Master Adipogenic Transcription Factors Research Articles

Regulation of White adipogenesis by long non coding RNA NEAT1

Obesity is a global health problem manifested due to various adipose tissue dysfunctions. Insights on adipogenesis and adipocyte development may unravel new strategies for the treatment of obesity and related metabolic diseases. Adipogenesis is controlled by many transcription factors and epigenetic regulators; PPARγ and C/EBPα are master adipogenic transcription factors. A clear understanding of transcriptional regulation mechanism of adipogenesis seems crucial. Expanding facts and knowledge demonstrated that long non-coding RNAs (lncRNAs), a category of non-protein coding RNAs of size greater than 200 nucleotides are identified as potential regulators in obesity-related biological processes through diverse mechanisms. Nuclear enriched abundant transcript 1 (NEAT1), a nuclear lncRNA interacts with nuclear domains that controls sequestration of paraspeckle proteins. NEAT 1 has evolved as a vital regulator involved in different cellular processes, developmental processes and physiological responses along with disease development and progression. In this mini review, the potential role and the basic mechanisms of lncRNA NEAT1 in adipogenesis are discussed. Clear understanding of the recent advances of lncRNAs is emphasized that will recommend the future application of lncRNAs in the treatment of obesity.

Hop2 interacts with the transcription factor CEBPα and suppresses adipocyte differentiation

CCAAT enhancer binding protein (CEBP) transcription factors (TFs) are known to promote adipocyte differentiation; however, suppressors of CEBP TFs have not been reported thus far. Here, we find that homologous chromosome pairing protein 2 (Hop2) functions as an inhibitor for the TF CEBPα. We found that Hop2 mRNA is highly and specifically expressed in adipose tissue, and that ectopic Hop2 expression suppresses reporter activity induced by CEBP as revealed by DNA transfection. Recombinant and ectopically expressed Hop2 was shown to interact with CEBPα in pull-down and coimmunoprecipitation assays, and interaction between endogenous Hop2 and CEBPα was observed in the nuclei of 3T3 preadipocytes and adipocytes by immunofluorescence and coimmunoprecipitation of nuclear extracts. In addition, Hop2 stable overexpression in 3T3 preadipocytes inhibited adipocyte differentiation and adipocyte marker gene expression. These in vitro data suggest that Hop2 inhibits adipogenesis by suppressing CEBP-mediated transactivation. Consistent with a negative role for Hop2 in adipogenesis, ablation of Hop2 (Hop2−/−) in mice led to increased body weight, adipose volume, adipocyte size, and adipogenic marker gene expression. Adipogenic differentiation of isolated adipose-derived mesenchymal stem cells showed a greater number of lipid droplet–containing colonies formed in Hop2−/− adipose-derived mesenchymal stem cell cultures than in wt controls, which is associated with the increased expression of adipogenic marker genes. Finally, chromatin immunoprecipitation revealed a higher binding activity of endogenous CEBPα to peroxisome proliferator–activated receptor γ, a master adipogenic TF, and a known CEBPα target gene. Therefore, our study identifies for the first time that Hop2 is an intrinsic suppressor of CEBPα and thus adipogenesis in adipocytes.

MLL3/MLL4-Associated PAGR1 Regulates Adipogenesis by Controlling Induction of C/EBPβ and C/EBPδ.

Transcription factors C/EBPβ and C/EBPδ are induced within hours after initiation of adipogenesis in culture. They directly promote the expression of master adipogenic transcription factors peroxisome proliferator-activated receptor γ (PPARγ) and C/EBPα and are required for adipogenesis in vivo However, the mechanism that controls the induction of C/EBPβ and C/EBPδ remains elusive. We previously showed that histone methyltransferases MLL3/MLL4 and associated PTIP are required for the induction of PPARγ and C/EBPα during adipogenesis. Here, we show MLL3/MLL4/PTIP-associated protein PAGR1 (also known as PA1) cooperates with phosphorylated CREB and ligand-activated glucocorticoid receptor to directly control the induction of C/EBPβ and C/EBPδ in the early phase of adipogenesis. Deletion of Pagr1 in white and brown preadipocytes prevents the induction of C/EBPβ and C/EBPδ and leads to severe defects in adipogenesis. Adipogenesis defects in PAGR1-deficient cells can be rescued by the ectopic expression of C/EBPβ or PPARγ. Finally, the deletion of Pagr1 in Myf5+ precursor cells impairs brown adipose tissue and muscle development. Thus, by controlling the induction of C/EBPβ and C/EBPδ, PAGR1 plays a critical role in adipogenesis.

