Summary of Study ST004238

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 PR002673. The data can be accessed directly via it's Project DOI: 10.21228/M84P1D This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST004238
Study Title	Lipidomic Characterization of Wild-Type vs ATGL/HSL Double Knockout Adipocytes in Basal and Stimulated Lipolysis
Study Summary	This study investigates the role of canonical lipolytic enzymes in the hydrolysis of short- and medium-chain fatty acid–containing triglycerides (SMCFA-TGs) in primary mouse adipocytes. To this end, we generated adipocytes deficient in both adipose triglyceride lipase (ATGL/PNPLA2) and hormone-sensitive lipase (HSL/LIPE) using a tamoxifen-inducible CreERT2/LoxP system. Pre-adipocytes were isolated from stromal vascular fractions of white adipose tissue of mice expressing or not the CreERT2 and containing floxed HSL/LIPE and ATGL/PNPLA2 alleles. Adipocytes were differentiated in vitro, and subjected to 4-hydroxy-tamoxifen treatment to induce gene deletion. Successful depletion of ATGL and HSL was confirmed by RT-qPCR and Western blotting. Lipolysis was stimulated with the β3-adrenergic receptor agonist CL316343, and both glycerol release and lipidomic profiles were analyzed. Functional assays demonstrated that basal and stimulated lipolysis were significantly reduced in ATGL/HSL-deficient adipocytes. Following lipolysis stimulations, neutral lipids were extracted to perform lipidomics analysis by LC-MS/MS.
Institute	University of Szeged
Last Name	David
First Name	Kovacs
Address	Dóm tér 9, 6723
Email	kovacs.david@med.u-szeged.hu
Phone	+3662342665
Submit Date	2025-09-23
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-10-20
Release Version	1

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Project:

Project ID:	PR002673
Project DOI:	doi: 10.21228/M84P1D
Project Title:	Chain length-dependent mobilization and oxidation of fatty acids in adipocytes
Project Summary:	Fatty acids (FAs) are essential metabolites in energy homeostasis. Adipocytes store FAs as triacylglycerol (TG) in their lipid droplets and mobilize them upon demand in order to provide energy for peripheral tissues. While the mobilization of long-chain fatty acids (LCFAs) during lipolysis is largely documented, there is little information on the metabolism of short- and medium-chain fatty acids (SMCFA) in adipocytes. We demonstrate that adipocytes store SMCFAs in their lipid droplets and that following lipolysis initiation, TGs containing SMCFAs undergo rapid hydrolysis. We found that this process is facilitated by the preferential accumulation of SMCFA-containing TGs at the surface of lipid droplets, thereby enhancing their accessibility to lipases. Unlike LCFAs, SMCFAs are not released from the adipocytes following lipolysis but undergo oxidation within the cell. Our findings suggest that SMCFAs are preferentially mobilized and oxidized to provide energy for the adipocyte, highlighting a distinct metabolic fate compared to LCFAs.
Institute:	University of Szeged
Department:	Institute of Biochemistry
Last Name:	Kovacs
First Name:	David
Address:	Dóm tér 9, Szeged, Csongrád-Csanád, 6723, Hungary
Email:	kovacs.david@med.u-szeged.hu
Phone:	+3662342665

Subject:

