Elastin-derived peptide VGVAPG decreases differentiation of mouse embryo fibroblast (3T3-L1) cells into adipocytes.

Elastin is a highly elastic protein present in connective tissue. As a result of protease activity, elastin hydrolysis occurs, and during this process, elastin-derived peptides (EDPs) are released. One of the constitutively repeating elastin and EDP building sequences is the hexapeptide VGVAPG. Therefore, the aim of our research was to define the effect of VGVAPG peptide on adipogenesis in a mouse 3T3-L1 cell line. 3T3-L1 cells were differentiated according to a previously described protocol and exposed to increasing concentrations of VGVAPG or VVGPGA peptide. The obtained results showed that VGVAPG peptide does not stimulate reactive oxygen species (ROS) production, caspase-1 activation, and 3T3-L1 cell proliferation. In the second part of the experiments, it was proved that VGVAPG peptide decreased lipid accumulation as measured by oil red O staining, which was confirmed by the profile of increased expression markers of undifferentiated preadipocytes. In our experiments, 10 nM VGVAPG added for differentiating to adipocytes increased the expression of Pref-1, serpin E1, and adiponectin as compared to rosiglitazone (PPARγ agonist)-treated group and simultaneously decreased the expression of VEGF and resistin as compared to the rosiglitazone-treated group. The obtained results show that VGVAPG peptide sustains 3T3 cells in undifferentiated state. ABBREVIATIONS: DMSO: dimethyl sulphoxide; EBP: elastin-binding protein; EDPs: elastin-derived peptides; FBS: foetal bovine serum; Glb1: gene for beta-galactosidase; LDL: low-density-lipoprotein; PAI-1 (Serpin E1): plasminogen activator inhibitor-1; PBS: phosphate-buffered saline; PPARγ: peroxisome proliferator-activated receptor gamma; Pref-1: preadipocyte factor 1; ROS: reactive oxygen species; VEGF-A: vascular endothelial growth factor-A; VGVAPG: Val-Gly-Val-Ala-Pro-Gly; ß-Gal: beta-galactosidase; ORO: oil red O; IBMX: 3-isobutyl-1-methylxanthine; H2DCFDA: 2',7'-dichlorodihydrofluorescein diacetate; DMEM: Dulbecco's Modified Eagle's Medium; VVGPGA: Val-Val-Gly-Pro-Gly-Ala.

Inhibitory Effects of Indirubin-3'-oxime Derivatives on Lipid Accumulation in 3T3-L1 Cells.

Obesity elevates the risk of cardiovascular disease and has been strongly associated with increases in the incidence of many metabolic diseases. Therefore, prevention of obesity leads to the prevention of metabolic diseases. In light of this, substances that exert anti-obesity effects are crucial for the prevention of obesity. Indirubin, a 3,2' bisindole isomer of indigo, is the active component of the traditional Chinese medicine used for the treatment of chronic myelocytic leukemia. In particular, indirubin-3'-oxime (1) was shown to inhibit the differentiation of adipocytes. In this study, we investigated the inhibitory effects of nine indirubin-3'-oxime derivatives against lipid accumulation during differentiation in 3T3-L1 cells. Among the compounds tested, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) at 5 µM exhibited significantly stronger inhibitory activity than indirubin-3'-oxime (1). Furthermore, 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) markedly suppressed the expression of CCAAT/enhancer-binding protein α, peroxisome proliferator activator γ2, and adipocyte protein 2, both of which are key adipogenic regulators at the intermediate stage of adipocyte differentiation. Our results demonstrate that 5-methoxyindirubin-3'-oxime (2) and 6-bromoindirubin-3'-oxime (7) significantly down-regulated lipid accumulation during differentiation of 3T3-L1 cells, suggesting their potential as novel therapeutic drugs against the development of obesity.

Epigallocatechin gallate targets FTO and inhibits adipogenesis in an mRNA m6A-YTHDF2-dependent manner.

