Summary of Study ST000593
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR000433. The data can be accessed directly via it's Project DOI: 10.21228/M8330V This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST000593 |
| Study Title | PGD2 and other lipid mediator changes in mouse adipose associated with administration of an oral inhibitor of H-PGDS (HQL-79) |
| Study Summary | This is an additional experiment being added onto a previous mouse feeding study that aimed to identify changes in metabolites that occur in metabolic tissues in the obese state that are long-lasting and not reversed by weight loss. We observed in the previous mice feeding study that levels of PGD2 increased in HFD fed mice and stayed high after the diet switch. Other members of the Prostaglandin family followed a similar trend (15-deoxy PGJ2, PGJ2) and were specific to adipose tissue. Based on previously published data indicating that central injection of PGD2 stimulates food intake, we attempted to observe this effect using an oral PGD2 inhibitor of H-PGDS (HQL-79). In fact, the oral inhibitor of the H-PGDS (HQL-79) administered peripherally (oral gavage in mice at 30mg/kg dose) reduced daily food intake. Mice were divided into two groups termed Vehicle (Control) and HGL-79 (H-PGDS inhibitor). Each group was analyzed for lipid mediator changes (including PGD2) in adipose tissue by the Newman lab. Analytical results generally met quality control criterion with respect to surrogate recoveries and replicate precision. Surrogate recoveries were good for most oxylipins (58-76%), endocannabinoids (53-75%), and fatty acids (36%). Recovery precision was good for most analytes in these profiles, ranging from 6-28% RSD for most surrogates. The precision for the LTB4 surrogate was higher than most others (38%). Analytical precision was assessed by duplicate analysis of two separate study samples. Analytical precision was 62 - 69% of analytes having <30% RSD for all profiles and correlation analysis for the analytes within these samples ranged from 0.90-0.99 R2. The complete data set is in the associated excel file (Osborn HQL-79 – Deliverable Data Newman Lab.xls). There were few statistically significant differences observed when comparing concentrations (pmol/gr) between the control and HGL-79 treatment groups. However, when we compared ratios we saw numerous differences between PGD2 and its metabolite d15-PGJ2 versus other prostaglandins. Specifically, ratios between PGD2 and other connected pathway metabolites indicate a shift toward PGE2 and PGF2a production instead of PGD2 (Figure 1) with HQL-79 treatment. The PGD2 and PGE2 metabolites ratio of d15-PGJ2/15-keto PGE2 was statistically significant (P<0.01) using a two-tailed t-test. The ratios of PGD2/PGE2 and PGD2/PGF2 had p values of P<0.09 and P=0.07), respectively. Considering that we were predicting changes that indicated less PGD2 production it may be justifiable to use one-tailed tests instead. In order to maintain consistency with the metabolomic data analysis in the previous study, I followed the same statistical protocol that Johannes preformed for the main Pilot study. Using R and Devium log transformed data. Since this was a two group comparision, if the data was normal a 2 tailed t-test was used and if not normal then Mann-Whitney was used. A far as the significance of a shift from PGD2 to PGE2 production, I found a nice review article that discusses in detail the role of prostaglandins in white adipose tissue (Flachs et al. 2013). In the review it cites articles that have shown PGE2 to induce UCP1, modulate lipolysis adipogenesis, and stimulate leptin release. On the other hand, PGD2 was shown to increase adipogenesis and weight gain. Its downstream product d15-PGJ2 has been shown to increase adipogenesis, adipocyte differentiation, and decrease leptin production. This is significant since I also observed that the ratio of d15-PGJ2 to 15-keto PGE2 (the downstream product of PGE2) was also decreased. Another prostaglandin whose ratio versus PGD2 was different in the inhibitor group was PGF2a which has been shown to increase glucose transport in adipose tissue. |
| Institute | University of California, Davis |
| Department | USDA Western Human Nutrition Research Center |
| Last Name | Newman |
| First Name | John |
| Address | 430 W. Health Sciences Dr., Davis, CA 95616 |
