Summary of Study ST004021

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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Study IDST004021
Study TitleAnalysis of the effects of MINCH on the metabolism of human preadipocytes and mature adipocytes by 13C metabolic tracing with [U-13C]glutamine
Study SummaryIn the third part of the project, we investigated the influence of MINCH on the activity of central carbon metabolism in human preadipocytes and mature adipocytes using [U-13C]glutamine. As in studies 1 and 2, the effects were compared with rosiglitazone-treated cells and untreated control cells. In preadipocytes, increased acetyl-CoA production from glutamine via transiently increased IDH activity as well as transiently decreased oxidative activity and decreased cycling of metabolites through the TCA cycle confirmed the switch of the TCA cycle to lipid synthesis. In mature adipocytes, [U-13C]glutamine labeling showed an increased glyceroneogenesis flux for the production of glycerol-3-phosphate, an important precursor for triglyceride synthesis. In addition, the transient increased flux of the oxidative TCA cycle and the cycling of metabolites through the TCA cycle indicate an overall increase in the activity of the oxidative TCA cycle metabolism. Since similar changes were observed in rosiglitazone-induced browning in human adipocytes, this confirms the browning effect in mature SGBS cells by MINCH treatment.
Institute
Helmholtz Centre for Environmental Research
Last NameGoerdeler
First NameCornelius
AddressPermoserstr. 15
Emailcornelius.goerdeler@ufz.de
Phone004934160252713
Submit Date2025-06-18
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:SU004159
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
SA462402Adult_Ctrl_24h_U_Gln_1SGBS mature adipocytes Control cell pellet 24 h
SA462403Adult_Ctrl_24h_U_Gln_3SGBS mature adipocytes Control cell pellet 24 h
SA462404Adult_Ctrl_24h_U_Gln_4SGBS mature adipocytes Control cell pellet 24 h
SA462405Adult_Ctrl_24h_U_Gln_2SGBS mature adipocytes Control cell pellet 24 h
SA462406Adult_Ctrl_3h_U_Gln_4SGBS mature adipocytes Control cell pellet 3 h
SA462407Adult_Ctrl_3h_U_Gln_3SGBS mature adipocytes Control cell pellet 3 h
SA462408Adult_Ctrl_3h_U_Gln_2SGBS mature adipocytes Control cell pellet 3 h
SA462409Adult_Ctrl_3h_U_Gln_1SGBS mature adipocytes Control cell pellet 3 h
SA462410Adult_Ctrl_SN_24h_U_Gln_4SGBS mature adipocytes Control cell supernatant 24 h
SA462411Adult_Ctrl_SN_24h_U_Gln_3SGBS mature adipocytes Control cell supernatant 24 h
SA462412Adult_Ctrl_SN_24h_U_Gln_2SGBS mature adipocytes Control cell supernatant 24 h
SA462413Adult_Ctrl_SN_24h_U_Gln_1SGBS mature adipocytes Control cell supernatant 24 h
SA462414Adult_Ctrl_SN_3h_U_Gln_1SGBS mature adipocytes Control cell supernatant 3 h
SA462415Adult_Ctrl_SN_3h_U_Gln_2SGBS mature adipocytes Control cell supernatant 3 h
SA462416Adult_Ctrl_SN_3h_U_Gln_3SGBS mature adipocytes Control cell supernatant 3 h
SA462417Adult_Ctrl_SN_3h_U_Gln_4SGBS mature adipocytes Control cell supernatant 3 h
SA462418Adult_DINCH_10µM_24h_U_Gln_3SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462419Adult_DINCH_10µM_24h_U_Gln_2SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462420Adult_DINCH_10µM_24h_U_Gln_4SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462421Adult_DINCH_10µM_24h_U_Gln_1SGBS mature adipocytes DINCH 10µM cell pellet 24 h
SA462422Adult_DINCH_10µM_3h_U_Gln_4SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462423Adult_DINCH_10µM_3h_U_Gln_3SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462424Adult_DINCH_10µM_3h_U_Gln_2SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462425Adult_DINCH_10µM_3h_U_Gln_1SGBS mature adipocytes DINCH 10µM cell pellet 3 h
