{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002920","ANALYSIS_ID":"AN004790","VERSION":"1","CREATED_ON":"October 10, 2023, 2:18 am"},

"PROJECT":{"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of the use of the phthalate plasticizer di-(2-ethylhexyl) phthalate (DEHP) due to its adverse health effects, it has been replaced by new substitutes such as the plasticizer diisononylcyclohexane-1,2-dicarboxylate (DINCH).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"},

"STUDY":{"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"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606","GENDER":"Male"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_1",
"Factors":{"Treatment":"Control GW9662 treatment"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_2",
"Factors":{"Treatment":"Control GW9662 treatment"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_3",
"Factors":{"Treatment":"Control GW9662 treatment"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_4",
"Factors":{"Treatment":"Control GW9662 treatment"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_1",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_2",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_3",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_4",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW_1",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW_2",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW_3",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW_4",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_1",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_2",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_3",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_4",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230418_SGBS_DINCH_MINCH_GW_intracellular_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_SN1",
"Factors":{"Treatment":"Control GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_SN2",
"Factors":{"Treatment":"Control GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_SN3",
"Factors":{"Treatment":"Control GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Ctrl_GW_SN4",
"Factors":{"Treatment":"Control GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_SN1",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_SN2",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_SN3",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"DINCH_GW_SN4",
"Factors":{"Treatment":"DINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW__SN1",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW_SN2",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW_SN3",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"MINCH_GW_SN4",
"Factors":{"Treatment":"MINCH 10µM + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_SN1",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"1","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_SN2",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"2","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_SN3",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"3","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
},
{
"Subject ID":"-",
"Sample ID":"Rosi_GW_SN4",
"Factors":{"Treatment":"Rosi (d0-d4) + GW9662 treatment supernatant"},
"Additional sample data":{"Replicate":"4","Species":"Homo sapiens","RAW_FILE_NAME":"230420_SGBS_DINCH_MINCH_GW_SN_CG.wiff"}
}
],
"COLLECTION":{"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_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)."},

"SAMPLEPREP":{"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."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1290 Infinity II","COLUMN_NAME":"Waters Xselect XP HSS T3 (150 x 2.1mm, 2.5µm)","SOLVENT_A":"10mM TBA, 10mM acetic acid, 5% MeOH, 2% IPA in water","SOLVENT_B":"100% IPA","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","COLUMN_TEMPERATURE":"40"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"ABI Sciex 6500+","INSTRUMENT_TYPE":"QTRAP","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","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)."},

"MS_METABOLITE_DATA":{
"Units":"Peak AUC",

"Data":[{"Metabolite":"Glucose 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"Metabolites":[{"Metabolite":"Glucose 6-phosphate","m/z":"259","PubChem ID":"5958","KEGG_ID":"C00668 "},{"Metabolite":"Fructose 6-phosphate","m/z":"259","PubChem ID":"69507","KEGG_ID":"C00085 "},{"Metabolite":"Fructose 1,6-bisphosphate","m/z":"339","PubChem ID":"10267","KEGG_ID":"C05378 "},{"Metabolite":"Glycerol 3-phosphate","m/z":"171","PubChem ID":"439162","KEGG_ID":"C00093"},{"Metabolite":"Glyceraldehyde 3-phosphate","m/z":"169","PubChem ID":"439168","KEGG_ID":"C00118   "},{"Metabolite":"3-Phosphoglycerate","m/z":"185","PubChem ID":"724","KEGG_ID":"C00197 "},{"Metabolite":"Phosphoenolpyruvate","m/z":"167","PubChem ID":"1005","KEGG_ID":"C00074"},{"Metabolite":"Pyruvate","m/z":"87","PubChem ID":"107735","KEGG_ID":"C00022"},{"Metabolite":"Lactate","m/z":"89","PubChem ID":"91435","KEGG_ID":"C00186"},{"Metabolite":"Alanine","m/z":"88","PubChem ID":"5950","KEGG_ID":"C00041"},{"Metabolite":"Acetyl-CoA","m/z":"808","PubChem ID":"444493","KEGG_ID":"C00024"},{"Metabolite":"Malonyl-CoA","m/z":"852","PubChem ID":"644066","KEGG_ID":"C00083"},{"Metabolite":"Citrate","m/z":"191","PubChem ID":"311","KEGG_ID":"C00158"},{"Metabolite":"cis-Aconitate","m/z":"173","PubChem ID":"643757","KEGG_ID":"C00417 "},{"Metabolite":"a-Ketoglutarate","m/z":"145","PubChem ID":"51","KEGG_ID":"C00026 "},{"Metabolite":"Aspartate","m/z":"132","PubChem ID":"5960","KEGG_ID":"C00049"},{"Metabolite":"Asparagine","m/z":"131","PubChem ID":"6267","KEGG_ID":"C00152"},{"Metabolite":"Succinate","m/z":"117","PubChem ID":"1110","KEGG_ID":"C00042"},{"Metabolite":"Fumarate","m/z":"115","PubChem ID":"444972","KEGG_ID":"C00122"},{"Metabolite":"Malate","m/z":"133","PubChem ID":"525","KEGG_ID":"C00711"},{"Metabolite":"Oxaloacetate","m/z":"131","PubChem ID":"970","KEGG_ID":"C00036"},{"Metabolite":"Glutamate","m/z":"146","PubChem ID":"33032","KEGG_ID":"C00025"},{"Metabolite":"Glutamine","m/z":"145","PubChem ID":"5961","KEGG_ID":"C00064"},{"Metabolite":"Ribulose 5-phosphate","m/z":"229","PubChem ID":"439184","KEGG_ID":"C00199"},{"Metabolite":"Ribose 5-phosphate","m/z":"229","PubChem ID":"439167","KEGG_ID":"C00117"},{"Metabolite":"Sedoheptulose 7-phosphate","m/z":"289","PubChem ID":"65007","KEGG_ID":"C05382"}]
}

}