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Dimethylfumarate Suppresses Adipogenic Differentiation in 3T3-L1 Preadipocytes through Inhibition of STAT3 Activity  [PDF]
Hyeon-Ji Kang, Hyun-Ae Seo, Younghoon Go, Chang Joo Oh, Nam Ho Jeoung, Keun-Gyu Park, In-Kyu Lee
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0061411
Abstract: The excessive accumulation of adipocytes contributes to the development of obesity and obesity-related diseases. The interactions of several transcription factors, such as C/EBPβ, PPARγ, C/EBPα, Nrf2, and STAT3, are required for adipogenic differentiation. Dimethylfumarate (DMF), an immune modulator and antioxidant, may function as an inhibitor of STAT3 and an activator of Nrf2. This study examined whether DMF inhibits adipogenic differentiation of 3T3-L1 preadipocytes by inhibiting STAT3 or activating Nrf2. DMF suppressed 3T3-L1 preadipocyte differentiation to mature adipocytes in a dose-dependent manner as determined by Oil Red O staining. The mRNA and protein levels of adipogenic genes, including C/EBPβ, C/EBPα, PPARγ, SREBP-1c, FAS, and aP2, were significantly lower in DMF-treated 3T3-L1 preadipocytes. Suppression of adipogenic differentiation by DMF treatment resulted primarily from inhibition of the early stages of differentiation. DMF inhibits clonal expansion during adipogenic differentiation through induction of a G1 cell cycle arrest. Additionally, DMF regulates cell cycle-related proteins, such as p21, pRb, and cyclin D. DMF treatment markedly inhibited differentiation medium-induced STAT3 phosphorylation and inhibited STAT3 transcriptional activation of a reporter construct composed of four synthetic STAT3-response elements. Moreover, inhibition of endogenous Nrf2 activity using a dominant negative Nrf2 did not abolish the DMF-induced inhibition of adipogenic differentiation of 3T3-L1 preadipocytes. In summary, DMF is a negative regulator of adipogenic differentiation based on its regulation of adipogenic transcription factors and cell cycle proteins. This negative regulation by DMF is mediated by STAT3 inhibition, but is unlikely to involve Nrf2 activation.
Mitochondrial Dysfunction and Adipogenic Reduction by Prohibitin Silencing in 3T3-L1 Cells  [PDF]
Dong Liu, Yiming Lin, Ting Kang, Bo Huang, Wei Xu, Minerva Garcia-Barrio, Moshood Olatinwo, Roland Matthews, Y. Eugene Chen, Winston E. Thompson
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0034315
Abstract: Increase in mitochondrial biogenesis has been shown to accompany brown and white adipose cell differentiation. Prohibitins (PHBs), comprised of two evolutionarily conserved proteins, prohibitin-1 (PHB1) and prohibitin-2 (PHB2), are present in a high molecular-weight complex in the inner membrane of mitochondria. However, little is known about the effect of mitochondrial PHBs in adipogenesis. In the present study, we demonstrate that the levels of both PHB1 and PHB2 are significantly increased during adipogenesis of 3T3-L1 preadipocytes, especially in mitochondria. Knockdown of PHB1 or PHB2 by oligonucleotide siRNA significantly reduced the expression of adipogenic markers, the accumulation of lipids and the phosphorylation of extracellular signal-regulated kinases. In addition, fragmentation of mitochondrial reticulum, loss of mitochondrial cristae, reduction of mitochondrial content, impairment of mitochondrial complex I activity and excessive production of ROS were observed upon PHB-silencing in 3T3-L1 cells. Our results suggest that PHBs are critical mediators in promoting 3T3-L1 adipocyte differentiation and may be the potential targets for obesity therapies.
The roots of Atractylodes japonica Koidzumi promote adipogenic differentiation via activation of the insulin signaling pathway in 3T3-L1 cells  [cached]
Han Yunkyung,Jung Hyo,Park Yong-Ki
BMC Complementary and Alternative Medicine , 2012, DOI: 10.1186/1472-6882-12-154
Abstract: Background Type 2 diabetes (T2D) is a complex metabolic disorder characterized by insulin resistance and hyperglycemia. Peroxisome proliferator-activated receptor gamma (PPARγ) is a key transcription factor and plays an important role in the regulation of genes involved in adipogenic differentiation, glucose metabolism and insulin signal transduction. Methods In this study, the effects of the root extract of Atractylodes japonica Koidzumi (Atractylodis Rhizoma Alba, ARA) on the differentiation of 3T3-L1 preadipocytes and the possible mechanism of glucose transport were investigated. 3T3-L1 cells were cultured with insulin and ARA extract. Results In 3T3-L1 cells, ARA extract significantly enhanced adipogenic differentiation and upregulated the expression of PPARγ genes and protein in a dose-dependent manner. ARA also promoted glucose transport by increasing the glucose transporter 4 (GLUT-4), phosphatidylinositol 3-kinase (PI3K) and insulin receptor substrates-1 (IRS-1) levels. Conclusion Our results suggest that ARA extract may be an attractive therapeutic agent for managing T2D via promoting the differentiation of adipocytes with the upregulation of PPARγ levels and the activation of the insulin signaling pathway.