BACKGROUND/OBJECTIVE: N6-methyladenosine (m6A) modification of mRNA plays a role in regulating adipogenesis. However, its underlying mechanism remains largely unknown. Epigallocatechin gallate (EGCG), the most abundant catechin in green tea, plays a critical role in anti-obesity and anti-adipogenesis. METHODS: High-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry (HPLC-QqQ-MS/MS) was performed to determine the m6A levels in 3T3-L1 preadipocytes. The effects of EGCG on the m6A levels in specific genes were determined by methylated RNA immunoprecipitation coupled with quantitative real-time PCR (meRIP-qPCR). Several adipogenesis makers and cell cycle genes were analyzed by quantitative real-time PCR (qPCR) and western blotting. Lipid accumulation was evaluated by oil red O staining. All measurements were performed at least for three times. RESULTS: Here we showed that EGCG inhibited adipogenesis by blocking the mitotic clonal expansion (MCE) at the early stage of adipocyte differentiation. Exposing 3T3-L1 cells to EGCG reduced the expression of fat mass and obesity-associated (FTO) protein, an m6A demethylase, which led to increased overall levels of RNA m6A methylation. Cyclin A2 (CCNA2) and cyclin dependent kinase 2 (CDK2) play vital roles in MCE. The m6A levels of CCNA2 and CDK2 mRNA were dramatically enhanced by EGCG. Interestingly, EGCG increased the expression of YTH N6-methyladenosine RNA binding protein 2 (YTHDF2), which recognized and decayed methylated mRNAs, resulting in decreased protein levels of CCNA2 and CDK2. As a result, MCE was blocked and adipogenesis was inhibited. FTO overexpression and YTHDF2 knockdown in 3T3-L1 cells significantly increased CCNA2 and CDK2 protein levels and ameliorated the EGCG-induced adipogenesis inhibition. Thus, m6A-dependent CCNA2 and CDK2 expressions mediated by FTO and YTHDF2 contributed to EGCG-induced adipogenesis inhibition. CONCLUSION: Our findings provide mechanistic insights into how m6A is involved in the EGCG regulation of adipogenesis and shed light on its anti-obesity effect.

Magnolol promotes thermogenesis and attenuates oxidative stress in 3T3-L1 adipocytes.

OBJECTIVE: The aim of this study was to explore the browning and antioxidative effects of magnolol in 3T3-L1 adipocytes, as recruitment of beige-like adipocytes (browning) by natural compounds is being considered as a promising strategy to fight against obesity. METHODS: Magnolol-induced browning effect was evaluated by determining the expression levels of specific marker genes and proteins using real-time polymerase chain reaction and immunoblotting, respectively. Induction of thermogenesis and suppression of oxidative stress in 3T3-L1 adipocytes were further validated by immunofluorescence. RESULTS: Magnolol significantly enhanced expression of a core set of brown fat-specific marker genes (Ucp1, Cd137, Prdm16, Cidea, and Tbx1) and proteins (UCP1, PRDM16, and PGC-1α). Increased expression of UCP1 and other brown fat-specific markers contributed to the browning of 3T3-L1 adipocytes possibly via activation of the AMPK, PPARγ, and protein kinase A (PKA) pathways. In addition, magnolol up-regulated key fatty acid oxidation and lipolytic markers (CPT1, ACSL1, SIRT1, and PLIN) and down-regulated lipogenic markers (FAS and SREBP1). Magnolol also reduced the production and release of reactive oxygen species. CONCLUSION: The current data suggest possible roles for magnolol in browning of white adipocytes, augmentation of lipolysis, and thermogenesis, as well as repression of oxidative stress and lipogenesis. Thus, magnolol may be explored as a potentially promising therapeutic agent for the prevention of obesity and other metabolic disorders.

Volumetric chemical imaging by stimulated Raman projection microscopy and tomography.

