Treatment Of 3T3-L1 Preadipocytes Research Articles

An Evaluation of the Anti-Inflammatory Effects of a Thai Traditional Polyherbal Recipe TPDM6315 in LPS-Induced RAW264.7 Macrophages and TNF-α-Induced 3T3-L1 Adipocytes.

TPDM6315 is an antipyretic Thai herbal recipe that contains several herbs with anti-inflammatory and anti-obesity activities. This study aimed to investigate the anti-inflammatory effects of TPDM6315 extracts in lipopolysaccharide (LPS)-induced RAW264.7 macrophages and TNF-α-induced 3T3-L1 adipocytes, and the effects of TPDM6315 extracts on lipid accumulation in 3T3-L1 adipocytes. The results showed that the TPDM6315 extracts reduced the nitric oxide production and downregulated the iNOS, IL-6, PGE2, and TNF-α genes regulating fever in LPS-stimulated RAW264.7 macrophages. The treatment of 3T3-L1 pre-adipocytes with TPDM6315 extracts during a differentiation to the adipocytes resulted in the decreasing of the cellular lipid accumulation in adipocytes. The ethanolic extract (10 µg/mL) increased the mRNA level of adiponectin (the anti-inflammatory adipokine) and upregulated the PPAR-γ in the TNF-α induced adipocytes. These findings provide evidence-based support for the traditional use of TPDM6315 as an anti-pyretic for fever originating from inflammation. The anti-obesity and anti-inflammatory actions of TPDM6315 in TNF-α induced adipocytes suggest that this herbal recipe could be useful for the treatment of metabolic syndrome disorders caused by obesity. Further investigations into the modes of action of TPDM6315 are needed for developing health products to prevent or regulate disorders resulting from inflammation.

Antiobesity, hepatoprotective and anti-hyperglycemic effects of a pharmaceutical formulation containing Cecropia pachystachya Trécul in mice fed with a hypercaloric diet

Ethnopharmacological relevanceThe leaves of Cecropia pachystachya Trécul (Urticaceae), known as embaúba, are used as hypoglycemic and for weight reduction in Brazilian traditional medicine. Aim of the studyThis study investigated the effects of a pharmaceutical formulation (ECP20) containing C. pachystachya extract on some metabolic alterations caused by a hypercaloric diet in mice. Material and methodsMice were randomly fed with a standard or hypercaloric diet and orally treated with ECP20 or vehicle for 13 weeks. Subsequently, adiposity, glucose intolerance, and the presence of nonalcoholic fatty liver disease were assessed. Adipose tissue and liver were collected after euthanasia and frozen at −80 °C for histological and antioxidant analyzes. The effect of ECP20 on the differentiation of 3T3-L1 pre-adipocytes was also investigated. ResultsAnimals treated with ECP20 showed less weight gain, reduced glycemia, glucose tolerance restored, and hepatoprotective effect. Also, ECP20 presented significant in vivo antioxidant activity. Treatment of 3T3-L1 preadipocytes with ECP20 did not inhibit cellular differencing. ConclusionsTherefore, ECP20 presented promising effects in the control of obesity and related disorders. Considering that glucose intolerance and hyperglycemia are strong evidence for the development of type 2 diabetes, the findings corroborated the traditional use of C. pachystachya to treat this disease. The chlorogenic acid and the flavonoids orientin and iso-orientin, present in the extract, might be involved in the activities found.

