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Identifying adipogenic chemicals: Disparate effects in 3T3-L1, OP9 and primary mesenchymal multipotent cell models.

ABSTRACT: 3T3-L1 pre-adipocytes are used commonly to identify new adipogens, but this cell line has been shown to produce variable results. Here, potential adipogenic chemicals (identified in the ToxCast dataset using the Toxicological Priority Index) were tested for their ability to induce adipocyte differentiation in 3T3-L1 cells, OP9 cells and primary mouse bone marrow multipotent stromal cells (BM-MSC). Ten of the 36 potential adipogens stimulated lipid accumulation in at least one model (novel: fenthion, quinoxyfen, prallethrin, allethrin, pyrimethanil, tebuconzaole, 2,4,6-tris (tert-butyl)phenol; known: fentin, pioglitazone, 3,3',5,5'-tetrabromobisphenol A). Only prallethrin and pioglitazone enhanced lipid accumulation in all models. OP9 cells were significantly more sensitive to chemicals known to activate PPARγ through RXR than the other models. Coordinate effects on adipocyte and osteoblast differentiation were investigated further in BM-MSCs. Lipid accumulation was correlated with the ability to stimulate expression of the PPARγ target gene, Plin1. Induction of lipid accumulation also was associated with reduction in alkaline phosphatase activity. Allethrin, prallethrin, and quinoxyfen strongly suppressed osteogenic gene expression. BM-MSCs were useful in coordinately investigating pro-adipogenic and anti-osteogenic effects. Overall, the results show that additional models should be used in conjunction with 3T3-L1 cells to identify a broader spectrum of adipogens and their coordinate effects on osteogenesis.

SUBMITTER: Andrews FV

PROVIDER: S-EPMC9432570 | biostudies-literature | 2020 Sep

REPOSITORIES: biostudies-literature

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Identifying adipogenic chemicals: Disparate effects in 3T3-L1, OP9 and primary mesenchymal multipotent cell models.

Andrews Faye V FV Kim Stephanie M SM Edwards Lariah L Schlezinger Jennifer J JJ

Toxicology in vitro : an international journal published in association with BIBRA 20200528

3T3-L1 pre-adipocytes are used commonly to identify new adipogens, but this cell line has been shown to produce variable results. Here, potential adipogenic chemicals (identified in the ToxCast dataset using the Toxicological Priority Index) were tested for their ability to induce adipocyte differentiation in 3T3-L1 cells, OP9 cells and primary mouse bone marrow multipotent stromal cells (BM-MSC). Ten of the 36 potential adipogens stimulated lipid accumulation in at least one model (novel: fenth ...[more]

PMID: 32473317

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Project description:The 3T3-L1 murine pre-adipocyte line is an established cell culture model for screening Metabolism Disrupting Chemicals (MDCs). Despite a need to accurately identify MDCs for further evaluation, relatively little research has been performed to comprehensively evaluate reproducibility across laboratories, assess factors that might contribute to varying degrees of differentiation between laboratories (media additives, plastics, cell source, etc.), or to standardize protocols. As such, the goals of this study were to assess interlaboratory variability of efficacy and potency outcomes for triglyceride accumulation and pre-adipocyte proliferation using the mouse 3T3-L1 pre-adipocyte cell assay to test chemicals. Ten laboratories from five different countries participated. Each laboratory evaluated one reference chemical (rosiglitazone) and three blinded test chemicals (tributyltin chloride, pyraclostrobin, and bisphenol A) using: 1) their Laboratory-specific 3T3-L1 Cells (LC) and their Laboratory-specific differentiation Protocol (LP), 2) Shared 3T3-L1 Cells (SC) with LP, 3) LC with a Shared differentiation Protocol (SP), and 4) SC with SP. Blinded test chemical responses were analyzed by the coordinating laboratory. The magnitude and range of bioactivities reported varied considerably across laboratories and test conditions, though the presence or absence of activity for each tested chemical was more consistent. Triglyceride accumulation activity determinations for rosiglitazone ranged from 90 to 100% across test conditions, but 30-70 % for pre-adipocyte proliferation; this was 40-80 % for triglyceride accumulation induced by pyraclostrobin, 80-100 % for tributyltin, and 80-100 % for bisphenol A. Consistency was much lower for pre-adipocyte proliferation, with 30-70 % active determinations for pyraclostrobin, 30-50 % for tributyltin, and 20-40 % for bisphenol A. Greater consistency was observed for the SC/SP assessment. As such, working to develop a standardized adipogenic differentiation protocol represents the best strategy for improving consistency of adipogenic responses using the 3T3-L1 model to reproducibly identify MDCs and increase confidence in reported outcomes.

Project description:Excess body weight is a major risk factor for type 2 diabetes (T2D) and associated metabolic complications, and weight loss has been shown to improve glycemic control and decrease morbidity and mortality in T2D patients. Weight-loss strategies using dietary interventions produce a significant decrease in diabetes-related metabolic disturbance. We have previously reported that the supplementation of low molecular chitosan oligosaccharide (GO2KA1) significantly inhibited blood glucose levels in both animals and humans. However, the effect of GO2KA1 on obesity still remains unclear. The aim of the study was to evaluate the anti-obesity effect of GO2KA1 on lipid accumulation and adipogenic gene expression using 3T3-L1 adipocytes in vitro and plasma lipid profiles using a Sprague-Dawley (SD) rat model. Murine 3T3-L1 preadipocytes were stimulated to differentiate under the adipogenic stimulation in the presence and absence of varying concentrations of GO2KA1. Adipocyte differentiation was confirmed by Oil Red O staining of lipids and the expression of adipogenic gene expression. Compared to control group, the cells treated with GO2KA1 significantly decreased in intracellular lipid accumulation with concomitant decreases in the expression of key transcription factors, peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT/enhancer-binding protein alpha (CEBP/α). Consistently, the mRNA expression of downstream adipogenic target genes such as fatty acid binding protein 4 (FABP4), fatty acid synthase (FAS), were significantly lower in the GO2KA1-treated group than in the control group. In vivo, male SD rats were fed a high fat diet (HFD) for 6 weeks to induced obesity, followed by oral administration of GO2KA1 at 0.1 g/kg/body weight or vehicle control in HFD. We assessed body weight, food intake, plasma lipids, levels of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) for liver function, and serum level of adiponectin, a marker for obesity-mediated metabolic syndrome. Compared to control group GO2KA1 significantly suppressed body weight gain (185.8 ± 8.8 g vs. 211.6 ± 20.1 g, p < 0.05) with no significant difference in food intake. The serum total cholesterol, triglyceride, and low-density lipoprotein (LDL) levels were significantly lower in the GO2KA1-treated group than in the control group, whereas the high-density lipoprotein (HDL) level was higher in the GO2KA1 group. The GO2KA1-treated group also showed a significant reduction in ALT and AST levels compared to the control. Moreover, serum adiponectin levels were significantly 1.5-folder higher than the control group. These in vivo and in vitro findings suggest that dietary supplementation of GO2KA1 may prevent diet-induced weight gain and the anti-obesity effect is mediated in part by inhibiting adipogenesis and increasing adiponectin level.

Dataset Information

Identifying adipogenic chemicals: Disparate effects in 3T3-L1, OP9 and primary mesenchymal multipotent cell models.

Publications

Identifying adipogenic chemicals: Disparate effects in 3T3-L1, OP9 and primary mesenchymal multipotent cell models.

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