Volumetric imaging allows global understanding of three-dimensional (3D) complex systems. Light-sheet fluorescence microscopy and optical projection tomography have been reported to image 3D volumes with high resolutions and at high speeds. Such methods, however, usually rely on fluorescent labels for chemical targeting, which could perturb the biological functionality in living systems. We demonstrate Bessel-beam-based stimulated Raman projection (SRP) microscopy and tomography for label-free volumetric chemical imaging. Our SRP microscope enables fast quantitation of chemicals in a 3D volume through a two-dimensional lateral scan. Furthermore, combining SRP and sample rotation, we demonstrate the SRP tomography that can reconstruct the 3D distribution of chemical compositions with optical spatial resolution at a higher speed than the Gaussian-beam-based stimulated Raman scattering sectioning imaging can. We explore the potential of our SRP technology by mapping polymer particles in 3D volumes and lipid droplets in adipose cells.

MiR-21a-5p suppresses bisphenol A-induced pre-adipocyte differentiation by targeting map2k3 through MKK3/p38/MAPK.

Childhood obesity is a metabolic disease characterized by accumulation of excessive fat. Bisphenol A (BPA), a potential obesogen compound, possesses an estrogen mimetic activity and endocrine disruption effect. MicroRNA-21a-5p (miR-21a-5p) is reported to regulate the adipogenic differentiation. Our study showed that miR-21a-5p overexpression significantly decreased the red lipid droplets and triglyceride level in BPA-induced 3T3-L1 cells. BPA induced the mRNA and protein expression levels of PPARγ, C/EBPα and adiponectin, and the induction was inhibited by miR-21a-5p mimics transfection. MiR-21a-5p mimics inhibited the GR activity, GR phosphorylation (S220, S21a-5p2, and S234), and the activation of p38/MAPK pathway, which are elevated by BPA treatment in 3T3-L1 cells. MiR-21a-5p overexpression inhibited the protein level of MKK3, but not in the mRNA level. Luciferase activity assay showed that miR-21a-5p directly targeted map2k3 3'-UTR. MKK3 overexpression attenuated the effect of miR-21a-5p mimics transfection on 3T3-L1 differentiation. We also assessed the body weight, fat mass and the content of serum lipid in rats subcutaneous injected with BPA and miR-21a-5p mimics. MiR-21a-5p overexpression attenuated BPA-induced obesity in vivo. These findings suggested that miR-21a-5p inhibited BPA induced adipocyte differentiation by targeting map2k3 through MKK3/p38/MAPK in 3T3-L1 cells, providing a potential therapeutic strategy for BPA induced obesity.

c-Jun regulates adipocyte differentiation via the KLF15-mediated mode.

Abnormal adipocyte differentiation is implicated in the development of metabolic disorders such as obesity and type II diabetes. Thus, an in-depth understanding of the molecular mechanisms associated with adipocyte differentiation is the first step in overcoming obesity and its related metabolic diseases. Here, we examined the role of c-Jun as a transcription factor in adipocyte differentiation. c-Jun overexpression in murine 3T3-L1 preadipocytes significantly inhibited adipocyte differentiation. In addition, the expression level of KLF15, an upstream effector of the key adipogenic factors C/EBPα and PPARγ, was decreased upon the ectopic expression of c-Jun. We found that c-Jun inhibited basal and glucocorticoid receptor (GR)-induced promoter activities of KLF15. c-Jun directly bound near the glucocorticoid response element (GRE) sites in the KLF15 promoter and inhibited adjacent promoter occupancies of GR. Furthermore, the restoration of KLF15 expression in 3T3-L1 cells with the stable ectopic expression of c-Jun partially rescued adipocyte differentiation. Our results demonstrate that c-Jun can suppress adipocyte differentiation through the down-regulation of KLF15 at the transcriptional level. This study proposes a novel mechanism by which c-Jun regulates adipocyte differentiation.

Differentiation of 3T3-L1 preadipocytes is inhibited under a modified ceiling culture.