SA462426Adult_DINCH_10µM_SN_24h_U_Gln_3SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462427Adult_DINCH_10µM_SN_24h_U_Gln_4SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462428Adult_DINCH_10µM_SN_24h_U_Gln_2SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462429Adult_DINCH_10µM_SN_24h_U_Gln_1SGBS mature adipocytes DINCH 10µM cell supernatant 24 h
SA462430Adult_DINCH_10µM_SN_3h_U_Gln_2SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462431Adult_DINCH_10µM_SN_3h_U_Gln_3SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462432Adult_DINCH_10µM_SN_3h_U_Gln_4SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462433Adult_DINCH_10µM_SN_3h_U_Gln_1SGBS mature adipocytes DINCH 10µM cell supernatant 3 h
SA462434Adult_MINCH_10nM_24h_U_Gln_1SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462435Adult_MINCH_10nM_24h_U_Gln_2SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462436Adult_MINCH_10nM_24h_U_Gln_3SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462437Adult_MINCH_10nM_24h_U_Gln_4SGBS mature adipocytes MINCH 10nM cell pellet 24 h
SA462438Adult_MINCH_10nM_3h_U_Gln_2SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462439Adult_MINCH_10nM_3h_U_Gln_1SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462440Adult_MINCH_10nM_3h_U_Gln_4SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462441Adult_MINCH_10nM_3h_U_Gln_3SGBS mature adipocytes MINCH 10nM cell pellet 3 h
SA462442Adult_MINCH_10nM_SN_24h_U_Gln_3SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462443Adult_MINCH_10nM_SN_24h_U_Gln_1SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462444Adult_MINCH_10nM_SN_24h_U_Gln_2SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462445Adult_MINCH_10nM_SN_24h_U_Gln_4SGBS mature adipocytes MINCH 10nM cell supernatant 24 h
SA462446Adult_MINCH_10nM_SN_3h_U_Gln_1SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462447Adult_MINCH_10nM_SN_3h_U_Gln_4SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462448Adult_MINCH_10nM_SN_3h_U_Gln_3SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462449Adult_MINCH_10nM_SN_3h_U_Gln_2SGBS mature adipocytes MINCH 10nM cell supernatant 3 h
SA462450Adult_MINCH_10µM_24h_U_Gln_1SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462451Adult_MINCH_10µM_24h_U_Gln_2SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462452Adult_MINCH_10µM_24h_U_Gln_4SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462453Adult_MINCH_10µM_24h_U_Gln_3SGBS mature adipocytes MINCH 10µM cell pellet 24 h
SA462454Adult_MINCH_10µM_3h_U_Gln_4SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462455Adult_MINCH_10µM_3h_U_Gln_2SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462456Adult_MINCH_10µM_3h_U_Gln_3SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462457Adult_MINCH_10µM_3h_U_Gln_1SGBS mature adipocytes MINCH 10µM cell pellet 3 h
SA462458Adult_MINCH_10µM_SN_24h_U_Gln_3SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462459Adult_MINCH_10µM_SN_24h_U_Gln_2SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462460Adult_MINCH_10µM_SN_24h_U_Gln_1SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462461Adult_MINCH_10µM_SN_24h_U_Gln_4SGBS mature adipocytes MINCH 10µM cell supernatant 24 h
SA462462Adult_MINCH_10µM_SN_3h_U_Gln_1SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462463Adult_MINCH_10µM_SN_3h_U_Gln_3SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462464Adult_MINCH_10µM_SN_3h_U_Gln_2SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462465Adult_MINCH_10µM_SN_3h_U_Gln_4SGBS mature adipocytes MINCH 10µM cell supernatant 3 h
SA462466Adult_Rosi_24h_U_Gln_1SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462467Adult_Rosi_24h_U_Gln_2SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462468Adult_Rosi_24h_U_Gln_3SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462469Adult_Rosi_24h_U_Gln_4SGBS mature adipocytes Rosiglitazone cell pellet 24 h