A Novel Regulatory Function of Sweet Taste-Sensing Receptor in Adipogenic Differentiation of 3T3-L1 Cells  [PDF]
Yosuke Masubuchi, Yuko Nakagawa, Jinhui Ma, Tsutomu Sasaki, Tadahiro Kitamura, Yoritsuna Yamamoto, Hitoshi Kurose, Itaru Kojima, Hiroshi Shibata
PLOS ONE , 2013, DOI: 10.1371/journal.pone.0054500
Abstract: Background Sweet taste receptor is expressed not only in taste buds but also in nongustatory organs such as enteroendocrine cells and pancreatic beta-cells, and may play more extensive physiological roles in energy metabolism. Here we examined the expression and function of the sweet taste receptor in 3T3-L1 cells. Methodology/Principal Findings In undifferentiated preadipocytes, both T1R2 and T1R3 were expressed very weakly, whereas the expression of T1R3 but not T1R2 was markedly up-regulated upon induction of differentiation (by 83.0 and 3.8-fold, respectively at Day 6). The α subunits of Gs (Gαs) and G14 (Gα14) but not gustducin were expressed throughout the differentiation process. The addition of sucralose or saccharin during the first 48 hours of differentiation considerably reduced the expression of peroxisome proliferator activated receptor γ (PPARγ and CCAAT/enhancer-binding protein α (C/EBPα at Day 2, the expression of aP2 at Day 4 and triglyceride accumulation at Day 6. These anti-adipogenic effects were attenuated by short hairpin RNA-mediated gene-silencing of T1R3. In addition, overexpression of the dominant-negative mutant of Gαs but not YM-254890, an inhibitor of Gα14, impeded the effects of sweeteners, suggesting a possible coupling of Gs with the putative sweet taste-sensing receptor. In agreement, sucralose and saccharin increased the cyclic AMP concentration in differentiating 3T3-L1 cells and also in HEK293 cells heterologously expressing T1R3. Furthermore, the anti-adipogenic effects of sweeteners were mimicked by Gs activation with cholera toxin but not by adenylate cyclase activation with forskolin, whereas small interfering RNA-mediated knockdown of Gαs had the opposite effects. Conclusions 3T3-L1 cells express a functional sweet taste-sensing receptor presumably as a T1R3 homomer, which mediates the anti-adipogenic signal by a Gs-dependent but cAMP-independent mechanism.
Beta-Mecaptoethanol Suppresses Inflammation and Induces Adipogenic Differentiation in 3T3-F442A Murine Preadipocytes  [PDF]
Wen Guo, Yahui Li, Wentao Liang, Siu Wong, Caroline Apovian, James L. Kirkland, Barbara E. Corkey
PLOS ONE , 2012, DOI: 10.1371/journal.pone.0040958
Abstract: Preadipocytes are present in adipose tissues throughout adult life that can proliferate and differentiate into mature adipocytes in response to environmental cues. Abnormal increase in adipocyte number or size leads to fat tissue expansion. However, it is now recognized that adipocyte hypertrophy is a greater risk factor for metabolic syndrome whereas fat tissue that continues to produce newer and smaller fat cells through preadipocyte differentiation is “metabolically healthy”. Because adipocyte hypertrophy is often associated with increased oxidant stress and low grade inflammation, both are linked to disturbed cellular redox, we tested how preadipocyte differentiation may be regulated by beta-mercaptoethanol (BME), a pharmacological redox regulator and radical scavenger, using murine 3T3-F442A preadipocytes as the cell model. Effects of BME on adipogenesis were measured by microphotography, real-time PCR, and Western analysis. Our data demonstrated that preadipocyte differentiation could be regulated by extracellular BME. At an optimal concentration, BME enhanced expression of adipogenic gene markers and lipid accumulation. This effect was associated with BME-mediated down-regulation of inflammatory cytokine expression during early differentiation. BME also attenuated TNFalpha-induced activation of NFkappaB in differentiating preadipocytes and partially restored TNFalpha-mediated suppression on adipogenesis. Using a non-adipogenic HEK293 cell line transfected with luciferase reporter genes, we demonstrated that BME reduced basal and TNFalpha-induced NFkappaB activity and increased basal and ciglitazone-induced PPARgamma activity; both may contribute to the pro-adipogenic effect of BME in differentiating F442A preadipocytes.
