Growth-arrested 3T3-L1 preadipocytes rapidly express CCAAT/enhancer-binding protein-␤ (C/EBP␤) upon hormonal induction of differentiation. However, the DNA binding activity of C/EBP␤ is not activated until the cells synchronously reenter S phase during the mitotic clonal expansion (MCE) phase of differentiation. In this period, C/EBP␤ is sequentially phosphorylated by MAPK and glycogen synthase kinase-3␤, inducing C/EBP␤ DNA binding activity and transcription of its target genes. Because the DNA binding activity of C/EBP␤ is further enhanced by oxidation in vitro, we investigated how redox state affects C/EBP␤ DNA binding and MCE during adipogenesis. When 3T3-L1 cells were treated with H 2 O 2 and hormonal stimuli, differentiation was accelerated with increased expression of peroxisome proliferator-activated receptor ␥. Interestingly, cell cycle progression (S to G 2 /M phase) was markedly enhanced by H 2 O 2 , whereas antioxidants caused an S phase arrest during the MCE. H 2 O 2 treatment resulted in the early appearance of a punctate pattern observed by immunofluorescent staining of C/EBP␤, which is a hallmark for C/EBP␤ binding to regulatory elements, whereas a short antioxidant treatment rapidly dispersed the centromeric localization of C/EBP␤. Consistently, reactive oxygen species production was increased during 3T3-L1 differentiation. Our results indicate that redox-induced C/EBP␤ DNA binding activity, along with the dual phosphorylation of C/EBP␤, is required for the MCE and terminal differentiation of adipocytes.

Recently, hepatic peroxisome proliferator-activated receptor (PPAR)γ has been implicated in hepatic lipid accumulation. We found that the C3H mouse strain does not express PPARγ in the liver and, when subject to a high-fat diet, is resistant to hepatic steatosis, compared with C57BL/6 (B6) mice. Adenoviral PPARγ2 injection into B6 and C3H mice caused hepatic steatosis, and microarray analysis demonstrated that hepatic PPARγ2 expression is associated with genes involved in fatty acid transport and the triglyceride synthesis pathway. In particular, hepatic PPARγ2 expression significantly increased the expression of monoacylglycerol O-acyltransferase 1 (MGAT1). Promoter analysis by luciferase assay and electrophoretic mobility shift assay as well as chromatin immunoprecipitation assay revealed that PPARγ2 directly regulates the MGAT1 promoter activity. The MGAT1 overexpression in cultured hepatocytes enhanced triglyceride synthesis without an increase of PPARγ expression. Importantly, knockdown of MGAT1 in the liver significantly reduced hepatic steatosis in 12-wk-old high-fat-fed mice as well as ob/ob mice, accompanied by weight loss and improved glucose tolerance. These results suggest that the MGAT1 pathway induced by hepatic PPARγ is critically important in the development of hepatic steatosis during dietinduced obesity.nonalcoholic fatty liver disease | adenoviral expression | SREBP1c | ChREBP | TLR4

Adipogenesis, the conversion of precursor cells into adipocytes, is associated with obesity and is mediated by glucocorticoids acting via hitherto poorly characterized mechanisms. Dexras1 is a small G protein of the Ras family discovered on the basis of its marked induction by the synthetic glucocorticoid dexamethasone. We show that Dexras1 mediates adipogenesis and diet-induced obesity. Adipogenic differentiation of 3T3-L1 cells is abolished with Dexras1 depletion, whereas overexpression of Dexras1 elicits adipogenesis. Adipogenesis is markedly reduced in mouse embryonic fibroblasts from Dexras1-deleted mice, whereas adiposity and dietinduced weight gain are diminished in the mutant mice.insulin | cyclic AMP | nitric oxide | Cushing disease

Snail belongs to the superfamily of zinc-finger transcription factors and plays a crucial role in processes regulating cell fate, such as the formation of mesoderm and initiation of epithelial-mesenchymal transition. We have previously discovered that Snail modulates adiponectin expression in 3T3-L1 cells during adipogenesis. In the present study, we elucidated the functional role of Snail in adipocyte differentiation and its underlying molecular mechanism. Snail expression was dramatically decreased during adipogenesis in 3T3-L1 cells. Overexpression of Snail blocked adipocyte differentiation by suppressing the expression of peroxisome proliferator-activated receptor gamma (PPARγ) and CCAAT-enhancer-binding protein alpha, while knockdown of Snail expression stimulated adipogenesis in 3T3-L1 cells. Chromatin immunoprecipitation assay and luciferase assay showed that Snail inhibits the transcriptional activity of the PPARγ gene by directly binding to the E-box motifs in the PPARγ promoter. Wnt10b induced phosphorylation of glycogen synthase kinase 3 beta (GSK3β), leading to inhibition of adipogenesis in 3T3-L1 cells in accordance with increased expression of Snail, whereas adipogenic capacity was restored in Snail siRNA-transfected preadipocytes. LiCl (a GSK3β inhibitor)-treated cells also showed increased expression of Snail, with a reduced adipogenic potential. Snail-overexpressing 3T3-F442A cells did not differentiate into mature adipocytes in immunodeficient nude mice. Taken together, Snail is a novel regulator of adipocyte differentiation, which acts by direct suppression of PPARγ expression. Our data also indicate that the expression of Snail is mediated by the Wnt-GSK3β signaling pathway.

