#METABOLOMICS WORKBENCH michaelsa93_20170406_113855 DATATRACK_ID:880 STUDY_ID:ST000593 ANALYSIS_ID:AN000909 PROJECT_ID:PR000433 VERSION 1 CREATED_ON April 17, 2017, 3:39 pm #PROJECT PR:PROJECT_TITLE PGD2 and other lipid mediator changes in mouse adipose associated with PR:PROJECT_TITLE administration of an oral inhibitor of H-PGDS (HQL-79) PR:PROJECT_SUMMARY This is an additional experiment being added onto a previous mouse feeding study PR:PROJECT_SUMMARY that aimed to identify changes in metabolites that occur in metabolic tissues in PR:PROJECT_SUMMARY the obese state that are long-lasting and not reversed by weight loss. We PR:PROJECT_SUMMARY observed in the previous mice feeding study that levels of PGD2 increased in HFD PR:PROJECT_SUMMARY fed mice and stayed high after the diet switch. Other members of the PR:PROJECT_SUMMARY Prostaglandin family followed a similar trend (15-deoxy PGJ2, PGJ2) and were PR:PROJECT_SUMMARY specific to adipose tissue. Based on previously published data indicating that PR:PROJECT_SUMMARY central injection of PGD2 stimulates food intake, we attempted to observe this PR:PROJECT_SUMMARY effect using an oral PGD2 inhibitor of H-PGDS (HQL-79). In fact, the oral PR:PROJECT_SUMMARY inhibitor of the H-PGDS (HQL-79) administered peripherally (oral gavage in mice PR:PROJECT_SUMMARY at 30mg/kg dose) reduced daily food intake. Mice were divided into two groups PR:PROJECT_SUMMARY termed Vehicle (Control) and HGL-79 (H-PGDS inhibitor). Each group was analyzed PR:PROJECT_SUMMARY for lipid mediator changes (including PGD2) in adipose tissue by the Newman lab. PR:PROJECT_SUMMARY Analytical results generally met quality control criterion with respect to PR:PROJECT_SUMMARY surrogate recoveries and replicate precision. Surrogate recoveries were good for PR:PROJECT_SUMMARY most oxylipins (58-76%), endocannabinoids (53-75%), and fatty acids (36%). PR:PROJECT_SUMMARY Recovery precision was good for most analytes in these profiles, ranging from PR:PROJECT_SUMMARY 6-28% RSD for most surrogates. The precision for the LTB4 surrogate was higher PR:PROJECT_SUMMARY than most others (38%). Analytical precision was assessed by duplicate analysis PR:PROJECT_SUMMARY of two separate study samples. Analytical precision was 62 - 69% of analytes PR:PROJECT_SUMMARY having <30% RSD for all profiles and correlation analysis for the analytes PR:PROJECT_SUMMARY within these samples ranged from 0.90-0.99 R2. The complete data set is in the PR:PROJECT_SUMMARY associated excel file (Osborn HQL-79 – Deliverable Data Newman Lab.xls). There PR:PROJECT_SUMMARY were few statistically significant differences observed when comparing PR:PROJECT_SUMMARY concentrations (pmol/gr) between the control and HGL-79 treatment groups. PR:PROJECT_SUMMARY However, when we compared ratios we saw numerous differences between PGD2 and PR:PROJECT_SUMMARY its metabolite d15-PGJ2 versus other prostaglandins. Specifically, ratios PR:PROJECT_SUMMARY between PGD2 and other connected pathway metabolites indicate a shift toward PR:PROJECT_SUMMARY PGE2 and PGF2a production instead of PGD2 (Figure 1) with HQL-79 treatment. The PR:PROJECT_SUMMARY PGD2 and PGE2 metabolites ratio of d15-PGJ2/15-keto PGE2 was statistically PR:PROJECT_SUMMARY significant (P<0.01) using a two-tailed t-test. The ratios of PGD2/PGE2 and PR:PROJECT_SUMMARY PGD2/PGF2 had p values of P<0.09 and P=0.07), respectively. Considering