| john.newman@ars.usda.gov | |
| Phone | +1-530-752-1009 |
| Submit Date | 2017-04-06 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | wiff |
| Analysis Type Detail | LC-MS |
| Release Date | 2017-10-03 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR000433 |
| Project DOI: | doi: 10.21228/M8330V |
| Project Title: | PGD2 and other lipid mediator changes in mouse adipose associated with administration of an oral inhibitor of H-PGDS (HQL-79) |
| Project Summary: | This is an additional experiment being added onto a previous mouse feeding study that aimed to identify changes in metabolites that occur in metabolic tissues in the obese state that are long-lasting and not reversed by weight loss. We observed in the previous mice feeding study that levels of PGD2 increased in HFD fed mice and stayed high after the diet switch. Other members of the Prostaglandin family followed a similar trend (15-deoxy PGJ2, PGJ2) and were specific to adipose tissue. Based on previously published data indicating that central injection of PGD2 stimulates food intake, we attempted to observe this effect using an oral PGD2 inhibitor of H-PGDS (HQL-79). In fact, the oral inhibitor of the H-PGDS (HQL-79) administered peripherally (oral gavage in mice at 30mg/kg dose) reduced daily food intake. Mice were divided into two groups termed Vehicle (Control) and HGL-79 (H-PGDS inhibitor). Each group was analyzed for lipid mediator changes (including PGD2) in adipose tissue by the Newman lab. Analytical results generally met quality control criterion with respect to surrogate recoveries and replicate precision. Surrogate recoveries were good for most oxylipins (58-76%), endocannabinoids (53-75%), and fatty acids (36%). Recovery precision was good for most analytes in these profiles, ranging from 6-28% RSD for most surrogates. The precision for the LTB4 surrogate was higher than most others (38%). Analytical precision was assessed by duplicate analysis of two separate study samples. Analytical precision was 62 - 69% of analytes having <30% RSD for all profiles and correlation analysis for the analytes within these samples ranged from 0.90-0.99 R2. The complete data set is in the associated excel file (Osborn HQL-79 – Deliverable Data Newman Lab.xls). There were few statistically significant differences observed when comparing concentrations (pmol/gr) between the control and HGL-79 treatment groups. However, when we compared ratios we saw numerous differences between PGD2 and its metabolite d15-PGJ2 versus other prostaglandins. Specifically, ratios between PGD2 and other connected pathway metabolites indicate a shift toward PGE2 and PGF2a production instead of PGD2 (Figure 1) with HQL-79 treatment. The PGD2 and PGE2 metabolites ratio of d15-PGJ2/15-keto PGE2 was statistically significant (P<0.01) using a two-tailed t-test. The ratios of PGD2/PGE2 and PGD2/PGF2 had p values of P<0.09 and P=0.07), respectively. Considering that we were predicting changes that indicated less PGD2 production it may be justifiable to use one-tailed tests instead. In order to maintain consistency with the metabolomic data analysis in the previous study, I followed the same statistical protocol that Johannes preformed for the main Pilot study. Using R and Devium log transformed data. Since this was a two group comparision, if the data was normal a 2 tailed t-test was used and if not normal then Mann-Whitney was used. A far as the significance of a shift from PGD2 to PGE2 production, I found a nice review article that discusses in detail the role of prostaglandins in white adipose tissue (Flachs et al. 2013). In the review it cites articles that have shown PGE2 to induce UCP1, modulate lipolysis adipogenesis, and stimulate leptin release. On the other hand, PGD2 was shown to increase adipogenesis and weight gain. Its downstream product d15-PGJ2 has been shown to increase adipogenesis, adipocyte differentiation, and decrease leptin production. This is significant since I also observed that the ratio of d15-PGJ2 to 15-keto PGE2 (the downstream product of PGE2) was also decreased. Another prostaglandin whose ratio versus PGD2 was different in the inhibitor group was PGF2a which has been shown to increase glucose transport in adipose tissue. |
| Institute: | University of California, San Diego |
| Department: | Department of Medicine |