Buckwheat (Fagopyrum esculentum M.) Sprout Treated with Methyl Jasmonate (MeJA) Improved Anti-Adipogenic Activity Associated with the Oxidative Stress System in 3T3-L1 Adipocytes  [PDF]
Young-Jun Lee,Kui-Jin Kim,Kee-Jai Park,Bo-Ra Yoon,Jeong-Ho Lim,Ok-Hwan Lee
International Journal of Molecular Sciences , 2013, DOI: 10.3390/ijms14011428
Abstract: Buckwheat sprouts contain various bioactive compounds including rutin which have a number of biological activities. We have previously shown that buckwheat sprouts (TBWE) treated with methyl jasmonate (MeJA) significantly increased the amount of phenolics and the antioxidant activity. The aim of this study was to demonstrate the effect of TBWE on anti-adipogenesis and pro-oxidant enzyme in 3T3-L1 adipocytes. We also evaluated the anti-oxidative activity of TBWE in adipocytes by using the nitroblue tetrazolium assay. Our data showed that TBWE markedly inhibited adipocyte differentiation and ROS production in 3T3-L1 cells compared with control groups. Moreover, TBWE has strongly shown the inhibition of adipogenic transcription factor as well as pro-oxidant enzymes. Together, we demonstrate that the MeJA treatment significantly increased the amount of phenolic compound, resulting in the suppression of adipogenesis and ROS production in the 3T3-L1 cells. These findings indicate that TBWE has the potential for anti-adipogenesis activity with anti-oxidative properties.
Colocynth (Citrullus colocynthis) Flesh Extract Suppresses Adipogenesis by Down-Regulating Adipogenic Transcription Factors and Their Target Genes in 3T3-L1 Preadipocytes  [PDF]
Raoua Jemai, Riadh Drira, Hamadi Fetoui, Mohamed Makni, Kazuichi Sakamoto
Food and Nutrition Sciences (FNS) , 2018, DOI: 10.4236/fns.2018.98074
Abstract: Citrullus colocynthis, a member of the Cucurbitaceae family, is widely distributed in North Africa. The fruits are recognized for their wide range of medicinal uses and promising pharmaceutical potential. The present study aimed to investigate the anti-obesity effect of the ethanol extract of colocynth flesh (FCEE) in 3T3-L1 cells following treatment at different doses. The viability of 3T3-L1 preadipocytes was analyzed via MTT assay and triglycerides were stained with Oil red O to assess lipid accumulation. Additionally, adipogenesis-related gene expression was quantified via qRT-PCR. FCEE (0 - 150 μg/mL) dose-dependently suppressed intracellular triglyceride accumulation during the adipogenesis by 23% and 66% at 100 and 150 μg/mL, respectively, but did not affect cell viability. Analysis of the time-dependence of the effect of FCEE demonstrated that the greatest anti-adipogenic activity was observed during the early stages of differentiation. FCEE also decreased GPDH activity in a dose-dependent manner, with 98% decrease observed at 150 μg/mL. In addition, at same range of FCEE concentrations, the main transcription factors, including CCAAT/enhancer binding protein α (C/EBPα), peroxisome proliferator activated receptor γ (PPARγ), and sterol regulatory element-binding protein 1c (SREBP-1c), were downregulated by 90%, 89%, and 89%, respectively at 150 μg/mL. As these are the master regulators of adipogenesis. The inhibition of their downstream target genes was also observed. Colocynth may be useful in the treatment of obesity owing to its powerful effects on fat, which result in changes to adipocyte differentiation and fat mobilization.