Chlorogenic acid promotes development of brown adipocyte-like phenotype in 3T3-L1 adipocytes

An emerging strategy for reducing obesity is induction of brown fat type phenotype in white adipose tissue. Chlorogenic acid (CGA), a dietary polyphenol, has been reported to modulate lipid and glucose metabolism, and improve glucose tolerance. However, information on its role in adipocyte browning is lacking. The effect of CGA on browning of 3T3-L1 pre-adipocytes and differentiated white adipocytes was investigated. Treatment of 3T3-L1 pre-adipocytes with CGA produced more adipocytes with multilocular phenotype, typical of brown adipocytes. Increase in uncoupler protein-1(UCP1)(1.7-fold) and Peroxisome proliferator-activated receptor-γ co-activator-1α (PGC-1α)(2-fold) and decrease in triacyl glycerol content (50%) suggested that CGA promotes browning. Morphological analysis and expression of UCP1 further showed that CGA induced trans-differentiation of 3T3-L1 white adipocytes cells to beige adipocytes. Increased phosphorylation of AMP-dependent kinase (AMPK) in cells treated with CGA and reversal of CGA stimulated browning by dorsomorphin, an inhibitor of AMPK, suggested that AMPK pathway is involved in mediating the effect of CGA.

The up-regulation of markers of adipose tissue fibrosis by visfatin in pre-adipocytes as well as obese children and adolescents

Adipocytes are surrounded by a three-dimensional network of extracellular matrix (ECM) proteins. Aberrant ECM accumulation and remodeling leads to adipose tissue fibrosis. Visfatin is one of the adipocytokines that is increased in obesity and is implicated in insulin resistance. The objective of this study was to investigate the effect of visfatin on major components of ECM remodeling. In this study, plasma levels of both endotrophin and visfatin in obese children and adolescents were significantly higher than those in control subjects and they showed a positive correlation with each other. Treatment of 3T3-L1 pre-adipocytes with visfatin caused significant up-regulation of Osteopontin (Opn), Collagen type VI (Col6), matrix metalloproteinases MMP-2 and MMP-9. By using inhibitors of major signaling pathways it was shown that visfatin exerted its effect on Col6a3 gene expression through PI3K, JNK, and NF-кB pathways, while induced Opn gene expression via PI3K, JNK, MAPK/ERK, and NOTCH1. Our conclusion is that, the relationship between visfatin, endotrophin and insulin resistance parameters in obesity as well as increased expression of ECM proteins by visfatin suggests adipose tissue fibrosis as a mechanism for devastating effects of visfatin in obesity.

Self-assembled hyaluronic acid nanoparticle suppresses fat accumulation via CD44 in diet-induced obese mice

Obesity, a major risk factor for type 2 diabetes and cardiovascular diseases, is characterized by an abnormal expansion of adipose tissue. Herein, we investigated the potential of hyaluronic acid nanoparticles (HA-NPs) as therapeutics to treat obesity-related diseases by assessing the in vitro and in vivo effects of HA-NPs on adipogenesis and lipogenesis. Treatment of 3T3-L1 preadipocytes with HA-NPs resulted in a dose-dependent suppression of adipogenesis and lipid accumulation, and decreased the expression of key adipogenic and lipogenic regulators. However, these HA-NPs mediated effects were not observed in 3T3-L1 cells transfected with siRNAs against CD44, a major HA receptor. Further, HA-NP treatment of diet-induced obese (DIO) mice reduced the epididymal fat mass and suppressed the induction of adipogenic and lipogenic regulators, while these effects were attenuated in the CD44-null mice. Thus, our study provides a better understanding of how HA-NP modulates fat accumulation and presents a potential anti-obesity strategy targeting CD44.

KCTD1: A novel modulator of adipogenesis through the interaction with the transcription factor AP2α

Adipogenesis has an important role in regulating energy balance, tissue homeostasis and disease pathogenesis. 3T3-L1 preadipocytes have been widely used as an in vitro model for studying adipocyte differentiation. We here show that KCTD1, a member of the potassium channel containing tetramerization domain proteins, plays an active role in adipogenesis. In particular, we show KCTD1 expression 3T3-L1 cells increases upon adipogenesis induction. Treatment of 3T3-L1 preadipocytes with Kctd1-specific siRNA inhibited the differentiation, as indicated by reduction of expression of the specific adipogenic markers C/ebpα, Pparγ2, Glut4, and Adiponectin. Moreover, we also show that the protein physically interacts with the transcription factor AP2α, a known inhibitor of adipogenesis, both in vitro and in cells. Interestingly, our data indicate that KCTD1 promotes adipogenesis through the interaction with AP2α and by removing it from the nucleus. Collectively, these findings disclose a novel role for KCTD1 and pave the way for novel strategies aimed at modulating adipogenesis.

