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Dataset integration identifies transcriptional regulation of microRNA genes by PPAR? in differentiating mouse 3T3-L1 adipocytes.

ABSTRACT: Peroxisome proliferator-activated receptor ? (PPAR?) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPAR? binding sites in mouse adipocytes and PPAR? upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPAR?-targets. By integration of four separate datasets of genome-wide PPAR? binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPAR? binding within 50?kb from miRNA transcription start sites. Nineteen mature miRNAs were upregulated ?2-fold during adipogenesis and for six of these miRNA loci the PPAR? binding sites were confirmed by at least three datasets. The upregulation of five miRNA genes miR-103-1 (host gene Pank3), miR-148b (Copz1), miR-182/96/183, miR-205 and miR-378 (Ppargc1b) followed that of Pparg. The PPAR?-dependence of four of these miRNA loci was demonstrated by PPAR? knock-down and the loci of miR-103-1 (Pank3), miR-205 and miR-378 (Ppargc1b) were also responsive to the PPAR? ligand rosiglitazone. Finally, chromatin immunoprecipitation analysis validated in silico predicted PPAR? binding sites at all three loci and H3K27 acetylation was analyzed to confirm the activity of these enhancers. In conclusion, we identified 22 putative PPAR? target miRNA genes, showed the PPAR? dependence of four of these genes and demonstrated three as direct PPAR? target genes in mouse adipogenesis.

SUBMITTER: John E

PROVIDER: S-EPMC3378868 | biostudies-other | 2012 May

REPOSITORIES: biostudies-other

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Dataset integration identifies transcriptional regulation of microRNA genes by PPARγ in differentiating mouse 3T3-L1 adipocytes.

John Elisabeth E Wienecke-Baldacchino Anke A Liivrand Maria M Heinäniemi Merja M Carlberg Carsten C Sinkkonen Lasse L

Nucleic acids research 20120207 10

Peroxisome proliferator-activated receptor γ (PPARγ) is a key transcription factor in mammalian adipogenesis. Genome-wide approaches have identified thousands of PPARγ binding sites in mouse adipocytes and PPARγ upregulates hundreds of protein-coding genes during adipogenesis. However, no microRNA (miRNA) genes have been identified as primary PPARγ-targets. By integration of four separate datasets of genome-wide PPARγ binding sites in 3T3-L1 adipocytes we identified 98 miRNA clusters with PPARγ ...[more]

PMID: 22319216

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Project description:The differentiation of precursor cells into mature adipocytes (adipogenesis) has been an area of increased focus, spurred by a rise in obesity rates. Though our understanding of adipogenesis and its regulation at the cellular level is growing, many questions remain, especially regarding the regulation of the metabolome. The 3T3-L1 cell line is the most well characterized cellular model of adipogenesis. Using a time course metabolomics approach, we show that the 3T3-L1 preadipocyte metabolome is greatly altered during the first 48 hours of differentiation, where cells go through about two rounds of cell division, a process known as mitotic clonal expansion. Short-chain peptides were among several small molecules that were increased during mitotic clonal expansion. Additional indicators of protein turnover were also increased, including bilirubin, a degradation product of heme-containing proteins, and 3-methylhistidine, a post-translationally modified amino acid that is not reutilized for protein synthesis. To study the origin of the peptides, we treated differentiating preadipocytes with 18O labeled water and found that 18O was incorporated into the short chain peptides, confirming them, at least in part, as products of hydrolysis. Inhibitors of the proteasome or matrix metalloproteinases affected the peptide levels during differentiation, but inhibitors of autophagy or peptidases did not. 18O was also incorporated into several choline metabolites including cytidine 5'-diphosphocholine (CDP-choline), glycerophosphocholine, and several phosphatidylcholine species, indicative of phosphatidylcholine synthesis/degradation and of flux through the CDP-choline cycle, a hallmark of proliferating cells. 18O-Tracer metabolomics further showed metabolic labeling of glutamate, suggestive of glutaminolysis, also characteristic of proliferating cells. Together, these results highlight the utility of 18O isotope labeling in combination with metabolomics to uncover changes in cellular metabolism that are not detectable by time-resolved metabolomics.