Evaluating the effects of honey bee (Apis mellifera L.) venom on the expression of insulin sensitivity and inflammation‐related genes in co‐culture of adipocytes and macrophages

AbstractObese adipose tissue is characterized by adipocyte hypertrophy and a massive macrophage infiltration. The interaction between macrophages with mature adipocytes releases pro‐inflammatory cytokines. This chronic inflammatory state can contribute to obesity‐related complications, such as insulin resistance, type 2 diabetes and cardiovascular disease. Therefore, we can attempt to prevent and treat obesity‐related diseases by inhibiting macrophage infiltration and blocking their interaction with adipocytes. Honey bee (Apis mellifera) venom (BV) has been reported to have anti‐inflammatory effects. Although BV is used to treat chronic inflammatory diseases, few studies have addressed its use in obesity‐associated inflammation. This study examines the inhibitory effects of BV on lipid accumulation in differentiating preadipocytes, inflammation, and insulin resistance in macrophages and adipocyte‐macrophage co‐culture system. We treated 3 T3‐L1 preadipocytes with BV during differentiation. We later measured lipid accumulation and gene expression of master adipogenic transcription factors. After RAW264.7 and 3 T3‐L1 cells were pretreated with BV, RAW264.7 cells were activated with LPS or co‐cultured with pretreated 3 T3‐L1 cells. Gene expression of pro‐inflammatory cytokines and insulin sensitizing genes was measured in these cells. BV inhibited lipid accumulation and C/EBPα and PPARγ gene expression during intermediate and late 3 T3‐L1 cell differentiation. BV also suppressed gene expression of pro‐inflammatory cytokines (COX‐2, iNOS, MCP‐1, TNF‐α, IL‐1β and IL‐6) in LPS‐stimulated macrophages, and in co‐culture of 3 T3‐L1 adipocytes and RAW264.7 macrophages. However, adiponectin and GLUT‐4 expression were both significantly increased by BV in co‐culture. These findings demonstrate that BV attenuates adipocyte hypertrophy and improves obesity‐related inflammation and insulin resistance in obese adipose tissue.

Regulation of adipocyte differentiation and metabolism by lansoprazole

AimsLansoprazole (LPZ) is one of the most commonly prescribed drugs for treatment of acid-related diseases, and it is increasingly recognized for its potential application as an anti-diabetic therapy. Although LPZ target tissues remain poorly understood, possible sites of action include adipose tissue. In this study, we assessed effects of LPZ on adipocyte differentiation and function by using 3T3-L1 preadipocytes and HFD-induced obesity mice as an in vitro and in vivo model, respectively. Main methodsOil red O staining and intracellular triacylglycerol content were used to determine lipid accumulation. Glucose uptake was performed to measure mature adipocyte function. Expression of adipocyte genes was determined by qRT-PCR and immunoblotting. Key findingsLPZ has dual effects on differentiation of 3T3-L1 cells. At low concentrations, LPZ enhanced adipocyte differentiation via induction of PPARγ and C/EBPα, two master adipogenic transcription factors, as well as lipogenic proteins, ACC1 and FASN. Increasing of adipocyte number subsequently increased basal and insulin-stimulated glucose uptake, and expression of Glut4 mRNA. Conversely, high concentrations of LPZ strongly inhibited differentiation and expression of PPARγ and C/EBPα, and maintained expression of preadipocytes markers, β-catenin and Pref-1. Inhibition of adipogenesis by LPZ reduced mature adipocyte number, Glut4 mRNA expression and insulin-stimulated glucose uptake. In addition, treatment with LPZ at 200 mg/kg significantly reduced body weight gain and total fat mass in HFD-induced obese mice. SignificanceThese results indicate that effects of LPZ on adipocyte differentiation are dependent on concentration and are correlated with PPARγ and C/EBPα.