Fibroblast Growth Factor 21 Exerts its Anti‐inflammatory Effects on Multiple Cell Types of Adipose Tissue in Obesity

Obesity-related, chronic, low-grade inflammation has been identified as a key factor in the development of many metabolic diseases, such as type 2 diabetes and cardiovascular diseases. Adipocytes, preadipocytes, and macrophages have been implicated in initiating inflammation in adipose tissue. This study aims to investigate the effects of fibroblast growth factor-21 (FGF-21) on obesity-related inflammation and its mechanisms in vivo and in vitro. Monosodium glutamate (MSG) was used to induce obesity in mice and subsequently treated the mice with or without FGF-21. Primary adipocytes and stromal vascular fraction cells were isolated from MSG-obesity mice for additional experiments. Results obtained by ELISA and real-time polymerase chain reaction showed that FGF-21 efficiently ameliorated obesity-related inflammation in MSG-obesity mice. This study demonstrated that preadipocytes and adipocytes responded to anti-inflammatory effects of FGF-21. In vitro, 3T3-L1 preadipocytes lacking β-klotho did not respond to FGF-21 under glucose uptake. Interestingly, the treatment of 3T3-L1 preadipocytes with FGF-21 significantly attenuated lipopolysaccharide-induced inflammatory response. Our study showed that FGF-21-induced glucose uptake and FGF-21-related anti-inflammatory effects are mediated by different signaling pathways. Moreover, FGF-21 showed anti-inflammatory effects on preadipocytes; these effects are mediated by the fibroblast growth factor receptor substrate 2/ERK1/2 signaling pathway.

Anti-adipogenic effect of Terminalia chebula fruit aqueous extract in 3T3-L1 preadipocytes

Pharmacognosy Magazine ,2019,15,64,197-204.DOI:10.4103/pm.pm_108_19Published:August 2019Type:Original Article Authors:Anuj Kumar Borah, Pooja Rani Kuri, Archana Singh, and Sougata Saha Author(s) affiliations:Anuj Kumar Borah1, Pooja Rani Kuri2, Archana Singh1, Sougata Saha3 1Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur, Assam, India 2Department of Molecular Biology and Biotechnology, Tezpur University, Tezpur; Department of Bioscience and Bioengineering, Indian Institute of Technology, Guwahati, Assam, India 3Department of Biotechnology, National Institute of Technology, Durgapur, West Bengal, India Abstract:Background: Phytoextracts, due to its complex nature of formulations yet little or no side effects, have been pursued as alternative medicine for the treatment of complex metabolic disorders such as obesity. One of the appealing strategies to achieve this is the modulation of adipocyte development and function with the treatment of phytoextracts. The current study explored the activity of Terminalia chebula fruit, a component of Ayurveda formulation “Triphala” on these aspects of adipogenesis. Materials and Methods: The effect of T. chebula aqueous fruit extract (CAFE) on the process of adipocyte development and function was investigated. To test the effect of CAFE on adipocyte development, 3T3-L1 preadipocytes were differentiated in the presence and absence of CAFE followed by estimation of lipid content and expression of adipogenic genes. To test its effect on adipocyte function, mature 3T3-L1 adipocytes were treated with the extract followed by estimation of lipolysis. Results: Treatment of 3T3-L1 preadipocytes with this extract had efficiently inhibited differentiation and lipid accumulation in these cells. Gene expression of key adipogenic regulators, peroxisome proliferative-activated receptor γ and C/CAAT enhancer-binding protein α, was suppressed due to the treatment with CAFE. Preadipocytes exposed to CAFE also showed suppressed expression of important adipogenic effector genes such as perilipin 1 and fatty acid synthase. Treatment of differentiated adipocytes with CAFE did not affect total lipid contents of the cells. However, CAFE treatment reduced lipolysis to a small extent. Conclusion: CAFE is an anti-adipogenic and anti-lipolytic agent which inhibits adipocyte differentiation by downregulating expression of key adipogenic genes. Keywords:3T3-L1 differentiation, adipogenesis, lipolysis, Obesity, Terminalia chebulaView:PDF (2.78 MB)

