Summary of Study ST002920

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 PR001814. The data can be accessed directly via it's Project DOI: 10.21228/M87D9M 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	ST002920
Study Title	Possible PPARG-independent effects of DINCH and MINCH on central carbon metabolism
Study Summary	In the third part of the project, we investigated the PPARG-independent effects of DINCH and MINCH on the central carbon metabolism of human SGBS cells. SGBS preadipocytes were treated for 12 days with 10 µM MINCH, 10 µM DINCH, or rosiglitazone in the presence of the PPARG inhibitor GW9662. Irreversible blocking of PPARG was achieved by incubating cells with 10 µM GW9662 1 hour before treatment and maintaining a regular treatment interval of 2 days. In conclusion, although the PPARG inhibitor GW9662 greatly reduced the effects of MINCH and rosiglitazone, slightly increased lipid accumulation along with increased lactate secretion and increased concentrations of pyruvate cycle metabolites were consistently induced by MINCH treatment even after PPARG inhibition. This suggests that MINCH exerts its effects on lipid accumulation and central carbon metabolism at least in part via a PPARG-independent mechanism.
Institute	Helmholtz Centre for Environmental Research
Last Name	Engelmann
First Name	Beatrice
Address	Permoserstr. 15
Email	beatrice.engelmann@ufz.de
Phone	00493412351099
Submit Date	2023-10-08
Raw Data Available	Yes
Raw Data File Type(s)	wiff
Analysis Type Detail	LC-MS
Release Date	2023-11-01
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001814
Project DOI:	doi: 10.21228/M87D9M
Project Title:	MINCH causes metabolic rewiring towards lipid accumulation and adipogenesis
Project Summary:	Humans are ubiquitously exposed to plastic additives, including plasticizers. There is growing evidence that exposure to certain plasticizers is associated with the development of obesity due to their metabolism-disrupting properties. Following the restriction of the use of the phthalate plasticizer di-(2-ethylhexyl) phthalate (DEHP) due to its adverse health effects, it has been replaced by new substitutes such as the plasticizer diisononylcyclohexane-1,2-dicarboxylate (DINCH). Despite recent studies suggesting that the primary metabolite monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) promotes human adipocyte differentiation, the adipogenic properties of MINCH remain controversial. Because the metabolome largely reflects the molecular phenotype and is sensitive to perturbation by external factors, we used targeted metabolomics to investigate the effects of DINCH and MINCH on key metabolic pathways of adipocytes. Analysis of central carbon metabolism is particularly relevant because it provides cellular energy through the degradation of organic compounds and metabolic precursors for anabolic functions that are critical for adipocyte function, such as de novo lipogenesis. The project consists of three main studies: analysis of the effects of DINCH and MINCH on central carbon metabolism of human SGSB cells, analysis of the insulin response of DINCH- and MINCH-treated SGSB cells, and analysis of the effects of DINCH and MINCH on central carbon metabolism of human SGSB cells in the presence of the PPARG inhibitor GW9662.
Institute:	Helmholtz Centre for Environmental Research
Last Name:	Engelmann
First Name:	Beatrice
Address:	Permoserstr. 15
Email:	beatrice.engelmann@ufz.de
Phone:	00493412351099

Subject:

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

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA316858	Ctrl_GW_1	Control GW9662 treatment
SA316859	Ctrl_GW_4	Control GW9662 treatment
SA316860	Ctrl_GW_3	Control GW9662 treatment
SA316861	Ctrl_GW_2	Control GW9662 treatment
SA316862	Ctrl_GW_SN2	Control GW9662 treatment supernatant
SA316863	Ctrl_GW_SN4	Control GW9662 treatment supernatant
SA316864	Ctrl_GW_SN3	Control GW9662 treatment supernatant
SA316865	Ctrl_GW_SN1	Control GW9662 treatment supernatant
SA316866	DINCH_GW_4	DINCH 10µM + GW9662 treatment
SA316867	DINCH_GW_3	DINCH 10µM + GW9662 treatment
SA316868	DINCH_GW_1	DINCH 10µM + GW9662 treatment
SA316869	DINCH_GW_2	DINCH 10µM + GW9662 treatment
SA316870	DINCH_GW_SN3	DINCH 10µM + GW9662 treatment supernatant
SA316871	DINCH_GW_SN4	DINCH 10µM + GW9662 treatment supernatant
SA316872	DINCH_GW_SN2	DINCH 10µM + GW9662 treatment supernatant
SA316873	DINCH_GW_SN1	DINCH 10µM + GW9662 treatment supernatant
SA316874	MINCH_GW_4	MINCH 10µM + GW9662 treatment
SA316875	MINCH_GW_1	MINCH 10µM + GW9662 treatment
SA316876	MINCH_GW_2	MINCH 10µM + GW9662 treatment
SA316877	MINCH_GW_3	MINCH 10µM + GW9662 treatment
SA316878	MINCH_GW_SN2	MINCH 10µM + GW9662 treatment supernatant
SA316879	MINCH_GW_SN3	MINCH 10µM + GW9662 treatment supernatant
SA316880	MINCH_GW_SN4	MINCH 10µM + GW9662 treatment supernatant
SA316881	MINCH_GW__SN1	MINCH 10µM + GW9662 treatment supernatant
SA316882	Rosi_GW_4	Rosi (d0-d4) + GW9662 treatment
SA316883	Rosi_GW_1	Rosi (d0-d4) + GW9662 treatment
SA316884	Rosi_GW_3	Rosi (d0-d4) + GW9662 treatment
SA316885	Rosi_GW_2	Rosi (d0-d4) + GW9662 treatment
SA316886	Rosi_GW_SN4	Rosi (d0-d4) + GW9662 treatment supernatant
SA316887	Rosi_GW_SN1	Rosi (d0-d4) + GW9662 treatment supernatant
SA316888	Rosi_GW_SN2	Rosi (d0-d4) + GW9662 treatment supernatant
SA316889	Rosi_GW_SN3	Rosi (d0-d4) + GW9662 treatment supernatant

Showing results 1 to 32 of 32

Showing results 1 to 32 of 32

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