Subject ID:	SU004390
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Genotype	Condition	Injection order
SA486528	28	Primary adipocyte	Double KO	Basal	1
SA486529	34	Primary adipocyte	Double KO	Basal	1
SA486530	31	Primary adipocyte	Double KO	Basal	1
SA486531	25	Primary adipocyte	Double KO	Basal	1
SA486532	29	Primary adipocyte	Double KO	Basal	2
SA486533	35	Primary adipocyte	Double KO	Basal	2
SA486534	26	Primary adipocyte	Double KO	Basal	2
SA486535	32	Primary adipocyte	Double KO	Basal	2
SA486536	27	Primary adipocyte	Double KO	Basal	3
SA486537	33	Primary adipocyte	Double KO	Basal	3
SA486538	30	Primary adipocyte	Double KO	Basal	3
SA486539	36	Primary adipocyte	Double KO	Basal	3
SA486540	46	Primary adipocyte	Double KO	Lipolysis	1
SA486541	43	Primary adipocyte	Double KO	Lipolysis	1
SA486542	40	Primary adipocyte	Double KO	Lipolysis	1
SA486543	37	Primary adipocyte	Double KO	Lipolysis	1
SA486544	47	Primary adipocyte	Double KO	Lipolysis	2
SA486545	44	Primary adipocyte	Double KO	Lipolysis	2
SA486546	41	Primary adipocyte	Double KO	Lipolysis	2
SA486547	38	Primary adipocyte	Double KO	Lipolysis	2
SA486548	39	Primary adipocyte	Double KO	Lipolysis	3
SA486549	42	Primary adipocyte	Double KO	Lipolysis	3
SA486550	45	Primary adipocyte	Double KO	Lipolysis	3
SA486551	48	Primary adipocyte	Double KO	Lipolysis	3
SA486552	4	Primary adipocyte	Wild type	Basal	1
SA486553	10	Primary adipocyte	Wild type	Basal	1
SA486554	7	Primary adipocyte	Wild type	Basal	1
SA486555	1	Primary adipocyte	Wild type	Basal	1
SA486556	5	Primary adipocyte	Wild type	Basal	2
SA486557	2	Primary adipocyte	Wild type	Basal	2
SA486558	8	Primary adipocyte	Wild type	Basal	2
SA486559	11	Primary adipocyte	Wild type	Basal	2
SA486560	12	Primary adipocyte	Wild type	Basal	3
SA486561	9	Primary adipocyte	Wild type	Basal	3
SA486562	6	Primary adipocyte	Wild type	Basal	3
SA486563	3	Primary adipocyte	Wild type	Basal	3
SA486564	19	Primary adipocyte	Wild type	Lipolysis	1
SA486565	16	Primary adipocyte	Wild type	Lipolysis	1
SA486566	13	Primary adipocyte	Wild type	Lipolysis	1
SA486567	22	Primary adipocyte	Wild type	Lipolysis	1
SA486568	20	Primary adipocyte	Wild type	Lipolysis	2
SA486569	14	Primary adipocyte	Wild type	Lipolysis	2
SA486570	23	Primary adipocyte	Wild type	Lipolysis	2
SA486571	17	Primary adipocyte	Wild type	Lipolysis	2
SA486572	18	Primary adipocyte	Wild type	Lipolysis	3
SA486573	24	Primary adipocyte	Wild type	Lipolysis	3
SA486574	15	Primary adipocyte	Wild type	Lipolysis	3
SA486575	21	Primary adipocyte	Wild type	Lipolysis	3

Showing results 1 to 48 of 48

Showing results 1 to 48 of 48

Collection:

Collection ID:	CO004383
Collection Summary:	Primary adipocytes were obtained from Hslfl/fl Atglfl/fl AdipoQ-Cre/ERT2- (Control) and Hslfl/fl Atglfl/fl AdipoQ-Cre/ERT2+ (Double KO) mice. Briefly, following isolation from adipose tissue, stromal vascular fraction was plated. After proliferation, pre-adipocytes were differentiated 2 days using rosiglitazone (170 nm, Cayman CAY-7140-100), IBMX (500 µm, Sigma I5879), dexamethasone (1 µM, Sigma D2915) and insulin (170 nM, Sigma I9278) in DMEM 1 mM glucose medium (Sigma, D5546) supplemented with 10% FBS. The deletion of HLS and ATGL was induced by the addition of 4-hydroxy-tamoxifen between day 2 and 4 post differentiation (Sigma H6278). After 7 days, the adipocytes were differentiated and used for the experiments.
Sample Type:	Primary adipocytes

Treatment:

Treatment ID:	TR004399
Treatment Summary:	To induce lipolysis, differentiated adipocytes were left to starve for a 24 h period without serum and insulin. Following this, lipolysis was stimulated for 2 h with the β3-adrenergic receptor agonist CL316343 at 100 nM (Sigma C5976). For basal lipolysis, an equal volume of DMSO used.

