Unknown,Transcriptomics,Genomics,Proteomics

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RNAseq analysis of 3T3-L1 adipocytes cultured in high and low media volumes

ABSTRACT: 3T3-L1 fibroblasts were cultured in high glucose DMEM supplemented with 10% foetal bovine serum (FBS) and 2 mM glutamax at 37°C in 10% CO2. For differentiation into adipocytes, fibroblasts were seeded into 12-well plates, and cultured in 1 mL/well of 10% FBS-supplemented DMEM containing an adipogenic cocktail (350 nM insulin, 0.5 mM 3-isobutyl-1-methylxanthine, and 250 nM dexamethasone) for 3 d, followed by 3 d in DMEM containing 10% FBS and 350 nM insulin. On day 9 post-differentiation, media volumes were either kept at 1 mL (high) or switched to 0.33 mL (low) for 16 h. Cells were then lysed in 350 µL RLT buffer for RNA extraction and sequencing.

INSTRUMENT(S): Illumina TruSeq® Stranded mRNA Library Preparation Kit (Illumina), Illumina HiSeq 4000

ORGANISM(S): Mus musculus

SUBMITTER: Ioannis Kamzolas

PROVIDER: E-MTAB-12298 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Metabolic syndrome is an important public concern and demand for effective therapeutic strategies. Abdominal obesity, especially an increase in the visceral adipose tissue, is the main cause of this syndrome. Flavonoids are expected to improve risk factors for metabolic syndrome. Bilberry, original species of blueberry containing anthocyanidin flavonoids have been used for centuries in Europe to ameliorate the symptoms of diabetes, but their effects and the mechanisms on lipid accumulation of adipocyte cells are not well defined. In the present study, we investigated effects of the Bilberry extract on differentiation of adipocytes using 3T3-L1 adipocyte cell line. Exposure to Bilberry extract during the early period of adipogenesis (6 days) was significantly inhibited adipocyte differentiation of 3T3-L1. During this period, Bilberry extract greatly down-regulated the mRNA levels of the key adipogenesis-associated markers peroxisome proliferator-activated receptor-γ (PPARγ). Furthermore, Bilberry extract significantly decreased expression of the transcription factor Sterol Regulatory Element Binding Protein 1c (SREBP1c), which plays a central role in adipocyte differentiation including the induction of PPARγ. The expression of SREBP1c is remarkably enhanced in response to insulin, thus raises the possibility that Bilberry extract might inhibit the Insulin pathway. So, We investigated whether Billberry extract and anthocyanidines turned the insulin signaling pathway using microarray. As a result, Gene Set Enrichment Analysis (GSEA) shows that these additives turned insulin signaling pathway certainly. We quantified expression profiles of whole mRNAs by microarray in the adipocyte from 3T3-L1 with or without additives. 3T3-L1 preadipocytes (American Type Culture Collection, Manassas, VA) were grown to confluence in DMEM media (Sigma Aldrich, Tokyo, Japan) with 10% calf serum and penicillin (100 U/ml)/streptomycin (100 ug/ml). Adipogenesis was induced using an adipogenesis assay kit (Chemicon International, Temecula, CA). On day 0, cells were induced with initiation media (10 ug/ml insulin, 1 uM dexamethasone and 0.5 uM IBMX in DMEM media added with 1: 0.1% DMSO, 2: 100 ug/ml Bilberry extract, 3: 100 nM Delphinidin or 4: 100 nM Cyanidin (total 4 samples). On day 2, harvested the cells, extracted total RNA and did microarray experiments.

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RNAseq analysis of 3T3-L1 adipocytes cultured in high and low media volumes

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