that we PR:PROJECT_SUMMARY were predicting changes that indicated less PGD2 production it may be PR:PROJECT_SUMMARY justifiable to use one-tailed tests instead. In order to maintain consistency PR:PROJECT_SUMMARY with the metabolomic data analysis in the previous study, I followed the same PR:PROJECT_SUMMARY statistical protocol that Johannes preformed for the main Pilot study. Using R PR:PROJECT_SUMMARY and Devium log transformed data. Since this was a two group comparision, if the PR:PROJECT_SUMMARY data was normal a 2 tailed t-test was used and if not normal then Mann-Whitney PR:PROJECT_SUMMARY was used. A far as the significance of a shift from PGD2 to PGE2 production, I PR:PROJECT_SUMMARY found a nice review article that discusses in detail the role of prostaglandins PR:PROJECT_SUMMARY in white adipose tissue (Flachs et al. 2013). In the review it cites articles PR:PROJECT_SUMMARY that have shown PGE2 to induce UCP1, modulate lipolysis adipogenesis, and PR:PROJECT_SUMMARY stimulate leptin release. On the other hand, PGD2 was shown to increase PR:PROJECT_SUMMARY adipogenesis and weight gain. Its downstream product d15-PGJ2 has been shown to PR:PROJECT_SUMMARY increase adipogenesis, adipocyte differentiation, and decrease leptin PR:PROJECT_SUMMARY production. This is significant since I also observed that the ratio of d15-PGJ2 PR:PROJECT_SUMMARY to 15-keto PGE2 (the downstream product of PGE2) was also decreased. Another PR:PROJECT_SUMMARY prostaglandin whose ratio versus PGD2 was different in the inhibitor group was PR:PROJECT_SUMMARY PGF2a which has been shown to increase glucose transport in adipose tissue. PR:INSTITUTE University of California, San Diego PR:DEPARTMENT Department of Medicine PR:LAST_NAME Osborn PR:FIRST_NAME Olivia PR:ADDRESS 9500 Gilman Dr., La Jolla, CA 92093 PR:EMAIL oosborn@ucsd.edu PR:PHONE 858-822-6645 PR:FUNDING_SOURCE NIH U24DK097154 #STUDY ST:STUDY_TITLE PGD2 and other lipid mediator changes in mouse adipose associated with ST:STUDY_TITLE administration of an oral inhibitor of H-PGDS (HQL-79) ST:STUDY_SUMMARY This is an additional experiment being added onto a previous mouse feeding study ST:STUDY_SUMMARY that aimed to identify changes in metabolites that occur in metabolic tissues in ST:STUDY_SUMMARY the obese state that are long-lasting and not reversed by weight loss. We ST:STUDY_SUMMARY observed in the previous mice feeding study that levels of PGD2 increased in HFD ST:STUDY_SUMMARY fed mice and stayed high after the diet switch. Other members of the ST:STUDY_SUMMARY Prostaglandin family followed a similar trend (15-deoxy PGJ2, PGJ2) and were ST:STUDY_SUMMARY specific to adipose tissue. Based on previously published data indicating that ST:STUDY_SUMMARY central injection of PGD2 stimulates food intake, we attempted to observe this ST:STUDY_SUMMARY effect using an oral PGD2 inhibitor of H-PGDS (HQL-79). In fact, the oral ST:STUDY_SUMMARY inhibitor of the H-PGDS (HQL-79) administered peripherally (oral gavage in mice ST:STUDY_SUMMARY at 30mg/kg dose) reduced daily food intake. Mice were divided into two groups ST:STUDY_SUMMARY termed Vehicle (Control) and HGL-79 (H-PGDS inhibitor). Each group was analyzed ST:STUDY_SUMMARY for lipid mediator changes (including PGD2) in adipose tissue by the Newman lab. ST:STUDY_SUMMARY Analytical results generally met quality control criterion with respect to ST:STUDY_SUMMARY surrogate recoveries