Epigenetic programming of Dnmt3a mediated by AP2α is required for granting preadipocyte the ability to differentiate.

Adipogenesis has an important role in regulating energy homeostasis in mammals. 3T3-L1 preadipocytes have been widely used as an in vitro model for analyzing the molecular mechanism of adipogenesis. Previous reports indicated that the stage of contact inhibition (CI), through which the proliferating cells exit from the cell cycle, was required for granting preadipocyte the ability to differentiate. While this kind of the granting mechanism remains elusive. In the present study, we showed that DNA (cytosine-5) methyltransferase 3a (Dnmt3a) was upregulated at both the mRNA and protein level during the CI stage, and resulted in increasing promoter methylation of adipogenic genes. We further identified that the expression of Activator protein 2α (AP2α), a member of the transcription factor activator protein 2 (AP2) family, was highly correlated with the expression of Dnmt3a during the CI stage. In addition, we showed that AP2α transcriptionally upregulated Dnmt3a by directly binding to its proximal promoter region. Importantly, treatment of 3T3-L1 preadipocytes with AP2α-specific siRNAs inhibited the preadipocyte differentiation in a stage-dependent manner, supporting the conclusion that AP2α has an important role during the CI stage. Furthermore, overexpression of Dnmt3a partially rescued the impairment of adipogenesis induced by AP2α knockdown. Collectively, our findings reveal that AP2α is an essential regulator for granting preadipocyte the ability to differentiate through the upregulation of Dnmt3a expression during the CI stage.

Effects of Pueraria lobata Root Ethanol Extract on Adipogenesis and Lipogenesis During 3T3-L1 Differentiation into Adipocytes.

We evaluated the inhibitory effect of Pueraria lobata root ethanol extract (PLREE) on lipid accumulation during 3T3-L1 differentiation to adipocytes by measuring the intracellular expression of adipogenic, lipogenic, and lipolytic markers and lipid accumulation. The total polyphenol and flavonoid content of PLREE were 47 and 29 mg/g, respectively. The electron donating capacity of PLREE at 1,000 μg/mL was 48.8%. Treatment of 3T3-L1 preadipocytes with 100, 250, or 500 μg/mL PLREE for 8 days dose-dependently promoted the differentiation of 3T3-L1 cells. In contrast, the lipid content of PLREE-treated cells was significantly reduced by 7.8% (p < 0.05), 35.6% (p < 0.001), and 42.2% (p < 0.001) following treatment with 100, 250, and 500 μg/mL PLREE, respectively, as compared to differentiated control cells. PLREE upregulated peroxisome proliferator-activated receptor γ mRNA and protein, and sterol regulator element-binding protein-1c mRNA levels, but did not affect CCAAT/enhancer binding-protein β and α mRNA levels. PLREE also downregulated acetyl-CoA carboxylase mRNA and protein, fatty acid synthase (FAS) protein, and leptin mRNA levels, but did not affect FAS mRNA expression. PLREE upregulated adipose triglyceride lipase mRNA and protein expression, and hormone-sensitive lipase (HSL) protein expression, but did not affect HSL mRNA expression. In conclusion, we found that PLREE enhanced adipogenesis, but reduced lipogenesis, resulting in decreased lipid accumulation in 3T3-L1 cells.