The fruits of Gleditsia sinensis Lam. inhibits adipogenesis through modulation of mitotic clonal expansion and STAT3 activation in 3T3-L1 cells

Ethnopharmacological relevanceGleditsia sinensis Lam. (G. sinensis) has been used in Oriental medicine for tumor, thrombosis, inflammation-related disease, and obesity. Aim of the studyThe pharmacological inhibitory effects of fruits of G. sinensis (GFE) on hyperlipidemia have been reported, but its inhibitory effects on adipogenesis and underlying mechanisms have not been elucidated. Herein we evaluated the anti-adipogenic effects of GFE and described the underlying mechanisms. Materials and methodsThe effects of ethanol extracts of GFE on adipocyte differentiation were examined in 3T3-L1 cells using biochemical and molecular analyses. ResultsDuring the differentiation of 3T3-L1 cells, GFE significantly reduced lipid accumulation and downregulated master adipogenic transcription factors, including CCAAT/enhancer-binding protein-α and peroxisome proliferator-activated receptor-γ, at mRNA and protein levels. These changes led to the suppression of several adipogenic-specific genes and proteins, including fatty acid synthase, sterol regulatory element-binding protein 1, stearoyl-CoA desaturase-1, and acetyl CoA carboxylase. However, the inhibitory effects of GFE on lipogenesis were only shown when GFE is treated in the early stage of adipogenesis within the first two days of differentiation. As a potential mechanism, during the early stages of differentiation, GFE inhibited cell proliferation by a decrease in the expression of DNA synthesis-related proteins and increased p27 expression and suppressed signal transducer and activator of transcription 3 (STAT3) activation induced in a differentiation medium. ConclusionsGFE inhibits lipogenesis by negative regulation of adipogenic transcription factors, which is associated with GFE-mediated cell cycle arrest and STAT3 inhibition.

Pingmu Decoction Induces Orbital Preadipocytes Apoptosis In Vitro.

Pingmu Decoction is the Traditional Chinese Medicine which has treated Graves' Ophthalmopathy (GO) in the inactive stage for more than ten years. This study was to explore the mechanism of Pingmu Decoction of inhibiting preadipocytes in GO patients from differentiating into mature adipocytes. Human orbital preadipocytes were isolated and cultured through tissue explant method. Orbital preadipocytes were induced into mature adipocytes. The medicinal serum was prepared from rats. The cells were treated with medicinal serum which were divided into three groups, low dose group (5%), medium dose group (10%), and high dose group (20%). The cells viabilities were observed by Oil Red O staining, MTT method, and Annexin V/propidium iodide (PI) double staining. Effect of Pingmu Decoction on cell apoptosis rate of orbital matured adipocytes was measured by flow cytometry. The genes Fas and Fas L from cell groups were tested by RT-PCR and Western blotting. The expression of master adipogenic transcription factors, including peroxisome proliferation-activity receptor (PPAR) γ and CCAAT/enhancer binding protein (C/EBP) α, was tested by Western blotting. Pingmu Decoction could reduce orbital preadipocytes viability and induce apoptosis of mature adipocyte via Fas/Fas L signaling pathway. Pingmu Decoction reduced lipid accumulation and downregulated the expression of PPAR γ and C/EBP α. Pingmu Decoction may play a therapeutic effect by reducing the accumulation of orbital adipocytes.

The Herbal Medicine KBH-1 Inhibits Fat Accumulation in 3T3-L1 Adipocytes and Reduces High Fat Diet-Induced Obesity through Regulation of the AMPK Pathway.

