Summary of Study ST004019

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 PR002515. The data can be accessed directly via it's Project DOI: 10.21228/M8JN9T This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST004019
Study TitleAnalysis of the effects of MINCH on the metabolism of human preadipocytes and mature adipocytes by 13C metabolic tracing with [U-13C]glucose
Study SummaryIn the first part of the project, we investigated the effects of MINCH on the pathway activity of central carbon metabolism in human preadipocytes and mature adipocytes by [U-13C]glucose. For this purpose, human SGBS preadipocytes and mature SGBS adipocytes were exposed to 10 nM and 10 µM MINCH and compared to rosiglitazone-treated cells and cells treated without rosiglitazone (untreated control). In mature adipocytes, the effects of 10 µM DINCH were additionally tested, as previous proteomic analyses indicated changes after 10 nM DINCH treatment in mature SGBS cells, but not in preadipocytes. In MINCH-treated preadipocytes, the induction of lipid accumulation was accompanied by increased glycolysis, pentose phosphate pathway activity as well as a switch of TCA cycle metabolism towards lipid synthesis, confirming the adipogenic effect of MINCH. In MINCH-treated mature adipocytes, [U-13C]glucose labeling indicated increased glycolysis and a switch in acetyl-CoA synthesis away from glucose and glutamine, probably toward the utilization of fatty acids via fatty acid oxidation. The metabolic changes were similar to those observed with rosiglitazone treatment, which has already been described to induce browning of mature adipocytes. Indeed, increased UCP-1 expression confirmed the induction of browning by MINCH treatment as well. No effect of DINCH treatment was observed in mature adipocytes.
Institute
Helmholtz Centre for Environmental Research
Last NameGoerdeler
First NameCornelius
AddressPermoserstr. 15
Emailcornelius.goerdeler@ufz.de
Phone004934160252713
Submit Date2025-06-16
Raw Data AvailableYes
Raw Data File Type(s)mzML, wiff
Analysis Type DetailLC-MS
Release Date2025-07-24
Release Version1
Cornelius Goerdeler Cornelius Goerdeler
https://dx.doi.org/10.21228/M8JN9T
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR002515
Project DOI:doi: 10.21228/M8JN9T
Project Title:13C Metabolic Tracing in Human SGBS Cells Provides a Potential New Approach Methodology for Assessing Metabolism-Disrupting Properties
Project Summary:Due to the increased use and production of plastic materials worldwide, humans are ubiquitously exposed to plastic additives, including plasticizers. Recent research suggests that exposure to certain plasticizers promotes obesity due to their metabolism-disrupting properties. Following the ban on di-(2-ethylhexyl) phthalate (DEHP) and other phthalate plasticizers due to their reproductive toxicity, substitutes such as the plasticizer diisononylcyclohexane-1,2-dicarboxylate (DINCH) have been increasingly used. However, in vitro, studies indicate that the primary metabolite monoisononylcyclohexane-1,2-dicarboxylic acid ester (MINCH) promotes differentiation of human adipocytes. In contrast, no obesogenic effect has been observed in application studies in vivo. The absence of weight-promoting effects of DINCH was confirmed in a recent study with DINCH-exposed C57BL/6N mice, but an increase in adipocyte size in visceral adipose tissue and sex-specific effects on serum lipid levels together with impaired insulin sensitivity were observed. Therefore, as there is still limited information about the potential metabolism-disrupting properties of MINCH, we used 13C tracing as a novel method to investigate the effects of MINCH on the pathway activity of central carbon metabolism in human adipocytes. In contrast to metabolomics, which provides information on changes in the abundance of metabolites, 13C metabolic tracing provides an overview of changes in metabolic pathway activity, enabling an in-depth understanding of how metabolism-disrupting chemicals might disrupt cellular metabolism. The changes in central carbon metabolism activity following MINCH treatment were analyzed after insulin stimulation using three carbon tracers. The project consists of three main studies, depending on the use of the carbon tracer: 1. Analysis of the effects of MINCH on glycolysis and pentose phosphate pathway (PPP) activity, the contribution of glucose to the tricarboxylic acid cycle (TCA) and pyruvate carboxylase-mediated anaplerosis in human preadipocytes and mature adipocytes using [U-13C]glucose; 2. Validation of the effects of MINCH on glycolysis and PPP activity and discrimination of their contribution to glucose metabolism in human preadipocytes and mature adipocytes using [1,2-13C]glucose; 3. Assessment of glyceroneogenesis activity, glutamine contribution to the TCA cycle, oxidative flux through the TCA, reductive carboxylation via isocitrate dehydrogenase (IDH) for lipid synthesis and cycling of metabolites through the TCA cycle in human preadipocytes and mature adipocytes using [U-13C]-glutamine.
Institute:Helmholtz Centre for Environmental Research
Last Name:Engelmann
First Name:Beatrice
Address:Permoserstr. 15
Email:beatrice.engelmann@ufz.de
Phone:004934160251099