and replicate precision. Surrogate recoveries were good for ST:STUDY_SUMMARY most oxylipins (58-76%), endocannabinoids (53-75%), and fatty acids (36%). ST:STUDY_SUMMARY Recovery precision was good for most analytes in these profiles, ranging from ST:STUDY_SUMMARY 6-28% RSD for most surrogates. The precision for the LTB4 surrogate was higher ST:STUDY_SUMMARY than most others (38%). Analytical precision was assessed by duplicate analysis ST:STUDY_SUMMARY of two separate study samples. Analytical precision was 62 - 69% of analytes ST:STUDY_SUMMARY having <30% RSD for all profiles and correlation analysis for the analytes ST:STUDY_SUMMARY within these samples ranged from 0.90-0.99 R2. The complete data set is in the ST:STUDY_SUMMARY associated excel file (Osborn HQL-79 – Deliverable Data Newman Lab.xls). There ST:STUDY_SUMMARY were few statistically significant differences observed when comparing ST:STUDY_SUMMARY concentrations (pmol/gr) between the control and HGL-79 treatment groups. ST:STUDY_SUMMARY However, when we compared ratios we saw numerous differences between PGD2 and ST:STUDY_SUMMARY its metabolite d15-PGJ2 versus other prostaglandins. Specifically, ratios ST:STUDY_SUMMARY between PGD2 and other connected pathway metabolites indicate a shift toward ST:STUDY_SUMMARY PGE2 and PGF2a production instead of PGD2 (Figure 1) with HQL-79 treatment. The ST:STUDY_SUMMARY PGD2 and PGE2 metabolites ratio of d15-PGJ2/15-keto PGE2 was statistically ST:STUDY_SUMMARY significant (P<0.01) using a two-tailed t-test. The ratios of PGD2/PGE2 and ST:STUDY_SUMMARY PGD2/PGF2 had p values of P<0.09 and P=0.07), respectively. Considering that we ST:STUDY_SUMMARY were predicting changes that indicated less PGD2 production it may be ST:STUDY_SUMMARY justifiable to use one-tailed tests instead. In order to maintain consistency ST:STUDY_SUMMARY with the metabolomic data analysis in the previous study, I followed the same ST:STUDY_SUMMARY statistical protocol that Johannes preformed for the main Pilot study. Using R ST:STUDY_SUMMARY and Devium log transformed data. Since this was a two group comparision, if the ST:STUDY_SUMMARY data was normal a 2 tailed t-test was used and if not normal then Mann-Whitney ST:STUDY_SUMMARY was used. A far as the significance of a shift from PGD2 to PGE2 production, I ST:STUDY_SUMMARY found a nice review article that discusses in detail the role of prostaglandins ST:STUDY_SUMMARY in white adipose tissue (Flachs et al. 2013). In the review it cites articles ST:STUDY_SUMMARY that have shown PGE2 to induce UCP1, modulate lipolysis adipogenesis, and ST:STUDY_SUMMARY stimulate leptin release. On the other hand, PGD2 was shown to increase ST:STUDY_SUMMARY adipogenesis and weight gain. Its downstream product d15-PGJ2 has been shown to ST:STUDY_SUMMARY increase adipogenesis, adipocyte differentiation, and decrease leptin ST:STUDY_SUMMARY production. This is significant since I also observed that the ratio of d15-PGJ2 ST:STUDY_SUMMARY to 15-keto PGE2 (the downstream product of PGE2) was also decreased. Another ST:STUDY_SUMMARY prostaglandin whose ratio versus PGD2 was different in the inhibitor group was ST:STUDY_SUMMARY PGF2a which has been shown to increase glucose transport in adipose tissue. ST:INSTITUTE U.S.D.A. Western Human Nutrition Research Center, University of California, ST:INSTITUTE Davis ST:DEPARTMENT Nutrition ST:LAST_NAME Newman ST:FIRST_NAME