The aim of this study was to investigate whether a novel formulation of an herbal extract, KBH-1, has an inhibitory effect on obesity. To determine its anti-obesity effects and its underlying mechanism, we performed anti-obesity-related experiments in vitro and in vivo. 3T3-L1 preadipocytes were analyzed for lipid accumulation as well as the protein and gene expression of molecular targets involved in fatty acid synthesis. To determine whether KBH-1 oral administration results in a reduction in high-fat diet (HFD)-induced obesity, we examined five groups (n = 9) of C57BL/6 mice as follows: 10% kcal fat diet-fed mice (ND), 60% kcal fat diet-fed mice (HFD), HFD-fed mice treated with orlistat (tetrahydrolipstatin, marketed under the trade name Xenical), HFD-fed mice treated with 150 mg/kg KBH-1 (KBH-1 150) and HFD-fed mice treated with 300 mg/kg KBH-1 (KBH-1 300). During adipogenesis of 3T3-L1 cells in vitro, KBH-1 significantly reduced lipid accumulation and down-regulated the expression of master adipogenic transcription factors, including CCAAT/enhancer binding protein (C/EBP) β, C/EBP α and peroxisome proliferation-activity receptor (PPAR) γ, which led to the suppression of the expression of several adipocyte-specific genes and proteins. KBH-1 also markedly phosphorylated the adenosine monophosphate-activated protein kinase (AMPK) and acetyl-CoA carboxylase (ACC). In addition, KBH-1-induced the inhibition effect on lipid accumulation and AMPK-mediated signal activation were decreased by blocking AMPK phosphorylation using AMPK siRNA. Furthermore, daily oral administration of KBH-1 resulted in dose-dependent decreases in body weight, fat pad mass and fat tissue size without systemic toxicity. These results suggest that KBH-1 inhibits lipid accumulation by down-regulating the major transcription factors of the adipogenesis pathway by regulating the AMPK pathway in 3T3-L1 adipocytes and in mice with HFD-induced obesity. These results implicate KBH-1, a safe herbal extract, as a potential anti-obesity therapeutic agent.

Enzymatic fragments of hyaluronan inhibit adipocyte differentiation in 3T3-L1 pre-adipocytes

Hyaluronan has diverse biological activities depending on its molecular size. High molecular weight hyaluronan (2000 kDa) is a major component of extracellular matrix, and has been used in wounding healing, extracellular matrix regeneration, and in the treatment of osteoarthritis. Hyaluronan fragments can stimulate inflammation or induce loss of extracellular matrix. Hyaluronan is expressed during adipocyte differentiation, and down regulation of hyaluronan synthesis can reduce adipogenic differentiation. However, the direct effects of hyaluronan fragments on adipocyte differentiation have not been elucidated. Therefore, we prepared hyaluronan fragments by enzymatic digestion, and examined the inhibitory effects of these hyaluronan fragments on the accumulation of lipid droplets and on adipogenic gene mRNA expression in differentiating 3T3-L1 pre-adipocytes. Medium sized hyaluronan fragments (50 kDa) decreased lipid droplet accumulation in a dose-dependent manner. However, high molecular weight hyaluronan did not inhibit lipid droplet accumulation when used at a concentration of 600 μg/ml. Two or 4 day treatments with medium molecular weight of hyaluronan resulted in similar inhibitory levels of lipid accumulation as did treatment for 8 days. Medium sized hyaluronan inhibited the differentiation of 3T3-L1 pre-adipocytes during the early stages of adipogenesis. When 3T3-L1 cells were treated with 180 μg/ml of medium sized hyaluronan, the mRNAs for the master adipogenic transcription factors PPAR-γ and C/EBP-α were inhibited. Additionally, medium molecular weight hyaluronan suppressed mRNA expression of PPAR-γ target genes, including aP2 and FAS. This study is the first to report that medium molecular weight hyaluronan fragments can inhibit adipocyte differentiation.