Design and synthesis of a potent inhibitor of class 1 DYRK kinases as a suppressor of adipogenesis

Dysregulation of dual-specificity tyrosine-phosphorylation-regulated kinase 1A (DYRK1A) has been demonstrated in several pathological conditions, including Alzheimer’s disease and cancer progression. It has been recently reported that a gain of function-mutation in the human DYRK1B gene exacerbates metabolic syndrome by enhancing obesity. In the previous study, we developed an inhibitor of DYRK family kinases (INDY) and demonstrated that INDY suppresses the pathological phenotypes induced by overexpression of DYRK1A or DYRK1B in cellular and animal models. In this study, we designed and synthesized a novel inhibitor of DYRK family kinases based on the crystal structure of the DYRK1A/INDY complex by replacing the phenol group of INDY with dibenzofuran to produce a derivative, named BINDY. This compound exhibited potent and selective inhibitory activity toward DYRK family kinases in an in vitro assay. Furthermore, treatment of 3T3-L1 pre-adipocytes with BINDY hampered adipogenesis by suppressing gene expression of the critical transcription factors PPARγ and C/EBPα. This study indicates the possibility of BINDY as a potential drug for metabolic syndrome.

The effects of cinnamon extract on 3T3‐L1 pre‐adipocyte differentiation and cell size.

Cinnamon has long been used as a supplement for decreasing blood glucose and lipid values. Type 2 diabetes and obesity are growing concerns of public health and a better understanding of anomalies within these metabolic conditions are essential. Literature largely supports correlations between glucose and lipid metabolism control but few distinct pathway mechanisms have been proven experimentally. We proses that treatment of 3T3-L1 pre-adipocytes with cinnamon extract alters both carbohydrate and lipid metabolism and that in doing so alter differentiation As well. We explored lipid droplet formation and quantitated lipid volume as a marker of adipogenesis using oil red O staining. Experiments were completed in both monolayer and three-dimensional agarose cell culture. Cultures were treated with a variety of concentrations of cinnamon extract and evaluated at various time points for growth, cell size, morphology and viability. Our results suggest that treatment with cinnamon extract in both monolayer and agarose culture promote adipogenesis from the fibroblast-like morphology to the round adipocyte like morphology. Likewise, we see significant lipid droplet formation in treated cells. The cells were treated only with cinnamon extract and media plus calf or fetal serum and no other components were added to enhance differentiation. Differentiation was initiated even in the absence of differentiating media. Additionally we observed a reduction in the cell size when treated with cinnamon extract at plating. Cells that were grown in a three-dimensional environment but not treated with cinnamon extract or differentiating media also appeared to initiate differentiation.

Leucine-rich glioma inactivated 3 and tumor necrosis factor-α regulate mutually through NF-κB

Leucine-rich glioma inactivated 3 (LGI3) is a secreted protein member of LGI family. We previously reported that LGI3 increased in obese adipose tissues and suppressed adipogenesis through its receptor, ADAM23. We proposed that LGI3 may be a pro-inflammatory adipokine secreted predominantly by preadipocytes and macrophages. In this study, we showed that LGI3 and tumor necrosis factor-α (TNF-α) upregulated each other in 3T3-L1 cells. Treatment of 3T3-L1 preadipocytes with LGI3 protein increased TNF-α mRNA and protein. LGI3 treatment led to NF-κB activation and binding to an NF-κB binding site (−523 to −514) in TNF-α promoter. TNF-α treatment increased mRNA and protein expression of LGI3 and ADAM23. TNF-α increased NF-κB binding to a predicted binding site (−40 to −31) in LGI3 promoter. High fat diet-fed mice showed that LGI3 and TNF-α were increased and colocalized in adipose tissue inflammation. Taken together, these results suggested that mutual upregulation of LGI3 and TNF-α may play a role in adipose tissue inflammation in obesity.