John ST:ADDRESS 430 W. Health Sciences Dr., Davis, CA 95616 ST:EMAIL john.newman@ars.usda.gov ST:PHONE +1-530-752-1009 #SUBJECT SU:SUBJECT_TYPE Animal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS Osb V1 Veh_01 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb V2 Veh_02 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb V3 Rep Avg. Veh_03 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb V4 Veh_04 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb V5 Veh_05 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb V6 Veh_06 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb V7 Veh_07 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb V8 Veh_08 Treatment:Vehicle Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H1 HQL_01 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H2 HQL_02 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H3 HQL_03 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H4 HQL_04 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H5 HQL_05 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H6 HQL_06 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H7 HQL_07 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus SUBJECT_SAMPLE_FACTORS Osb H8 Rep Avg. HQL_08 Treatment:HQL-79 Organ=Adipose Tissue; Species=Mus Musculus #COLLECTION CO:COLLECTION_SUMMARY Mice were sacrificed between 10am and noon (ad libitum fed) and adipose tissue CO:COLLECTION_SUMMARY collected and snap frozen and stored at –80oC. CO:SAMPLE_TYPE Adipose Tissue #TREATMENT TR:TREATMENT_SUMMARY C57BL6 male mice were treated with HQL-79 (H-PDGS inhibitor) or vehicle TR:TREATMENT_SUMMARY (control) by oral gavage at a dose of 30mg/kg for 5 days. TR:TREATMENT_DOSE 30mg/kg TR:TREATMENT_DOSEDURATION 5 days #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Oxylipins, endocannabinoids, and fatty acids were isolated using a Waters Ostro SP:SAMPLEPREP_SUMMARY Sample Preparation Plate (Milford, MA). Adipose samples were pulverized and SP:SAMPLEPREP_SUMMARY aliquoted (~10-15mg) were added to 2mL polypropylene tubes and spiked with a 5 SP:SAMPLEPREP_SUMMARY µL anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 SP:SAMPLEPREP_SUMMARY μL 1000nM analytical deuterated surrogates. A total of 50 µL of methanol was SP:SAMPLEPREP_SUMMARY added and the tube was placed in a Geno/Grinder for 30 sec. An additional 550µL SP:SAMPLEPREP_SUMMARY isopropanol w/ 10mM ammonium formate & 1% formic acid and 100 uL water was added SP:SAMPLEPREP_SUMMARY and the tube was placed in a Geno/Grinder for 30 sec before being centrifuged at SP:SAMPLEPREP_SUMMARY 10,000g for 5 min at room temp. The supernate was then transferred into the SP:SAMPLEPREP_SUMMARY plate wells and samples were eluted into glass inserts containing 10 μL 20% SP:SAMPLEPREP_SUMMARY glycerol by applying a vacuum at 15 Hg for 10 min. Eluent was dried by speed SP:SAMPLEPREP_SUMMARY vacuum for 35 min at the medium BP setting, before switching to an aqueous SP:SAMPLEPREP_SUMMARY setting for an additional 35 min. Once dry, samples were re-constituted with the SP:SAMPLEPREP_SUMMARY internal standard 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-Phenyl SP:SAMPLEPREP_SUMMARY 3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN), vortexed 1 min, SP:SAMPLEPREP_SUMMARY transferred to a spin filter (0.1 µm, Millipore, Billerica, MA), centrifuged SP:SAMPLEPREP_SUMMARY for 3 min at 6ºC at <4500g (rcf), before being transferred to 2 mL LC-MS amber SP:SAMPLEPREP_SUMMARY vials. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal SP:SAMPLEPREP_SUMMARY standard was used to quantify the recovery of surrogate standards. SP:SAMPLEPREP_PROTOCOL_FILENAME HQL-79_Lipid_Mediator_Data_Report.docx #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity BEH C18 (150 x 2.1mm, 1.7um) CH:FLOW_GRADIENT See protocol/methods file CH:FLOW_RATE 0.25 CH:COLUMN_TEMPERATURE 60 °C CH:SOLVENT_A 0.1% acetic acid CH:SOLVENT_B 90% ACN / 10% IPA CH:INTERNAL_STANDARD See protocol/methods file CH:RETENTION_TIME See protocol/methods file CH:SAMPLE_INJECTION 0.5 uL CH:ANALYTICAL_TIME 16 min CH:WEAK_WASH_SOLVENT_NAME 20% methanol, 10% isopropanol CH:WEAK_WASH_VOLUME 600 µL CH:STRONG_WASH_SOLVENT_NAME 50:50 Acetonitrile:Methanol CH:STRONG_WASH_VOLUME 600 µL CH:SAMPLE_LOOP_SIZE 17 uL #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME ABI Sciex API 4000 QTrap MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Concentration pmol/g MS_METABOLITE_DATA_START Samples Veh_01 Veh_02 Veh_03 Veh_04 Veh_05 Veh_06 Veh_07 Veh_08 HQL_01 HQL_02 HQL_03 HQL_04 HQL_05 HQL_06 HQL_07 HQL_08 Factors Treatment:Vehicle Treatment:Vehicle Treatment:Vehicle Treatment:Vehicle Treatment:Vehicle Treatment:Vehicle Treatment:Vehicle Treatment:Vehicle Treatment:HQL-79 Treatment:HQL-79 Treatment:HQL-79 Treatment:HQL-79 Treatment:HQL-79 Treatment:HQL-79 Treatment:HQL-79 Treatment:HQL-79 1-OG 18400 3300 5500 7600 2630 4050 9680 14500 10100 7200 6740 2580 6840 16400 1290 2435 2-OG 87300 14900 20000 22800 7680 13500 39800 63100 37000 19500 25800 7650 19100 46800 3120 8585 1-LG 6740 1390 1585 1700 1010 1720 2960 5880 3490 2090 2820 638 1480 4450 572 1136.5 2-LG 54000 14900 15100 14400 8410 12100 36500 36000 36700 13600 16500 5910 17200 21600 2990 6635 1-AG 555 120 172.5 138 44.2 103 210 467 265 132 129 54.2 112 284 28 61.1 2-AG 5600 1170 1140.5 1160 307 686 1800 2910 2120 1090 969 439 663 2280 108 384 PEA 840 748 958.5 750 1400 973 1430 1180 460 783 944 853 1730 1250 579 907 SEA 1220 974 1019 818 1700 1150 1850 1920 577 1080 1480 883 1840 1500 769 1090.5 OEA 367 234 209 228 172 280 213 444 240 342 203 152 232 354 101 190.5 LEA 159 118 95.2 117 71.2 159 95.6 159 91.2 169 89.9 98.1 113 165 58.6 89.85 aLEA 2.41 2.1 1.53 2.19 1.24 3.33 1.66 2.74 1.73 2.59 1.6 1.81 2.01 2.57 0.954 1.415 Dihomo GLA EA 3.42 1.98 1.445 2.02 1.47 2.82 1.84 3.55 1.94 2.6 1.78 1.24 1.88 2.91 0.87 1.38 AEA 20.8 11.3 10.215 12.8 7.51 18.4 14.3 23.1 10.3 17.4 9.52 8.23 11 17 6.92 9.69 DEA 15 2.53 2.875 6.24 1.71 6.13 7.1 14.6 4.21 10.7 4.35 2.94 2.99 13.3 2.91 3.84 DHEA 17.2 9.73 9.58 12.1 7.12 17.9 10.8 18.6 8.24 14.4 8.26 8.76 10.7 20.7 5.42 8.74 PGF2a EA 0.606 1.02 0.899 0.733 0.781 0.804 1.04 0.977 0.5 1.06 0.716 0.854 0.77 1.09 0.719 0.933 NO-Gly 0.944 0.978 1.63 0.81 0.77 1.06 0.787 2.19 0.946 0.865 1.22 1.83 0.656 0.587 0.77 1.089 PGE2 1G PGF2a 1G PGE2 EA PGD2 EA 15-HETE EA 11(12)-EpETre EA NA-Gly MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID CID 1-OG 12178130 2-OG 5319879 1-LG 6436630 2-LG 5365676 1-AG 16019980 2-AG C13856 5282280 PEA C16512 4671 SEA 27902 OEA 5283454 LEA 5283446 aLEA 5283449 Dihomo GLA EA C13828 5282272 AEA C11695 5281969 DEA C13829 5282273 DHEA 53245830 PGF2a EA 53481911 NO-Gly 6436908 PGE2 1G 52193688 PGF2a 1G 24778485 PGE2 EA PGD2 EA 15-HETE EA 11(12)-EpETre EA 53480479 NA-Gly 5283389 METABOLITES_END #END