The anti-adipogenic effect of PGRN on porcine preadipocytes involves ERK1,2 mediated PPARγ phosphorylation

Recent researches indicate that PGRN is closely related to diabetes and is regarded as a novel adipokine associated with obesity development, affecting adipocyte biology. In the present study, we investigated the effects and mechanisms of PGRN on porcine preadipocytes differentiation. Porcine preadipocytes were induced to differentiation with the addition of lentivirius-expressed PGRN shRNA at the early or late stage of induction period, and in the presence or absence of recombinant PGRN protein. The effects of PGRN on adipogenic genes expression and ERK activation were investigated. At the early stage of induction, knockdown of PGRN promoted differentiation, evidenced by enhanced lipid accumulation, upregulation of adipocyte markers, as well as master adipogenic transcription factors, PPARγ and C/EBPα. While, decreasing PGRN expression at the late stage of induction (day 3) had no effect on differentiation. These results suggested that PGRN functions in the early adipogenic events. Conversely, porcine preadipocytes differentiation was impaired by MDI and recombinant PGRN protein induction, the expressions of adipocyte markers were decreased. Further studies revealed that PGRN can specifically facilitate ERK1,2 activation, and this activation can be abolished by U0126. Moreover, PPARγ phosphorylation at serine 112 site was increased by PGRN treatment, which could reduce the transcriptional activity of PPARγ. We conclude that PGRN inhibits adipogenesis in porcine preadipocytes partially through ERK activation mediated PPARγ phosphorylation.

Scutellarin from Scutellaria baicalensis Suppresses Adipogenesis by Upregulating PPARα in 3T3-L1 Cells

Adipocyte dysfunction is a major cause of obesity, which is associated strongly with many disorders including psychological and medical morbidities, metabolic abnormalities, and cardiovascular diseases as well as a series of cancers. This study investigated the antiadipogenic activity of scutellarin (1) in 3T3-L1 preadipocytes as well as the underlying molecular mechanisms. It was observed that 1 reduced adipocyte differentiation of 3T3-L1 cells potently, as evidenced by a decrease in cellular lipid accumulation. At the molecular level, mRNA expression of the master adipogenic transcription factors, PPARγ and C/EBPα, was decreased markedly. However, mRNA levels of C/EBPβ, the upstream regulator of PPARγ and C/EBPα, were not decreased by 1. Moreover, a dose-dependent upregulation of PPARα was observed for 1. Computational modeling indicated that 1 can bind to PPARα, γ, and δ each in a distinct manner, while it can activate PPARα only by forming a hydrogen bond with Y464, thus stabilizing the AF-2 helix and activating PPARα. Therefore, these results suggest that 1, a major component of Scutellaria baicalensis, attenuates fat cell differentiation by upregulating PPARα as well as downregulating the expression of PPARγ and C/EBPα, thus showing therapeutic potential for obesity-related diseases.

Piperine, a Component of Black Pepper, Inhibits Adipogenesis by Antagonizing PPARγ Activity in 3T3-L1 Cells

This study investigated the antiadipogenic activity of black pepper extract and its constituent piperine in 3T3-L1 preadipocytes as well as the underlying molecular mechanisms. Both black pepper extract and piperine, without affecting cytotoxicity, strongly inhibited the adipocyte differentiation of 3T3-L1 cells. The mRNA expression of the master adipogenic transcription factors, PPARγ, SREBP-1c, and C/EBPβ, was markedly decreased. Intriguingly, mRNA levels of PPARγ target genes were also down-regulated. Moreover, a luciferase reporter assay indicated that pipierine significantly represses the rosiglitazone-induced PPARγ transcriptional activity. Finally, GST-pull down assays demonstrated that piperine disrupts the rosiglitazone-dependent interaction between PPARγ and coactivator CBP. Genome-wide analysis using microarray further supports the role of piperine in regulating genes associated with lipid metabolism. Overall, these results suggest that piperine, a major component of black pepper, attenuates fat cell differentiation by down-regulating PPARγ activity as well as suppressing PPARγ expression, thus leading to potential treatment for obesity-related diseases.