Project description:Gegen Qinlian Decoction (GQD), a well-documented traditional Chinese Medicine (TCM) formula, was reported with convincing anti-diabetic effects in clinical practice. However, the precise antidiabetic mechanism of GQD remains unknown. In this study, the anti-hyperglycemic and/or lipid lowering effects of GQD were demonstrated in high-fat diet with a low dose of streptozotocin induced diabetic Sprague-Dawley rats and insulin resistance (IR)-3T3-L1 adipocytes. GQD treatment increased expression and activity levels of both PPAR? and PPAR? in adipocytes, which transcriptionally affected an ensemble of glucose and lipid metabolic genes in vivo and in vitro. The results clearly indicated that GQD treatment intervened with multiple pathways controlled by concomitantly downstream effects of adipocytic PPAR? and PPAR?, to influence two opposite lipid pathways: fatty acid oxidation and lipid synthesis. Antagonist GW9662 decreased the mRNA expression of Ppar? and target genes Adpn and Glut4 whereas GW6471 decreased the mRNA expression of Ppar? and target genes Cpt-1?, Lpl, Mcad, Lcad, Acox1, etc. Nuclear location and activity experiments showed that more PPAR? and PPAR? shuttled into nuclear to increase its binding activities with target genes. GQD decreased the phosphorylation level of ERK1/2 and/or CDK5 to elevate PPAR? and PPAR? activities in IR-3T3-L1 adipocytes through post-translational modification. The increase in p-p38MAPK and SIRT1 under GQD treatment may be attributed to partially reduce PPAR? adipogenesis activity and/or activate PPAR? activity. Compared with the rosiglitazone-treated group, GQD elevated Cpt-1? expression, decreased diabetic biomarker Fabp4 expression, which produced an encouraging lipid profile with triglyceride decrease partially from combined effects on upregulated adipocytic PPAR? and PPAR? activities. These results suggested that GQD improved diabetes by intervening a diverse array of PPAR? and PPAR? upstream and downstream signaling transduction cascades, which jointly optimized the expression of target gene profiles to promote fatty acid oxidation and accelerate glucose uptake and utilization than PPAR? full agonist rosiglitazone without stimulating PPAR? activity. Thus, GQD showed anti-diabetic/or antihyperglycemic effects, partially through regulating adipocytic PPAR? and PPAR? signaling systems to maintaining balanced glucose and lipid metabolisms. This study provides a new insight into the anti-diabetic effect of GQD as a PPAR?/? dual agonist to accelerate the clinical use.

Project description:Purpose: Mitochondrial dysfunction in adipose tissue has emerged as key to the development of obesity and diabetes. Salvianolic acid B (SalB) is a water-soluble ingredient derived from Salvia miltiorrhiza that has been shown to possess potential anti-obese and anti-diabetic activities. However, the cellular mechanism of SalB on mitochondrial function with respect to these metabolic disorders has not been elucidated. Therefore, we aim to investigate the effects of SalB on mitochondrial function in 3T3-L1 adipocytes and analyze the underlying molecular mechanism. Methods: The effects of SalB on adipocyte differentiation, glucose uptake, and glycerol release were evaluated in 3T3-L1 adipocytes. Differentiated adipocytes were treated with SalB (50 ?M) with or without PPAR? antagonist (GW9662, 20 ?M) for 48 h, and mitochondrial oxygen consumption rate (OCR) as well as extracellular acidification rate (ECAR) were assessed using an XF Extracellular Flux Analyzer. The mitochondrial distribution of adipocytes was assessed using Mito Tracker Green (MTG) and observed under a fluorescent microscope. In addition, the mRNA expression levels of peroxisome proliferators-activated receptor ?/? (PPAR?/?), CCAAT/enhancer binding protein? (C/EBP?), Nuclear respiratory factor 1/2 (NRF1/2), Uncoupling protein 2 (UCP2), and phosphofructokinase 2/fructose-2, 6-bisphosphatase 2 (PFKFB2) were detected by RT-PCR. Finally, changes in the protein levels of peroxisome proliferators-activated receptor ? coactivator-1? (PGC-1?) were determined by western blotting and immunofluorescence analysis. Results: Treatment with SalB increased glucose uptake and mitochondrial respiration, reduced glycerol release and promoted adipocyte differentiation by increasing mRNA expression of adipogenic transcription factors including PPAR?, C/EBP?, and PPAR?. Furthermore, SalB enhanced adipocytes mitochondrial content, mitochondrial respiration and glycolysis capacity, which had been attenuated by GW9662 treatment through the increased expression of PGC-1?. Conclusion: Our results provide novel insights into the role of PGC-1? and mitochondria as probable mediators of SalB activity in the regulation of adipocyte differentiation in 3T3-L1 adipocytes.

Dataset Information

Dataset integration identifies transcriptional regulation of microRNA genes by PPAR? in differentiating mouse 3T3-L1 adipocytes.

Publications

Dataset integration identifies transcriptional regulation of microRNA genes by PPARγ in differentiating mouse 3T3-L1 adipocytes.

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