Cyanidine-3-O-Galactoside Enriched Aronia melanocarpa Extract Inhibits Adipogenesis and Lipogenesis via Down-Regulation of Adipogenic Transcription Factors and Their Target Genes in 3T3-L1 Cells  [PDF]
Su-Min Lim, Jae In Jung, Nam Young Kim, Jung-Shik Bae, Hyun Sook Lee, Eun Ji Kim
Food and Nutrition Sciences (FNS) , 2019, DOI: 10.4236/fns.2019.102011
Abstract: Aronia melamocarpa (AM) is a rich source of anthocyanins, which are known to help prevent obesity. The cyanidine-3-O-galactoside enriched AM extract (AM-Ex) containing more cyanidine-3-O-galactoside than conventional AM extract was recently developed. The objective of this study was to examine the effect of AM-Ex on adipogenesis and its action mechanisms in vitro using 3T3-L1 adipocytes. To examine the anti-obesity effect of AM-Ex, 3T3-L1 cells were induced adipocyte differentiation and incubated with various concentration of AM-Ex. Lipid accumulation, cellular triglyceride content, mRNA expression of transcription factors and adipogenic genes were analyzed. Treatment with 100 - 400 μg/mL of AM-Ex resulted in a dose-dependent decrease in adipocyte differentiation and triglyceride accumulation. mRNA expression of adipogenic transcription factors, such as peroxisome proliferator-activated receptor gamma, CCAAT/enhancer binding protein α, sterol regulatory element-binding protein 1 were decreased. The level of gene expression of adipogenesis and lipogenesis-related genes, such as adipocyte protein 2, lipoprotein lipase, acetyl-CoA carboxylase, ATP-citrate lyase and fatty acid synthase were decreased. These results suggest that AM-Ex alleviated risk factors related to obesity by modulating multiple pathways associated with adipogenesis.
Centipede grass exerts anti-adipogenic activity through inhibition of C/EBPβ, C/EBPα, and PPARγ expression and the AKT signaling pathway in 3T3-L1 adipocytes  [cached]
Park Hyoung Joon,Chung Byung Yeoup,Lee Min-Kwon,Song Yuno
BMC Complementary and Alternative Medicine , 2012, DOI: 10.1186/1472-6882-12-230
Abstract: Background Centipede grass (CG) originates from China and South America and is reported to contain several C-glycosyl flavones and phenolic constituents, including maysin and luteolin derivatives. This study aimed to investigate, for the first time, the antiobesity activity of CG and its potential molecular mechanism in 3T3-L1 cells. Methods To study the effect of CG on adipogenesis, differentiating 3T3-L1 cells were treated every day with CG at various concentrations (0–100 μg/ml) for six days. Oil-red O staining and triglyceride content assay were performed to determine the lipid accumulation in 3T3-L1 cells. The expression of mRNAs or proteins associated with adipogenesis was measured using RT-PCR and Western blotting analysis. We examined the effect of CG on level of phosphorylated Akt in 3T3-L1 cells treated with CG at various concentration s during adipocyte differentiation. Results Differentiation was investigated with an Oil-red O staining assay using CG-treated 3T3-L1 adipocytes. We found that CG suppressed lipid droplet formation and adipocyte differentiation in 3T3-L1 cells in a dose-dependent manner. Treatment of the 3T3-L1 adipocytes with CG resulted in an attenuation of the expression of adipogenesis-related factors and lipid metabolic genes. The expression of C/EBPα and PPARγ, the central transcriptional regulators of adipogenesis, was decreased by the treatment with CG. The expression of genes involved in lipid metabolism, aP2 were significantly inhibited following the CG treatment. Moreover, the CG treatment down-regulated the phosphorylation levels of Akt and GSK3β. Conclusions Taken collectively, these data indicated that CG exerts antiadipogenic activity by inhibiting the expression of C/EBPβ, C/EBPα, and PPARγ and the Akt signaling pathway in 3T3-L1 adipocytes.
Ginsenoside Rc Promotes Anti-Adipogenic Activity on 3T3-L1 Adipocytes by Down-Regulating C/EBPα and PPARγ  [PDF]
Ji-Won Yang,Sung Soo Kim
Molecules , 2015, DOI: 10.3390/molecules20011293
Abstract: Panax ginseng and its major components, the ginsenosides, are widely used in oriental medicine for the prevention of various disorders. In the present study, the inhibitory activity of ginsenoside Rc on adipogenesis was investigated using the 3T3-L1 cell line. The results obtained showed that Rc reduced the proliferation and viability of 3T3-L1 preadipocytes in a dose-dependent manner. Treatment with Rc decreased the number of adipocytes and reduced lipid accumulation in maturing 3T3-L1 preadipocytes, demonstrating an inhibitory effect on lipogenesis. Moreover, it was found that Rc directly induced lipolysis in adipocytes and down-regulated the expression of major transcription factors of the adipogenesis pathway, such as PPARγ and C/EBPα. These findings indicate that Rc is capable of suppressing adipogenesis and therefore they seem to be natural bioactive factors effective in adipose tissue mass modulation.
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