Antiobesity and Inflammatory Cytokines Effect of Lactobacillus sp. OPK‐3 Isolated from Kimchi on 3T3‐L1 Preadipocytes

This study investigated the inhibitory effect Lactobacillus sp. OPK‐3 isolated from kimchi (Korean traditional fermented food) (L. sp. OPK‐3) on cellular lipid accumulation and inflammatory cytokines in preadipocyte 3T3L‐1 cells. 3T3‐L1 cells were treated with KOLAB exhibited less lipid accumulation, as well as GPDH activity, and mRNA levels of PPARγ and C/EBPα, the master adipogenic transcription factors, were regulated by L. sp. OPK‐3. Moreover, mRNA levels of PPARγ target genes (fat‐specific marker genes), such as aP2 and CD36 are also down‐regulated by L. sp. OPK‐3. We also assessed the effects of L. sp. OPK‐3 on inflammatory cytokines production in preadipocytes. White adipose tissue is now recognized as an important endocrine organ which secretes various signal factors and protein termed adipockines. In this study, we investigated the effects of L. sp. OPK‐3 on TNF‐α‐induced changes in pro‐atherogenic adipokines in 3T3‐L1 cells. The results suggested that treatment of fat cell using lactic acid bacteria from kimchi might play a role in the prevention of fat accumulation and improve obesity‐induced cardiovascular disease, particularly atherosclerosis, by attenuating the TNF‐α‐induced changes in adipokine production. Furthermore, animal research suggests the possible action of lactic acid bacteria from kimchi as a modulator of lipid metabolism. (This work was supported by grant No. R01‐2008‐000‐20979‐0 from Korea Science and engineering foundation)

Pigment epithelium-derived factor suppresses adipogenesis via inhibition of the MAPK/ERK pathway in 3T3-L1 preadipocytes

We previously reported that circulating levels of pigment epithelium-derived factor (PEDF), a newly identified adipokine, are increased in patients with type 2 diabetes, correlating with body mass index. However, the role of PEDF in adipogenesis remains elusive. In the present study, we have investigated the effects and mechanisms of PEDF on adipocyte differentiation in 3T3-L1 preadipocytes. Differentiation of 3T3-L1 preadipocytes was induced in the presence or absence of human recombinant PEDF protein. The effects of PEDF on adipogenic gene expression, mitotic clonal expansion (MCE), and MAPK activation were investigated. Physiological concentrations of human PEDF protein inhibited adipocyte differentiation, evidenced by decreased lipid accumulation, downregulation of adipocyte markers, and inhibition of master adipogenic transcription factors such as C/EBP-alpha and PPARgamma. The antiadipogenic effects of PEDF were observed only when PEDF was added to the cells on day 0, but not on day 3 during differentiation, suggesting that PEDF targets some early adipogenic events. Similarly, overexpression of PEDF by adenovirus attenuated adipocyte differentiation. Further studies revealed that PEDF, or U-0126, a specific MAPK/ERK inhibitor, sequentially inhibited the early activation of ERK and MCE. Moreover, PEDF attenuated expression and the phosphorylation of C/EBP-beta at Thr(188), an essential step for transcriptional activation of C/EBP-beta. In addition, PEDF expression was decreased significantly in the first 24 h during adipocyte differentiation, suggesting that downregulation of PEDF may be essential for the initiation of MCE and adipogenesis. We conclude that PEDF inhibits adipogenesis in 3T3-L1 preadipocytes partially because of inhibition of the MAPK/ERK signaling pathway and MCE.