| Last Name: | Osborn |
| First Name: | Olivia |
| Address: | 9500 Gilman Dr., La Jolla, CA 92093 |
| Email: | oosborn@ucsd.edu |
| Phone: | 858-822-6645 |
| Funding Source: | NIH U24DK097154 |
Subject:
| Subject ID: | SU000616 |
| Subject Type: | Animal |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Species Group: | Mammal |
Factors:
Subject type: Animal; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | Treatment |
|---|---|---|
| SA032636 | HQL_04 | HQL-79 |
| SA032637 | HQL_03 | HQL-79 |
| SA032638 | HQL_05 | HQL-79 |
| SA032639 | HQL_07 | HQL-79 |
| SA032640 | HQL_08 | HQL-79 |
| SA032641 | HQL_02 | HQL-79 |
| SA032642 | HQL_06 | HQL-79 |
| SA032643 | HQL_01 | HQL-79 |
| SA032644 | Veh_04 | Vehicle |
| SA032645 | Veh_03 | Vehicle |
| SA032646 | Veh_02 | Vehicle |
| SA032647 | Veh_05 | Vehicle |
| SA032648 | Veh_06 | Vehicle |
| SA032649 | Veh_08 | Vehicle |
| SA032650 | Veh_07 | Vehicle |
| SA032651 | Veh_01 | Vehicle |
| Showing results 1 to 16 of 16 |
Collection:
| Collection ID: | CO000610 |
| Collection Summary: | Mice were sacrificed between 10am and noon (ad libitum fed) and adipose tissue collected and snap frozen and stored at –80oC. |
| Sample Type: | Adipose Tissue |
Treatment:
| Treatment ID: | TR000630 |
| Treatment Summary: | C57BL6 male mice were treated with HQL-79 (H-PDGS inhibitor) or vehicle (control) by oral gavage at a dose of 30mg/kg for 5 days. |
| Treatment Dose: | 30mg/kg |
| Treatment Doseduration: | 5 days |
Sample Preparation:
| Sampleprep ID: | SP000623 |
| Sampleprep Summary: | Oxylipins, endocannabinoids, and fatty acids were isolated using a Waters Ostro Sample Preparation Plate (Milford, MA). Adipose samples were pulverized and aliquoted (~10-15mg) were added to 2mL polypropylene tubes and spiked with a 5 µL anti-oxidant solution (0.2 mg/ml solution BHT/EDTA in 1:1 MeOH:water) and 10 μL 1000nM analytical deuterated surrogates. A total of 50 µL of methanol was added and the tube was placed in a Geno/Grinder for 30 sec. An additional 550µL isopropanol w/ 10mM ammonium formate & 1% formic acid and 100 uL water was added and the tube was placed in a Geno/Grinder for 30 sec before being centrifuged at 10,000g for 5 min at room temp. The supernate was then transferred into the plate wells and samples were eluted into glass inserts containing 10 μL 20% glycerol by applying a vacuum at 15 Hg for 10 min. Eluent was dried by speed vacuum for 35 min at the medium BP setting, before switching to an aqueous setting for an additional 35 min. Once dry, samples were re-constituted with the internal standard 1-cyclohexyl ureido, 3-dodecanoic acid (CUDA) and 1-Phenyl 3-Hexadecanoic Acid Urea (PHAU) at 100 nM (50:50 MeOH:CAN), vortexed 1 min, transferred to a spin filter (0.1 µm, Millipore, Billerica, MA), centrifuged for 3 min at 6ºC at <4500g (rcf), before being transferred to 2 mL LC-MS amber vials. Extracts were stored at -20ºC until analysis by UPLC-MS/MS. The internal standard was used to quantify the recovery of surrogate standards. |
| Sampleprep Protocol Filename: | HQL-79_Lipid_Mediator_Data_Report.docx |
Combined analysis:
| Analysis ID | AN000909 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity |
| Column | Waters Acquity BEH C18 (150 x 2.1mm,1.7um) |
| MS Type | ESI |
| MS instrument type | Triple quadrupole |
| MS instrument name | ABI Sciex API 4000 QTrap |
| Ion Mode | POSITIVE |
| Units | Concentration pmol/g |
Chromatography:
| Chromatography ID: | CH000646 |
| Instrument Name: | Waters Acquity |
| Column Name: | Waters Acquity BEH C18 (150 x 2.1mm,1.7um) |
| Column Temperature: | 60 °C |
| Flow Gradient: | See protocol/methods file |
| Flow Rate: | 0.25 |
| Internal Standard: | See protocol/methods file |
| Retention Time: | See protocol/methods file |
| Sample Injection: | 0.5 uL |
| Solvent A: | 100% water; 0.1% acetic acid |
| Solvent B: | 90% acetonitrile/ 10% isopropanol |
| Analytical Time: | 16 min |
| Weak Wash Solvent Name: | 20% methanol, 10% isopropanol |
| Weak Wash Volume: | 600 µL |
| Strong Wash Solvent Name: | 50:50 Acetonitrile:Methanol |
| Strong Wash Volume: | 600 µL |
| Sample Loop Size: | 17 uL |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS000808 |
| Analysis ID: | AN000909 |
| Instrument Name: | ABI Sciex API 4000 QTrap |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| Ion Mode: | POSITIVE |
