Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA316886

Local Sample ID:	Rosi_GW_SN4
Subject ID:	SU003033
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male

Select appropriate tab below to view additional metadata details:

Subject:

Subject ID:	SU003033
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Rosi_GW_SN4	SA316886	FL037785	Rosi (d0-d4) + GW9662 treatment supernatant	Treatment

Collection:

Collection ID:	CO003026
Collection Summary:	The SGBS cells were obtained from Prof. Martin Wabitsch laboratory at the University Clinic Ulm. SGBS preadipocytes were differentiated according to the standard protocol described previously (Wabitsch et al., 2001).
Sample Type:	Adipose tissue
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR003042
Treatment Summary:	SGSB preadipocytes were maintained at 37°C and 5% CO2 in 95% humidity. To assess the PPARG-independent effects of DINCH and MINCH on central carbon metabolism, SGBS preadipocytes were exposed to differentiation media containing DINCH (10 µM DINCH+GW) or MINCH (10 µM MINCH+GW) supplemented with the PPARG antagonist GW9662 for 12 days. Irreversible blocking of PPARG prior to treatment was achieved by adding the antagonist 1 hour before the addition of the respective chemical. For comparison, SGBS cells were treated with rosiglitazone (d0-d4) as in the standard protocol but in the presence of GW9662 (Rosi+GW), and SGBS cells were treated with GW9662 only for 12 days (Ctrl+GW). During differentiation, a final concentration of 0.01% (v/v) MeOH and 0.02% (v/v) DMSO was added to all differentiation media. Continuous exposure was mimicked by replacing the cell culture medium every other day. Each treatment was performed in four biological replicates (n=4).

Treatment ID:

TR003042

Treatment Summary:

SGSB preadipocytes were maintained at 37°C and 5% CO2 in 95% humidity. To assess the PPARG-independent effects of DINCH and MINCH on central carbon metabolism, SGBS preadipocytes were exposed to differentiation media containing DINCH (10 µM DINCH+GW) or MINCH (10 µM MINCH+GW) supplemented with the PPARG antagonist GW9662 for 12 days. Irreversible blocking of PPARG prior to treatment was achieved by adding the antagonist 1 hour before the addition of the respective chemical. For comparison, SGBS cells were treated with rosiglitazone (d0-d4) as in the standard protocol but in the presence of GW9662 (Rosi+GW), and SGBS cells were treated with GW9662 only for 12 days (Ctrl+GW). During differentiation, a final concentration of 0.01% (v/v) MeOH and 0.02% (v/v) DMSO was added to all differentiation media. Continuous exposure was mimicked by replacing the cell culture medium every other day. Each treatment was performed in four biological replicates (n=4).

Sample Preparation:

Sampleprep ID:	SP003039
Sampleprep Summary:	Extraction of intracellular and extracellular metabolites was performed by a 1:1:1 methanol:water:chloroform extraction protocol. For the extraction of intracellular metabolites, the culture medium was removed and the cells were rinsed twice with 1 ml of 0.9% ice-cold NaCl. The rinsing solution was removed, and the metabolism of the cells was stopped by adding 400 µL of MeOH (-20 °C) followed by 400 µL of ice-cold H2O containing 10 µM d6-glutarate. Cells were collected using a cell lifter and 400 µL of chloroform was added. After shaking at 1,400 rpm and 4 °C for 20 min, the extraction mixture was centrifuged at 18,000 g and 4 °C for 5 min. Subsequently, 300 µL volume of the polar upper phase was collected and evaporated to complete dryness. For the extraction of extracellular metabolites, 300 µL of the supernatant was extracted by adding 400 µL MeOH (-20 °C) containing 100 nM MEHP, 100 µL ice-cold H2O containing 40 µM d6-glutarate, and 400 µL chloroform (-20 °C). Subsequent sample preparation was identical to the extraction of intracellular metabolites. Note: After measurement of the samples by LC-MS, the raw AUC values uploaded here were normalized to the internal standard (d6-glutarate, if applicable) and DNA content per well (measured by DAPI fluorescence). After normalization, log2 fold changes were calculated by dividing the normalized peak area from each replicate of each treatment by the normalized peak area from each control. Insulin data were not normalized to DAPI because fold changes were calculated by dividing the intensities of the insulin-stimulated cells by the noninsulin-stimulated cells from each treatment.

Sampleprep ID:

SP003039

Sampleprep Summary:

Extraction of intracellular and extracellular metabolites was performed by a 1:1:1 methanol:water:chloroform extraction protocol. For the extraction of intracellular metabolites, the culture medium was removed and the cells were rinsed twice with 1 ml of 0.9% ice-cold NaCl. The rinsing solution was removed, and the metabolism of the cells was stopped by adding 400 µL of MeOH (-20 °C) followed by 400 µL of ice-cold H2O containing 10 µM d6-glutarate. Cells were collected using a cell lifter and 400 µL of chloroform was added. After shaking at 1,400 rpm and 4 °C for 20 min, the extraction mixture was centrifuged at 18,000 g and 4 °C for 5 min. Subsequently, 300 µL volume of the polar upper phase was collected and evaporated to complete dryness. For the extraction of extracellular metabolites, 300 µL of the supernatant was extracted by adding 400 µL MeOH (-20 °C) containing 100 nM MEHP, 100 µL ice-cold H2O containing 40 µM d6-glutarate, and 400 µL chloroform (-20 °C). Subsequent sample preparation was identical to the extraction of intracellular metabolites. Note: After measurement of the samples by LC-MS, the raw AUC values uploaded here were normalized to the internal standard (d6-glutarate, if applicable) and DNA content per well (measured by DAPI fluorescence). After normalization, log2 fold changes were calculated by dividing the normalized peak area from each replicate of each treatment by the normalized peak area from each control. Insulin data were not normalized to DAPI because fold changes were calculated by dividing the intensities of the insulin-stimulated cells by the noninsulin-stimulated cells from each treatment.

Combined analysis:

Analysis ID	AN004790
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity II
Column	Waters Xselect XP HSS T3 (150 x 2.1mm, 2.5µm)
MS Type	ESI
MS instrument type	QTRAP
MS instrument name	ABI Sciex 6500+
Ion Mode	NEGATIVE
Units	Peak AUC

Chromatography:

Chromatography ID:	CH003621
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Waters Xselect XP HSS T3 (150 x 2.1mm, 2.5µm)
Column Temperature:	40
Flow Gradient:	0-5 min 0% B, 5-9 min 0%- 2% B, 9-9.5 min 2-6% B, 9.5-11.5 min 6% B, 11.5-12 min 6-11% B, 12-13.5 min 11% B, 13.5-15.5 min 11-28% B, 15.5-16.5 min 28-53% B, 16.5-22.5 53% B, 22.5-23 min 53-0% B, 23-33 min 0% B
Flow Rate:	0-15.5 min 0.4 mL/min, 15.5-16.5 min 0.4-0.15 mL/min, 16.5-23 min 0.15 mL/min, 23-27 min 0.15-0.4 mL/min, 27-33 min 0.4 mL/min
Solvent A:	10mM TBA, 10mM acetic acid, 5% MeOH, 2% IPA in water
Solvent B:	100% IPA
Chromatography Type:	Reversed phase

MS:

MS ID:	MS004536
Analysis ID:	AN004790
Instrument Name:	ABI Sciex 6500+
Instrument Type:	QTRAP
MS Type:	ESI
MS Comments:	For identification and quantitation, a scheduled MRM method was used, with specific transitions for every metabolite. Data acquisition and peak integration were performed in Analyst® software (Version 1.7.1).
Ion Mode:	NEGATIVE