KLF8 (Krüppel-like factor 8) is a zinc-finger transcription factor known to play an essential role in the regulation of the cell cycle, apoptosis, and differentiation. However, its physiological roles and functions in adipogenesis remain unclear. In the present study, we show that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3-L1 preadipocytes, we found that KLF8 expression was induced during differentiation, which was followed by expression of peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα). Adipocyte differentiation was significantly attenuated by the addition of siRNA against KLF8, whereas overexpression of KLF8 resulted in enhanced differentiation. Furthermore, luciferase reporter assays demonstrated that overexpression of KLF8 induced PPARγ2 and C/EBPα promoter activity, suggesting that KLF8 is an upstream regulator of PPARγ and C/EBPα. The KLF8 binding sites were localized by site mutation analysis to −191 region in C/EBPα promoter and −303 region in PPARγ promoter, respectively. Taken together, these data reveal that KLF8 is a key component of the transcription factor network that controls terminal differentiation during adipogenesis.

Adipogenesis is largely dependent on the signal transducers and activators of transcription (STAT) pathway. However, the molecular mechanism of the STAT pathway in the adipogenesis of human bone marrow-derived stromal cells (hBMSCs) remains not well understood. The purpose of this research was to characterize the transcriptional regulation involved in expression of STAT5A and STAT5B during adipogenesis in hBMSCs and 3T3-L1 cells. The expression of STAT5A and STAT5B increases with the onset of adipogenesis in hBMSCs and 3T3-L1 cells. The PPAR response elements regulatory element of STAT5A exists at a promoter region ranging from -346 to -101, and the CCAAT/enhancer-binding protein (C/EBP) regulatory element is located at -196 to -118 of the STAT5B promoter. C/EBPb and C/EBPa bound to the STAT5B promoter region, whereas peroxisome proliferatoractivated receptor g (PPARg) bound to STAT5A. RNA interference of STAT5A completely blocked differentiation, whereas the inhibition of STAT5B only partially blocked differentiation. We propose that C/EBPa, C/EBPb, and PPARg control adipogenesis by regulating STAT5B and STAT5A and that STAT5A is necessary, whereas STAT5B plays a supplementary role during adipogenesis. Further, the regulation of PPARg-STAT5 by C/EBPb signaling seems to be the crucial adipogenesis pathway-initiating cascade of the various adipogenic genes.

The mesenchymal cell is a multipotent stem cell with the capacity to give rise to multiple cell types.Recently, a number of studies have demonstrated a role of BMP4 in the commitment of mesenchymal stem cells (C3H10T1/2) into the adipocyte lineage. Previously we investigated the genetic events underlying adipocyte commitment, for which C3H10T1/2 stem cells were treated with 5‐azaC and selected preadipocyte clonal lines, named A33 line. In this study, we analyzed the BMP4 promoter region in 10T1/2 and A33 cell. The activity was decreased dramatically to the level measured for the constructs between −197 and −60 bp promoter sequence. And then, we searched that global changes in gene expression of 10T1/2 and A33 cells were examined by microarray. We found that gene expression of scavenger receptor class A member 5 was significantly increased A33 compared 10T1/2. Further studies are underway to identify a transcription factor bound to this region, which will reveal the BMP4 upstream pathway essential for the adipocyte commitment of stem cells. And we will investigate the role of scara5 in mesenchymal stem cell development.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0030711 and 2011‐0015665) and by Korean Research WCU grant (R31‐2008‐000‐10086‐0) from the Korean Ministry of Education, Science, and Technology.

KLF8 is a zinc‐finger transcription factor known to play an essential role in cell cycle and differentiation. However, its physiological roles and functions in 3T3‐L1 cells remain relatively unclear. In the present study, we showed that KLF8 acts as a key regulator controlling adipocyte differentiation. In 3T3‐L1 preadipocytes, KLF8 expression was induced at an early stage of differentiation which was followed by expression of PPAR¥ã2 and C/EBP¥á. The mRNA level of KLF8 dramatically increased at 36 h and 48 h during differentiation and the protein level also gradually increased after 36 h. This induction and differentiation was attenuated significantly by the presence of small interfering RNA against KLF8, whereas overexpression of KLF8 induced 3T3‐L1 differentiation. Furthermore, overexpression of KLF8 induced PPAR¥ã2 and C/EBP¥á promoter activity and the direct binding site in C/EBP¥á promoter exists between (−205) and (−89) region. Taken together, this study provides KLF8 as a key component of the transcription factor network that controls the terminal differentiation during adipogenesis.This work was supported by the National Research Foundation of Korea (NRF) grant funded by the Korea government (MEST) (No. 2011‐0030711 and 2011‐0015665) and by Korean Research WCU grant (R31‐2008‐000‐10086‐0) from the Korean Ministry of Education, Science, and Technology.