Coregulator Cell Cycle and Apoptosis Regulator 1 (CCAR1) Positively Regulates Adipocyte Differentiation through the Glucocorticoid Signaling Pathway

Glucocorticoids contribute to adipocyte differentiation by cooperating with transcription factors, such as CCAAT/enhancer-binding protein β (C/EBPβ), to stimulate transcription of the gene encoding peroxisome proliferator-activated receptor (PPARγ), a master regulator of adipogenesis. However, the mechanism of PPARγ gene regulation by glucocorticoids, the glucocorticoid receptor (GR), and its coregulators is poorly understood. Here we show that two GR binding regions (GBRs) in the mouse PPARγ gene were responsive to glucocorticoid, and treatment of 3T3-L1 preadipocytes with glucocorticoid alone induced GR occupancy and chromatin remodeling at PPARγ GBRs, which also contain binding sites for C/EBP and PPARγ proteins. GR recruited cell cycle and apoptosis regulator 1 (CCAR1), a transcription coregulator, to the PPARγ gene GBRs. Notably, CCAR1 was required for GR occupancy and chromatin remodeling at one of the PPARγ gene GBRs. Moreover, depletion of CCAR1 markedly suppressed differentiation of mouse mesenchymal stem cells and 3T3-L1 preadipocytes to mature adipocytes and decreased induction of PPARγ, C/EBPα, and C/EBPδ. Although CCAR1 was required for stimulation of several GR-regulated adipogenic genes in 3T3-L1 preadipocytes by glucocorticoid, it was not required for GR-activated transcription of certain anti-inflammatory genes in human A549 lung epithelial cells. Overall, our results highlighted the novel and specific roles of GR and CCAR1 in adipogenesis.

Regulation Effects of Crataegus pinnatifida Leaf on Glucose and Lipids Metabolism

The leaf of Crataegus pinnatifida (Rosaceae) is commonly consumed either raw or cooked to improve digestion and promote blood circulation in China. To investigate the regulation effects of it on glucose and lipid metabolism, the flavonoids fraction was prepared and analyzed by HPLC and LC-MS. In vivo, at doses of 250 and 500 mg/kg, the flavonoids fraction showed inhibitory effects on TG and glucose absorption, accelerating effects on gastrointestinal transit but no effect on gastric emptying. In vitro, treatment of 3T3-L1 preadipocytes with 30 μg/mL flavonoids fraction significantly suppressed the accumulation of TG and free fatty acid. It also suppressed the gene expressions of C/EBPα, PPARγ, SREBP 1c, aP2 and adiponectin but did not affect that of leptin. C. pinnatifida leaf may be useful for type 2 diabetics and hyperlipidemics as a foodstuff.

Downregulated expression of the secreted glycoprotein follistatin-like 1 (Fstl1) is a robust hallmark of preadipocyte to adipocyte conversion

Obesity is a public health crisis in the United States. Targeting preadipocyte to adipocyte conversion may be an effective approach to regulate adipose mass. Using differential screening we identified Fstl1, a secreted glycoprotein with roles in immunomodulation, cell growth, cardioprotection, and vascularization, as a “preadipokine”. Fstl1 is highly expressed in 3T3-L1 preadipocytes and dramatically downregulated early in their differentiation to adipocytes. Northern blot analysis of murine tissues reveals white adipose tissue (WAT), lung and heart as primary sites of Fstl1 transcript expression. In WAT, Fstl1 transcript is restricted to the preadipocyte-containing stromal-vascular cell population. Time course studies in multiple adipogenesis models reveal downregulation of Fstl1 is a hallmark of white and brown adipocyte conversion. By Western blot, we show culture media of 3T3-L1 preadipocytes contains high levels of Fstl1 protein that rapidly decline in adipocyte conversion. Moreover, we observe a correlation between preadipocyte phenotype and Fstl1 expression in that TNFα-mediated de-differentiation of 3T3-L1 adipocytes is accompanied by re-expression of Fstl1 transcript and protein. Treatment of 3T3-L1 preadipocytes with a panel of 18 hormones and other agents revealed the demethylating agent 5-aza-cytidine decreases Fstl1 transcript and protein levels by ∼90%. Furthermore, of 10 additional preadipocyte-expressed genes analyzed we find Pref-1, Col1A1, Sca-1/Ly6a, Lox and Thbs2, are also downregulated by 5-aza-cytidine. Using luciferase reporter constructs containing 791 or 3922 bp of the Fstl1 5′ flanking region, we determine negative transcriptional regulation by Kruppel-like factor 15. Together, our data suggest downregulation of Fstl1 expression may be an important feature of preadipocyte to adipocyte conversion.

Effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes

The effects of ghrelin on the proliferation and differentiation of 3T3-L1 preadipocytes and the possible mechanisms were investigated in this study. 3T3-L1 preadipocytes were cultured in vitro and treated with different concentrations of ghrelin. Proliferation of 3T3-L1 preadipocytes was evaluated by MTT method and mRNA levels of c-myc and thymidine kinase were detected by RT-PCR. Morphological changes of 3T3-L1 preadipocytes were observed and cell differentiation was measured by oil red O staining. The mRNA levels of peroxisome proliferator-activated receptor gamma (PPARgamma) and CAAT/enhancer binding protein (C/EBPalpha) in the cells at different differentiation stages were detected by RT-PCR. The results showed that ghrelin at concentrations of 10(-7) to 10(-15) mol/L could significantly promote preadipocyte proliferation (P<0.05), with the most pronounced effect observed at 10(-11) mol/L (P<0.01). Treatment of 3T3-L1 preadipocytes with ghrelin significantly increased the mRNA levels of c-myc and thymidine kinase (P<0.01). Morphological findings demonstrated that the great amount of lipid droplets appeared in the 3T3-L1 preadipocytes treated with ghrelin. Ghrelin could morphologically induce the differentiation of 3T3-L1 preadipocytes into mature adipocytes. Ghrelin significantly increased the mRNA levels of PPARgamma and C/EBPalpha during the differentiation, when compared with control group (P<0.05). The mRNA levels of PPARgamma and C/EBPalpha were obviously up-regulated with the differentiation of preadipocytes after the treatment of ghrelin. There were significant difference in the mRNA levels of PPARgamma and C/EBPalpha on day 2 and day 8 of the differentiation of 3T3-L1 preadipocytes (P<0.01). In conclusion, ghrelin could promote the proliferation and differentiation of 3T3-L1 preadipocytes by increasing the mRNA levels of PPARgamma and C/EBPalpha and therefore enhance the sensitivity of adipocytes against insulin.

Regulation of Cyclin D1 and Wnt10b Gene Expression by cAMP-responsive Element-binding Protein during Early Adipogenesis Involves Differential Promoter Methylation

Cyclin D1 expression is elevated and Wnt10b is repressed by cAMP during the first few hours of adipogenesis. cAMP-responsive element-binding protein (CREB) is a primary target for cAMP signaling, and we have shown that activation of CREB promotes adipogenesis and adipocyte survival. Here we tested the impact of CREB on expression of cyclin D1 and wingless-related mouse mammary tumor virus integration site 10b (Wnt10b) in 3T3-L1 cells. Forced depletion of CREB blocked Bt(2)cAMP-stimulated cyclin D1 expression and basal Wnt10b gene expression. Two CREB-binding sites were identified in the Wnt10b promoter region. Ablation of either site partially blocked promoter activity, while mutation of both sites completely suppressed promoter activity. These results suggest that CREB activates transcription from both the cyclin D1 and Wnt10b gene promoters. What accounts for the differential regulation of cyclin D1 and Wnt10b genes by cAMP? Chromatin immunoprecipitation revealed CREB bound to the Wnt10b promoter in untreated preadipocytes but not following treatment with Bt(2)cAMP. CREB binding to the cyclin D1 promoter was detected in untreated cells and post-Bt(2)cAMP. Differences between CREB binding to the two genes correlated with increasing methylation of the Wnt10b promoter following Bt(2)cAMP treatment, whereas no methylation of the cyclin D1 promoter was observed. Treatment of cells with the methylase inhibitor 5-azacytidine restored CREB binding to the Wnt10b gene promoter and prevented the inhibition of Wnt10b RNA expression by Bt(2)cAMP. We conclude that cAMP stimulates phosphorylation and binding of CREB to the cyclin D1 gene promoter. Simultaneously, hypermethylation of the Wnt10b gene promoter suppresses binding of CREB, allowing adipogenesis to proceed.