Sample Preparation:

Sampleprep ID:	SP004396
Sampleprep Summary:	To extract lipids from primary adipocyte samples, a 3-phase liquid extraction was performed to separate neutral and polar lipids. First, cell collected in a minimal volume of PBS were transferred into glass tubes, containing already the internal standard mix (Splash) and TG 6:0/6:0/6:0. Then, to reduce sample loss, 0.75 mL of ACN were added to microtubes, vortexed, and transferred into the corresponding glass tubes. The remaining solvents were added to each sample: 0.75 mL Hex, 0.25 mL EtAc. The aqueous phase (sample) was completed with ultrapure water when needed, resulting in Hex:EtAc:ACN:Aqueous (3:1:3:2, V:V:V:V). Samples were first vortexed for 30 min at 4oC with glass beads, and only then centrifuged. The upper phase was collected into a new tube with a Hamilton glass syringe, which was washed three times in solvent between samples. Hex was added (half the volume of the first extraction) to the 2 remaining phases for re-extraction. Samples were again vortexed and centrifuged, and upper and middle phases were collected separately. To reduce phospholipid loss, a re-extraction of middle phase was done with ACN:EtAc (3:1, V:V) (half the volume of first extraction). Extraction (solvents and water) and experiment blanks (PBS) were included. All extraction solvents had 50 μg/mL BHT. Extracted samples were kept dried at -20oC under Ar.

Sampleprep ID:

SP004396

Sampleprep Summary:

To extract lipids from primary adipocyte samples, a 3-phase liquid extraction was performed to separate neutral and polar lipids. First, cell collected in a minimal volume of PBS were transferred into glass tubes, containing already the internal standard mix (Splash) and TG 6:0/6:0/6:0. Then, to reduce sample loss, 0.75 mL of ACN were added to microtubes, vortexed, and transferred into the corresponding glass tubes. The remaining solvents were added to each sample: 0.75 mL Hex, 0.25 mL EtAc. The aqueous phase (sample) was completed with ultrapure water when needed, resulting in Hex:EtAc:ACN:Aqueous (3:1:3:2, V:V:V:V). Samples were first vortexed for 30 min at 4oC with glass beads, and only then centrifuged. The upper phase was collected into a new tube with a Hamilton glass syringe, which was washed three times in solvent between samples. Hex was added (half the volume of the first extraction) to the 2 remaining phases for re-extraction. Samples were again vortexed and centrifuged, and upper and middle phases were collected separately. To reduce phospholipid loss, a re-extraction of middle phase was done with ACN:EtAc (3:1, V:V) (half the volume of first extraction). Extraction (solvents and water) and experiment blanks (PBS) were included. All extraction solvents had 50 μg/mL BHT. Extracted samples were kept dried at -20oC under Ar.

Chromatography:

Chromatography ID:	CH005357
Instrument Name:	Thermo Dionex Ultimate 3000
Column Name:	Thermo Accucore C18 (150 x 2.1mm,2.6um)
Column Temperature:	35°C
Flow Gradient:	The elution was carried out using the following stepwise gradient of solvent B: 0.0 min, 35%; 4.0 min, 60%; 8.0 min, 70%; 16.0 min, 85%; 25.0 min, 97%.
Flow Rate:	400 µl/min
Solvent A:	50% Acetonitrile/50% Water; 10 mM ammonium formate; 0.1% formic acid
Solvent B:	88% Isopropanol/10% Acetonitrile/2% Water; 2 mM ammonium formate; 0.02 % formic acid
Chromatography Type:	Reversed phase

Analysis:

Analysis ID:	AN007055
Laboratory Name:	CNRS Institute of Molecular and Cellular Pharmacology
Analysis Type:	MS
Chromatography ID:	CH005357
Num Factors:	12
Num Metabolites:	359
Units:	Peak intensity