SA462470Adult_Rosi_3h_U_Gln_2SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462471Adult_Rosi_3h_U_Gln_4SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462472Adult_Rosi_3h_U_Gln_3SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462473Adult_Rosi_3h_U_Gln_1SGBS mature adipocytes Rosiglitazone cell pellet 3 h
SA462474Adult_Rosi_SN_24h_U_Gln_1SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462475Adult_Rosi_SN_24h_U_Gln_2SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462476Adult_Rosi_SN_24h_U_Gln_3SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462477Adult_Rosi_SN_24h_U_Gln_4SGBS mature adipocytes Rosiglitazone cell supernatant 24 h
SA462478Adult_Rosi_SN_3h_U_Gln_1SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462479Adult_Rosi_SN_3h_U_Gln_2SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462480Adult_Rosi_SN_3h_U_Gln_3SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462481Adult_Rosi_SN_3h_U_Gln_4SGBS mature adipocytes Rosiglitazone cell supernatant 3 h
SA462482Pre_Ctrl_24h_U_Gln_4SGBS preadipocytes Control cell pellet 24 h
SA462483Pre_Ctrl_24h_U_Gln_3SGBS preadipocytes Control cell pellet 24 h
SA462484Pre_Ctrl_24h_U_Gln_1SGBS preadipocytes Control cell pellet 24 h
SA462485Pre_Ctrl_24h_U_Gln_2SGBS preadipocytes Control cell pellet 24 h
SA462486Pre_Ctrl_3h_U_Gln_3SGBS preadipocytes Control cell pellet 3 h
SA462487Pre_Ctrl_3h_U_Gln_2SGBS preadipocytes Control cell pellet 3 h
SA462488Pre_Ctrl_3h_U_Gln_1SGBS preadipocytes Control cell pellet 3 h
SA462489Pre_Ctrl_3h_U_Gln_4SGBS preadipocytes Control cell pellet 3 h
SA462490Pre_Ctrl_SN_24h_U_Gln_1SGBS preadipocytes Control cell supernatant 24 h
SA462491Pre_Ctrl_SN_24h_U_Gln_2SGBS preadipocytes Control cell supernatant 24 h
SA462492Pre_Ctrl_SN_24h_U_Gln_3SGBS preadipocytes Control cell supernatant 24 h
SA462493Pre_Ctrl_SN_24h_U_Gln_4SGBS preadipocytes Control cell supernatant 24 h
SA462494Pre_Ctrl_SN_3h_U_Gln_2SGBS preadipocytes Control cell supernatant 3 h
SA462495Pre_Ctrl_SN_3h_U_Gln_4SGBS preadipocytes Control cell supernatant 3 h
SA462496Pre_Ctrl_SN_3h_U_Gln_3SGBS preadipocytes Control cell supernatant 3 h
SA462497Pre_Ctrl_SN_3h_U_Gln_1SGBS preadipocytes Control cell supernatant 3 h
SA462498Pre_MINCH_10nM_24h_U_Gln_2SGBS preadipocytes MINCH 10nM cell pellet 24 h
SA462499Pre_MINCH_10nM_24h_U_Gln_4SGBS preadipocytes MINCH 10nM cell pellet 24 h
SA462500Pre_MINCH_10nM_24h_U_Gln_3SGBS preadipocytes MINCH 10nM cell pellet 24 h
SA462501Pre_MINCH_10nM_24h_U_Gln_1SGBS preadipocytes MINCH 10nM cell pellet 24 h
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Collection:

Collection ID:CO004152
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:TR004168
Treatment Summary:SGSB preadipocytes and mature adipocytes were maintained at 37°C and 5% CO2 in 95% humidity. The treatment of preadipocytes and mature adipocytes was performed as described in study 1. For isotopic labeling under insulin-stimulated conditions, cells were insulin-starved for 16 h, and the medium was replaced with 3FC medium prepared with customized DMEM/F12 in which glutamine was replaced with the stable isotope-labeled analog [U-13C]glutamine and conditioned according to the treatment (DINCH, MINCH rosiglitazone or control). Labeling incubation was performed for 3 and 24h 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:SP004165
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:CH005036
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:AN006627
Analysis Type:MS
Chromatography ID:CH005036
Num Factors:36
Num Metabolites:123
Units:Peak AUC
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