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Novel Transcriptome Profiling Analyses Demonstrate that Selective PPARg Modulators Display Attenuated and Selective Gene Regulatory Activity in Comparison with PPARg Full Agonists

ABSTRACT: We conducted extensive transcriptome profiling studies to characterize 70 SPPARgMs and seven PPARg full agonists in 3T3-L1 adipocytes, and a subset of these ligands in adipose tissue of diabetic db/db mice. In both cases, the SPPARgMs generated attenuated gene regulatory responses, and their gene expression signatures were more enriched in metabolic pathways that are likely to mediate anti-diabetic efficacy than those of PPARg full agonists. More importantly, our profiling results demonstrated that in both 3T3-L1 adipocytes and db/db mice, SPPARgMs regulate the expression of anti-diabetic efficacy-associated genes to a greater extent than that of adverse effect-associated genes, while PPARg full agonists regulate both gene sets proportionally.

ORGANISM(S): Mus musculus

PROVIDER: GSE31222 | GEO | 2012/06/30

SECONDARY ACCESSION(S): PRJNA146075

REPOSITORIES: GEO

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Project description:Obesity due to overnutrition causes adipose tissue dysfunction, serving as a critical pathological step on the road to Type 2 diabetes (T2D) and other metabolic disorders. Here, we performed an unbiased investigation into the fundamental molecular mechanisms by which adipocytes transition to an unhealthy state during obesity. We fed NuTRAP (Nuclear tagging and Translating Ribosome Affinity Purification) mice crossed with Adipoq-Cre with chow or high fat diet (HFD) for 10 weeks and determined adipocyte-specific transcriptomic profiles by RNA-seq, active promoter and enhancer activities by H3K27ac ChIP-seq, and the PPARg cistrome by ChIP-seq. We also assessed the impact of the PPARg agonist rosiglitazone (Rosi) on gene expression and cellular state of adipocytes from HFD-fed mice. We used bioinformatic approaches to find transcriptional pathways and performed functional studies using shRNA-mediated loss-of-function approaches in 3T3-L1 adipocytes. Adipocytes from HFD-fed mice exhibited reduced expression of adipocyte markers and metabolic genes while showing enhanced expression of myofibroblast marker genes involved in cytoskeletal organization, accompanied by the formation of actin filament structures within the cell. PPARg binding was globally reduced in adipocytes after HFD feeding, and Rosi restored the molecular and cellular phenotypes of adipocytes associated with HFD feeding. SMAD binding motifs were over-represented in HFD-induced promoters and enhancers, while TGFb1 expression was elevated in adipose tissues after HFD feeding. TGFb1 treatment of mature 3T3-L1 adipocytes induced gene expression and cellular changes similar to those seen after HFD in vivo, and knockdown of Smad3 blunted the effects of TGFb1. Our data demonstrate that adipocytes fail to maintain cellular identity after HFD, acquiring characteristics of a myofibroblast-like cell type through reduced PPARg activity and elevated TGFb-SMAD signaling. This cellular identity crisis may serve as a fundamental mechanism that drives functional decline of adipose tissues during obesity.

Project description:Visceral fat (VF) and subcutaneous fat (SF) are developmentally different tissues with different gene expression. Islet-1 (ISL1), a LIM-homeobox transcription factor with important developmental and regulatory function in islet, neural, and cardiac tissue, is virtually absent in SF but substantially expressed in the stromovascular [preadipocyte containing] fraction of VF; expression correlates negatively with adiposity in rodents and man. ISL1 expression is transiently increased in 3T3-L1 preadipocytes during early differentiation, suggesting a functional role. To examine the role of ISL1 in adipogenesis, we tested whether retroviral overexpression of ISL1 in 3T3-L1 preadipocytes affected their ability to differentiate into mature adipocytes. Terminal differentiation was assessed by Oil Red O [lipid droplet] staining and by immunoblot detection of adipocyte marker proteins, including aP2 and GLUT4. ISL1 significantly inhibited lipid droplet formation, reduced lipid accumulation (about 80% inhibition, p<0.05), and substantially inhibited aP2 and GLUT4 expression. ISL1 did not inhibit expression of C/EBPb and C/EBPd after induction of differentiation, but reduced PPARg and C/EBPa by >50% at both mRNA and protein level. In addition, the PPARg agonist, rosiglitazone, substantially rescued ISL1 inhibited adipogenesis in the absence of exogenous PPARg, and fully rescued in the presence of exogenous PPARg. In summary, ISL1 overexpression inhibited fat droplet formation, lipid accumulation, and adipocyte-specific gene expression; there was accompanying inhibition of C/EBPa, PPARg and downstream gene expression. We conclude that ISL1 overexpression inhibited adipocyte differentiation by inhibition of PPARg regulated gene expression. As abdominal obesity strongly correlates with insulin resistance, and cardiovascular risk, ISL1 up-regulation may impact abdominal obesity and its concomitant metabolic derangements. Total cellular RNA was isolated from 3T3-L1 cells expressing Flag-ISL1 or not at 48 h following treatment with differentiation cocktail. Individual RNA from biological triplicates was used for microarray analysis.

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Novel Transcriptome Profiling Analyses Demonstrate that Selective PPARg Modulators Display Attenuated and Selective Gene Regulatory Activity in Comparison with PPARg Full Agonists

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