mM ammonium formate were added. An Agilent 1200 series binary pump method was set to a flow rate of 0.1 ml/min for the first 5 min followed by 0.4 ml/min for the remainder of the gradient. At 5 min, concomitant with the increase in flow rate, the gradient was increased from 0% B to 20% B. The gradient increased linearly to 100% B at 45 min, followed by an 8 min wash at 0.5 ml/min with 100% B before re-equilibrating the column with 0% B for 7 min. MS analysis was performed using an Agilent 6220 ESI-TOF fitted with an electrospray ionization (ESI) source. The capillary voltage was set to 3,500 kV and the fragmentor voltage to 100 V. The drying gas temperature was set to 350°C at a flow rate of 10 l/min with a nebulizer pressure set to 45 psi. Untargeted data were collected using a mass-to-charge range of m/z 100–1,500.

MS Data Analysis Data analysis with XCMS was used to identify changing metabolites between samples. Raw data files from the TOF-MS were converted to mzXML files using the program mzStar for subsequent XCMS analysis. Samples were compared (i.e., AG4OX versus WT) and differences were ranked according to statistical significance as calculated by an unpaired Student’s t test. The data were then filtered based on a peak size (>5 × 104 counts) and statistical significance (p value < 0.05) prior to visual inspection of the remaining ions to ensure that the differences identified by XCMS were reflected in the raw data. Peak areas were normalized to the frozen wet mass of the extracted tissues and these corrected peak areas were used to calculate relative changes. For the volcano plot, data were obtained from the 60 min profiling analysis in negative mode and the data were filtered based on retention time range (10–50 min) and abundance (>1 × 105 counts).

Purification of PAHSAs from Mouse Tissue to Identify the Predominant PAHSA Isomer in SQ WAT by Tandem MS PAHSA in microgram quantities was obtained by purification from WAT (0.75 g) harvested from AG4OX mice. After homogenizing the tissue and extracting the homogenate with chloroform-methanol, triglycerides were removed from the lipid extract (0.6 g) by flash chromatography on an alumina column. Neutral lipids were eluted with 20% ethyl acetate in hexanes, and the remaining lipids were eluted with 2% acetic acid in ethyl acetate. The fractions were combined and the solvent was removed in vacuo. The oily residue was then purified by preparative reversed phase HPLC (Shimazu). The preparative column dimensions were 20 × 150 mm, the particle size was 10 μm, and the stationary phase was C18. Buffer A was 100% water and Buffer B was 100% methanol, both containing 10 mM ammonium acetate. The flow rate was 8 ml/min and the solvent composition started at 60% B, where it was held for 5 min. The composition was increased to 100% B over 60 min, after which the column was washed with 100% B for 20 min. The PAHSA-containing fractions, assessed by QQQ-MS in MRM mode, were combined. PAHSA eluted in minutes 68–70 on this gradient, and the methanol-water eluate was evaporated under a nitrogen gas stream, leaving an oily residue (6.8 mg). Analysis of the residue by TOF-MS demonstrated that PAHSA was the dominant component of the mixture and the absence of hydroxystearic acid was verified. For structural characterization by MS/MS, the PAHSA residue was reconstituted in chloroform for injection on the triple quadrupole mass spectrometer.