Subject:

Subject ID:SU004157
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 Sample source Treatment Sample Type Labeling time
SA462114Adult_Ctrl_24h_UGlc_1SGBS mature adipocytes Control cell pellet 24 h
SA462115Adult_Ctrl_24h_UGlc_3SGBS mature adipocytes Control cell pellet 24 h
SA462116Adult_Ctrl_24h_UGlc_4SGBS mature adipocytes Control cell pellet 24 h
SA462117Adult_Ctrl_24h_UGlc_2SGBS mature adipocytes Control cell pellet 24 h
SA462118Adult_Ctrl_3h_UGlc_4SGBS mature adipocytes Control cell pellet 3 h
SA462119Adult_Ctrl_3h_UGlc_3SGBS mature adipocytes Control cell pellet 3 h
SA462120Adult_Ctrl_3h_UGlc_2SGBS mature adipocytes Control cell pellet 3 h
SA462121Adult_Ctrl_3h_UGlc_1SGBS mature adipocytes Control cell pellet 3 h
SA462122Adult_Ctrl_SN_24h_UGlc_4SGBS mature adipocytes Control cell supernatant 24 h
SA462123Adult_Ctrl_SN_24h_UGlc_3SGBS mature adipocytes Control cell supernatant 24 h
SA462124Adult_Ctrl_SN_24h_UGlc_2SGBS mature adipocytes Control cell supernatant 24 h
SA462125Adult_Ctrl_SN_24h_UGlc_1SGBS mature adipocytes Control cell supernatant 24 h
SA462126Adult_Ctrl_SN_3h_UGlc_1SGBS mature adipocytes Control cell supernatant 3 h
SA462127Adult_Ctrl_SN_3h_UGlc_2SGBS mature adipocytes Control cell supernatant 3 h
SA462128Adult_Ctrl_SN_3h_UGlc_3SGBS mature adipocytes Control cell supernatant 3 h
SA462129Adult_Ctrl_SN_3h_UGlc_4SGBS mature adipocytes Control cell supernatant 3 h
SA462130Adult_DINCH_10µM_24h_UGlc_3SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462131Adult_DINCH_10µM_24h_UGlc_2SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462132Adult_DINCH_10µM_24h_UGlc_4SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462133Adult_DINCH_10µM_24h_UGlc_1SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462134Adult_DINCH_10µM_3h_UGlc_4SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462135Adult_DINCH_10µM_3h_UGlc_3SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462136Adult_DINCH_10µM_3h_UGlc_2SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462137Adult_DINCH_10µM_3h_UGlc_1SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462138Adult_DINCH_10µM_SN_24h_UGlc_3SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462139Adult_DINCH_10µM_SN_24h_UGlc_4SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462140Adult_DINCH_10µM_SN_24h_UGlc_2SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462141Adult_DINCH_10µM_SN_24h_UGlc_1SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462142Adult_DINCH_10µM_SN_3h_UGlc_2SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462143Adult_DINCH_10µM_SN_3h_UGlc_3SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462144Adult_DINCH_10µM_SN_3h_UGlc_4SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462145Adult_DINCH_10µM_SN_3h_UGlc_1SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462146Adult_MINCH_10nM_24h_UGlc_1SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462147Adult_MINCH_10nM_24h_UGlc_2SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462148Adult_MINCH_10nM_24h_UGlc_3SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462149Adult_MINCH_10nM_24h_UGlc_4SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462150Adult_MINCH_10nM_3h_UGlc_2SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462151Adult_MINCH_10nM_3h_UGlc_1SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462152Adult_MINCH_10nM_3h_UGlc_4SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462153Adult_MINCH_10nM_3h_UGlc_3SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462154Adult_MINCH_10nM_SN_24h_UGlc_3SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462155Adult_MINCH_10nM_SN_24h_UGlc_1SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462156Adult_MINCH_10nM_SN_24h_UGlc_2SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462157Adult_MINCH_10nM_SN_24h_UGlc_4SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462158Adult_MINCH_10nM_SN_3h_UGlc_1SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462159Adult_MINCH_10nM_SN_3h_UGlc_4SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462160Adult_MINCH_10nM_SN_3h_UGlc_3SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462161Adult_MINCH_10nM_SN_3h_UGlc_2SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462162Adult_MINCH_10µM_24h_UGlc_1SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462163Adult_MINCH_10µM_24h_UGlc_2SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462164Adult_MINCH_10µM_24h_UGlc_4SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462165Adult_MINCH_10µM_24h_UGlc_3SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462166Adult_MINCH_10µM_3h_UGlc_4SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462167Adult_MINCH_10µM_3h_UGlc_2SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462168Adult_MINCH_10µM_3h_UGlc_3SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462169Adult_MINCH_10µM_3h_UGlc_1SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462170Adult_MINCH_10µM_SN_24h_UGlc_3SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462171Adult_MINCH_10µM_SN_24h_UGlc_2SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462172Adult_MINCH_10µM_SN_24h_UGlc_1SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462173Adult_MINCH_10µM_SN_24h_UGlc_4SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462174Adult_MINCH_10µM_SN_3h_UGlc_1SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462175Adult_MINCH_10µM_SN_3h_UGlc_3SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462176Adult_MINCH_10µM_SN_3h_UGlc_2SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462177Adult_MINCH_10µM_SN_3h_UGlc_4SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462178Adult_Rosi_24h_UGlc_1SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462179Adult_Rosi_24h_UGlc_2SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462180Adult_Rosi_24h_UGlc_3SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462181Adult_Rosi_24h_UGlc_4SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462182Adult_Rosi_3h_UGlc_2SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462183Adult_Rosi_3h_UGlc_4SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462184Adult_Rosi_3h_UGlc_3SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462185Adult_Rosi_3h_UGlc_1SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462186Adult_Rosi_SN_24h_UGlc_1SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462187Adult_Rosi_SN_24h_UGlc_2SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462188Adult_Rosi_SN_24h_UGlc_3SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462189Adult_Rosi_SN_24h_UGlc_4SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462190Adult_Rosi_SN_3h_UGlc_1SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462191Adult_Rosi_SN_3h_UGlc_2SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462192Adult_Rosi_SN_3h_UGlc_3SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462193Adult_Rosi_SN_3h_UGlc_4SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462194Pre_Ctrl_24h_UGlc_4SGBS preadipocytes Control cell pellet 24 h
SA462195Pre_Ctrl_24h_UGlc_3SGBS preadipocytes Control cell pellet 24 h
SA462196Pre_Ctrl_24h_UGlc_1SGBS preadipocytes Control cell pellet 24 h
SA462197Pre_Ctrl_24h_UGlc_2SGBS preadipocytes Control cell pellet 24 h
SA462198Pre_Ctrl_3h_UGlc_3SGBS preadipocytes Control cell pellet 3 h
SA462199Pre_Ctrl_3h_UGlc_2SGBS preadipocytes Control cell pellet 3 h
SA462200Pre_Ctrl_3h_UGlc_1SGBS preadipocytes Control cell pellet 3 h
SA462201Pre_Ctrl_3h_UGlc_4SGBS preadipocytes Control cell pellet 3 h
SA462202Pre_Ctrl_SN_24h_UGlc_1SGBS preadipocytes Control cell supernatant 24 h
SA462203Pre_Ctrl_SN_24h_UGlc_2SGBS preadipocytes Control cell supernatant 24 h
SA462204Pre_Ctrl_SN_24h_UGlc_3SGBS preadipocytes Control cell supernatant 24 h
SA462205Pre_Ctrl_SN_24h_UGlc_4SGBS preadipocytes Control cell supernatant 24 h
SA462206Pre_Ctrl_SN_3h_UGlc_2SGBS preadipocytes Control cell supernatant 3 h
SA462207Pre_Ctrl_SN_3h_UGlc_4SGBS preadipocytes Control cell supernatant 3 h
SA462208Pre_Ctrl_SN_3h_UGlc_3SGBS preadipocytes Control cell supernatant 3 h
SA462209Pre_Ctrl_SN_3h_UGlc_1SGBS preadipocytes Control cell supernatant 3 h
SA462210Pre_MINCH_10nM_24h_UGlc_2SGBS preadipocytes MINCH 10nM cell pellet 24 h
SA462211Pre_MINCH_10nM_24h_UGlc_4SGBS preadipocytes MINCH 10nM cell pellet 24 h
SA462212Pre_MINCH_10nM_24h_UGlc_3SGBS preadipocytes MINCH 10nM cell pellet 24 h
SA462213Pre_MINCH_10nM_24h_UGlc_1SGBS preadipocytes MINCH 10nM cell pellet 24 h
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Collection:

Collection ID:CO004150
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. DOI: 10.1038/sj.ijo.0801520).
Sample Type:Adipose tissue

Treatment:

Treatment ID:TR004166
Treatment Summary:SGSB preadipocytes and mature adipocytes were maintained at 37 °C and 5 % CO2 at 95 % humidity. To investigate the effects of MINCH on preadipocytes during adipocyte differentiation, SGBS preadipocytes were treated with differentiation media (DMEM/F12 medium supplemented with 33 µM biotin, 17 µM pantothenate, 100 µg/mL streptomycin, 100 IU/mL penicillin, 25 nmol/L dexamethasone, 0.5 mmol/L 3-isobutyl-1-methylxanthine, 0.1 μmol/L cortisol, 0.01 mg/mL transferrin, 0.2 nmol/L triiodothyronine, and 20 nmol/L human insulin (QD d0-d4) or with 33 µM biotin, 17 µM pantothenate, 100 µg/mL streptomycin, 100 IU/mL penicillin, 0.1 μmol/L cortisol, 0.01 mg/mL transferrin, 0.2 nmol/L triiodothyronine, and 20 nmol/L human insulin (3FC medium d4-d12)) without the PPARG agonist rosiglitazone supplemented with MINCH (10 nM and 10 µM) for 12 days. To obtain a reference for adipogenesis, SGBS cells were differentiated in the presence of rosiglitazone according to the standard protocol; to obtain an untreated control, they were differentiated in the absence of rosiglitazone. To investigate the effects on mature adipocytes, all cells were differentiated according to the standard protocol until day 12 (Wabitsch et al., 2001), followed by treatment with 10 nM and 10 µM MINCH as well as 10 µM DINCH until day 20, in addition to rosiglitazone treatment and the untreated control. For isotope labeling under insulin-stimulated conditions, cells were kept insulin-deprived for 16 hours, and the medium was replaced with 3FC medium prepared with custom-made DMEM/F12 in which glucose was replaced with the stable isotope-labeled analog [U-13C]glucose, and conditioned according to the treatment (DINCH, MINCH, rosiglitazone or control). Labeling incubation was performed for 3 and 24 h, followed by metabolite extraction. A final concentration of 0.01% (v/v) MeOH and 0.02% (v/v) DMSO was added to all conditioned differentiation media. Continuous exposure was mimicked by replacing the cell culture medium every second day. Each treatment was performed in four biological replicates (n=4).

Sample Preparation:

Sampleprep ID:SP004163
Sampleprep Summary:Intracellular and extracellular metabolites were extracted with 1:1:1 methanol:water:chloroform. To extract the intracellular metabolites, the culture medium was removed, the cells were rinsed twice with 1 ml 0.9 % ice-cold NaCl, and the metabolism was stopped by adding MeOH (-20 °C) followed by the addition of ice-cold H2O containing 10 µM d6-glutarate in equal amounts. The cells were collected by cell lifter and chloroform was added to the lysate. After shaking for 20 minutes at 1,400 rpm and 4 °C, the extraction mixture was centrifuged at 18,000 g and 4 °C for 5 minutes. The polar upper phase was then collected and evaporated to complete dryness. For extraction of the extracellular metabolites, 300 µL of the supernatant was extracted in a 1:1 methanol:water:chloroform ratio, again with MeOH (-20 °C) containing 100 nM MEHP and ice-cold H2O containing 40 µM d6-glutarate. The following sample preparation was identical to the extraction of the intracellular metabolites. Note: After LC-MS measurement of the samples, the raw AUC isotopologue values provided here were corrected by 1.1 % of the 13C natural abundance using the R-based IsoCorrectoR tool (Heinrich et al., 2018). After correction, the relative 13C isotopolog abundances and the 13C fractional contributions from glucose and glutamine were calculated for each metabolite.

Chromatography:

Chromatography ID:CH005034
Chromatography Summary:Solvent A: 10mM tributylamine, 10mM acetic acid, 5% MeOH, 2% 2-propanol in water; Solvent B: 100% 2-propanol
Instrument Name:Agilent 1290 Infinity II
Column Name:Waters Xselect XP HSS T3 (150 x 2.1mm, 2.5um)
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:93% water/5% methanol/2% isopropanol; 10mM tributylamine; 10mM acetic acid
Solvent B:100% isopropanol
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN006625
Analysis Type:MS
Chromatography ID:CH005034
Num Factors:36
Num Metabolites:123
Units:Peak AUC
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