Effect of functional materials producing microbial strains isolated from Kimchi on antiobesity and inflammatory cytokines in 3T3‐L1 preadipocytes

This study investigated the inhibitory effect of lactic acid bacteria isolated from Kimchi (Korea traditional fermented food) (KLAB) treatment on cellular lipid accumulation and inflammatory cytokines in preadipocyte 3T3L‐1 cell lines. 3T3‐L1 cells were treated with GLAB significantly reduced lipid accumulation, as well as GPDH activity. And mRNA levels of PPAR? and C/EBPα, the master adipogenic transcription factors, are regulated by KLAB. Moreover, mRNA levels of PPAR? target genes (fat‐specific marker genes), such as aP2 are down‐regulated by GLAB. And, verify the role of KLAB on inflammatory cytokines in preadipocyte. White aipocyte tissue is now recognized as an important endocrine organ which secretes various signal factors and protein termed adipockine. In this study, we investigated the effects of KLAB on TNF‐α ‐induced atherogenic changes of the adipokines in 3T3‐L1 cells. The results suggested that treatment of lactic acid from Kimchi might play a role in the prevention of fat accumulation and improve obesity‐induced cardiovascular disease, particularly atherosclerosis, by attenuating the TNF‐α ‐induced changes of adipokines. Furthermore, animal research suggests the possible action of lactic acid from Kimchi as a facilitator of lipid profiles. (This work was supported by the Korea Science and Engineering Foundation (KOSEF) grant funded by the Korea government (MEST) (NO. R01‐2008‐000‐20979‐0))

The transcription factors Egr1 and Egr2 have opposing influences on adipocyte differentiation.

The zinc finger-containing transcription factors Egr1 (Krox24) and Egr2 (Krox20) have been implicated in the proliferation and differentiation of many cell types. Egr2 has previously been shown to play a positive role in adipocyte differentiation but the function of Egr1 in this context is unknown. We compared the roles of Egr1 and Egr2 in the differentiation of murine 3T3-L1 adipocytes. Egr1 protein was rapidly induced after addition of differentiation cocktail while Egr2 protein initially remained low before increasing on days 1 and 2, concomitant with the disappearance of Egr1. In marked contrast to the effects of Egr2, differentiation was inhibited by ectopic expression of Egr1 and potentiated by knockdown of Egr1. The pro-adipogenic effects of Egr1 knockdown were particularly notable when IBMX was omitted from the differentiation medium. However, knockdown of Egr1 did not affect C/EBPβ protein expression or phosphorylation of CREB Ser133. Further, Egr1 did not directly affect the activity of promoters for the master adipogenic transcription factors, C/EBPα or PPARγ2, as assessed in luciferase reporter assays. These data indicate that Egr1 and Egr2 exert opposing influences on adipocyte differentiation and that the careful regulation of both is required for maintaining appropriate levels of adipogenesis. Further, the pro-differentiation effects of IBMX involve suppression of the inhibitory influence of Egr1.

Critical role of phosphoinositide 3-kinase cascade in adipogenesis of human mesenchymal stem cells

Mesenchymal stem cells (MSCs) are multipotent stem cells capable of differentiating into adipocytes in the presence of a hormone cocktail. These cells thus provide a promising model for studying the early events of adipogenesis. Here, we examine the involvement of the PI3K/Akt and mTOR/p70S6K signaling pathways in human MSC adipogenesis. We found that the two pathways were strongly activated with a similar temporal profile under the adipogenesis-inducing hormone cocktail and this activation could be blocked by LY294002, a specific inhibitor of PI3K. Furthermore, rapamycin, a specific inhibitor of mTOR, blocked the activation of mTOR/p70S6K but not PI3K/Akt. Both LY294002 and rapamycin severely suppressed lipid accumulation, as well as the expression of adipogenic markers, including PPAR gamma 2 and C/EBP alpha, two master adipogenic transcription factors. Together, these data indicate that the mTOR/p70S6K pathway acts downstream of the PI3K/Akt pathway in mediating the adipogenic conversion of MSCs. In conclusion, our data suggest that the PI3K/Akt and mTOR/p70S6K signaling pathways are essential for adipogenesis of human MSCs.