Inhibition of adipocyte differentiation by Nur77, Nurr1, and Nor1.

Members of the nuclear receptor 4A (NR4A) subgroup of nuclear receptors have been implicated in the regulation of glucose and lipid metabolism in insulin-sensitive tissues such as liver and skeletal muscle. However, their function in adipocytes is not well defined. Previous studies have reported that these receptors are rapidly up-regulated after treatment of 3T3-L1 preadipocytes with an adipogenic cocktail. We show here that although Nur77 expression is acutely induced by cAMP agonists in 3T3-L1 cells, it is not induced by other adipogenic stimuli, such as peroxisome proliferator-activated receptor-gamma ligands, nor is it induced during the differentiation of 3T3-F442A preadipocytes, suggesting that Nur77 induction is not an obligatory feature of preadipocyte differentiation. We further demonstrate that inflammatory signals that antagonize differentiation, such as TNFalpha and lipopolysaccharide, acutely induce Nur77 expression both in vitro and in vivo. We also show that NR4A expression in adipose tissue is responsive to fasting/refeeding. Retroviral transduction of each of the NR4A receptors (Nur77, Nurr1, and NOR1) into either 3T3-L1 or 3T3-F442A preadipocytes potently inhibits adipogenesis. Interestingly, NR4A-mediated inhibition of adipogenesis cannot be rescued by peroxisome proliferator-activated receptor-gamma overexpression or activation. Transcriptional profiling of Nur77-expressing preadipocytes led to the identification of gap-junction protein alpha1 (Gja1) and tolloid-like 1 (Tll1) as Nur77-responsive genes. Remarkably, retroviral expression of either Gja1 or Tll1 in 3T3-L1 preadipocytes also inhibited adipocyte differentiation, implicating these genes as potential mediators of Nur77's effects on adipogenesis. Finally, we show that Nur77 expression inhibits mitotic clonal expansion of preadipocytes, providing an additional mechanism by which Nur77 may inhibit adipogenesis.

Self-assembly of Glut4 Storage Vesicles during Differentiation of 3T3-L1 Adipocytes

Glut4 storage vesicles (GSVs) represent translocation-competent vesicular carriers in fat and skeletal muscle cells that deliver Glut4 to the plasma membrane in response to insulin stimulation. GSVs include three major cargo proteins: Glut4, insulin-responsive aminopeptidase (IRAP), and sortilin. Previous work has suggested that the lumenal interaction between Glut4 and sortilin and the cytoplasmic interaction between sortilin and GGA adaptors play an important role in recruitment of Glut4 into the GSVs. However, the mechanism of IRAP targeting to this compartment remains unknown. To address this question, we show that in differentiating adipocytes IRAP enters the GSVs from the "donor" membranes on day 3 of differentiation. Forced expression of sortilin in undifferentiated cells does not recruit IRAP into the vesicles. However, double expression of sortilin and Glut4 reconstitutes functional GSVs that incorporate endogenous IRAP. To explain this process, we show by a yeast two-hybrid system and chemical cross-linking that the lumenal domain of IRAP can interact with the lumenal loop of Glut4. IRAP without the lumenal domain is faithfully targeted to the donor membranes but has significantly lower insulin responsiveness than full-length IRAP. We suggest that lumenal interactions between Glut4 and IRAP play an important role in the assembly of the GSVs.