Phloretin enhances adipocyte differentiation and adiponectin expression in 3T3-L1 cells

Adipocyte dysfunction is strongly associated with the development of cardiovascular risk factors and diabetes. It is accepted that the regulation of adipogenesis or adipokines expression, notably adiponectin, is able to prevent these disorders. In this report, we show that phloretin, a dietary flavonoid, enhances 3T3-L1 adipocyte differentiation as evidenced by increased triglyceride accumulation and GPDH activity. At a molecular level, mRNA expression levels of both PPARγ and C/EBPα, the master adipogenic transcription factors, are markedly increased by phloretin. Moreover, mRNA levels of PPARγ target genes such as LPL, aP2, CD36 and LXRα are up-regulated by phloretin. We also show that phloretin enhances the expression and secretion of adiponectin. Co-transfection studies suggest the induction of PPARγ transcriptional activity as a possible mechanism underlying the phloretin-mediated effects. Taken together, these results suggest that phloretin may be beneficial for reducing insulin resistance through its potency to regulate adipocyte differentiation and function.

Microarray analysis during adipogenesis identifies new genes altered by antiretroviral drugs

To elucidate the pathogenesis of HAART-associated lipodystrophy, by investigating the effects of antiretroviral drugs on adipocyte differentiation and gene expression profile. Analysis of gene expression profile by DNA microarrays and quantitative RT-PCR of 3T3-L1 preadipocytes treated with the nucleoside reverse transcriptase inhibitors (NRTI) lamivudine, zidovudine, stavudine, and zalcitabine, and with the protease inhibitors (PI) indinavir, saquinavir, and lopinavir during maturation into adipocytes. Under standard adipogenic differentiation protocols, PI significantly inhibited adipocyte differentiation, as demonstrated by cell viability assay and Oil Red O staining and quantification, whereas NRTI had mild effects on adipogenesis. Gene expression profile analysis showed that treatment with NRTI modulated the expression of transcription factors, such as Aebp1, Pou5f1 and Phf6, which could play a key role in the determination of the adipocyte phenotype. PI also modulated gene expression toward inhibition of adipocyte differentiation, with up-regulation of the Wnt signaling gene Wnt10a and down-regulation of the expression of genes encoding master adipogenic transcription factors (e.g., C/EBPalpha and PPARgamma), oestrogen receptor beta, and adipocyte-specific markers (e.g., Adiponectin, Leptin, Mrap, Cd36, S100A8). This study identifies new genes modulated by PI and NRTI in differentiating adipocytes. Abnormal expression of these genes, which include master adipogenic transcription factors and genes involved in lipid metabolism and cell cycle control, could contribute to the understanding of the pathogenesis of HAART-associated lipodystrophy.

Wnt Signaling Inhibits Adipogenesis through β-Catenin-dependent and -independent Mechanisms

Wnt signaling has been reported to block apoptosis and regulate differentiation of mesenchymal progenitors through inhibition of glycogen synthase kinase 3 and stabilization of beta-catenin. The effects of Wnt in preadipocytes may be mediated through Frizzled (Fz) 1 and/or Fz2 as these Wnt receptors are expressed in preadipocytes and their expression declines upon induction of differentiation. We ectopically expressed constitutively active chimeras between Wnt8 and Fz1 or Fz2 in preadipocytes and mesenchymal precursor cells. Our results indicated that activated Fz1 increases stability of beta-catenin, inhibits apoptosis, induces osteoblastogenesis, and inhibits adipogenesis. Although activated Fz2 does not influence apoptosis or osteoblastogenesis, it inhibits adipogenesis through a mechanism independent of beta-catenin. An important mediator of the beta-catenin-independent pathway appears to be calcineurin because inhibitors of this serine/threonine phosphatase partially rescue the block to adipogenesis caused by Wnt3a or activated Fz2. These data supported a model in which Wnt signaling inhibits adipogenesis through both beta-catenin-dependent and beta-catenin-independent mechanisms.