Ceiling culture is an inverted and closed cell culture system which represents a novel method for exploring adipocyte characteristics and function. Although the role of ceiling culture in mature adipocyte dedifferentiation has been extensively studied, its potential effects on preadipocyte differentiation remain unclear. In this study, we established a simplified dish ceiling culture method for 3T3-L1 preadipocytes and showed that our novel ceiling culture method could reproduce the function of the traditional flask ceiling culture. Then, we investigated the effects of ceiling culture on 3T3-L1 preadipocyte differentiation by Oil red O staining and RT-qPCR. The results showed that ceiling culture significantly impaired triglyceride accumulation and adipogenic marker genes expression in 3T3-L1 preadipocytes. These findings suggest that ceiling culture inhibited 3T3-L1 preadipocyte differentiation while inducing mature adipocytes dedifferentiation. Taken together, our data facilitate the understanding of the property of ceiling culture and promote the study of revealing the underlying mechanisms of mature adipocytes dedifferenatiation.

Aculeatin, a coumarin derived from Toddalia asiatica (L.) Lam., enhances differentiation and lipolysis of 3T3-L1 adipocytes.

Toddalia asiatica (L.) Lam. (T. asiatica) has been utilized traditionally for medicinal purposes such as the treatment of diabetes. Currently, the extract is considered to be a good source of anti-diabetic agents, but the active compounds have yet to be identified. In this study, we investigated the effects of fractionated T. asiatica extracts on the differentiation of 3T3-L1 preadipocytes and identified aculeatin as a potential active agent. When 3T3-L1 preadipocytes were treated with aculeatin isolated from T. asiatica in the presence of insulin, aculeatin increased cellular triglyceride levels and glycerol-3-phosphate dehydrogenase activity. This indicated that aculeatin could enhance the differentiation of preadipocytes into adipocytes. Further analyses using a DNA microarray and real-time quantitative reverse-transcription PCR showed an increase in the expression of peroxisome proliferator-activated receptor-γ target genes (Pparg, Ap2, Cd36, Glut4 and Adipoq) by aculeatin, suggesting that aculeatin enhances the differentiation of 3T3-L1 cells by modulating the expression of genes critical for adipogenesis. Interestingly, after treatment of differentiated adipocytes with aculeatin, glucose uptake and lipolysis were enhanced. Overall, our results suggested that aculeatin is an active compound in T. asiatica for enhancing both differentiation and lipolysis of adipocytes, which are useful for the treatment of lipid abnormalities as well as diabetes.

Role of p53 in preadipocyte differentiation.

Insulin resistance, diabetes and many kinds of cancers are common in overweight and obese individuals. The tumor suppressor p53 is important in securing genetic stability, but its role in the regulation of metabolic processes and cell differentiation remains unclear. We have investigated the role of p53 in adipocyte differentiation. Using 3T3-L1 cells, a mouse embryonic fibroblast preadipocyte model and DIO rat model, p53 expression and function during adipocyte differentiation were investigated. p53 expression increased on the second and fourth day of adipocyte differentiation and decreased thereafter. Its overexpression in 3T3-L1 preadipocytes markedly reduced adipogenesis and marker gene expression. p53 activity was weakened in DIO rat abdominal adipose tissue because of an decreased expression of its activated phosphorylated form. In contrast, p53 knockout enhanced adipogenesis and the expression of marker genes, but significantly reduced insulin-stimulated Akt phosphorylation. These results indicate that p53 partly suppresses preadipocyte differentiation and adipogenesis by regulating adipocyte gene expression and Akt signaling.

Upregulation of the thioredoxin-dependent redox system during differentiation of 3T3-L1 cells to adipocytes.

Hydrogen peroxide acts as a signaling molecule in early adipogenesis. In differentiating adipocytes, elevated hydrogen peroxide generation is balanced through induction of antioxidant enzymes such as catalase and peroxiredoxins. Thioredoxin reductases (TrxR) and glutathione peroxidases (GPx) are selenoenzymes that constitute part of the major thiol-dependent antioxidant systems in cells. Here we show that the protein levels of cytoplasmic/nuclear TrxR1 and mitochondrial TrxR2 increase in the course of adipocyte differentiation of 3T3-L1 cells together with the TrxR2 substrate thioredoxin 2 (Trx2), resulting in elevated TrxR activity in mature adipocytes. Gene and protein expression of the GPx isoenzyme GPx4 was also stimulated during adipogenesis. Chronic exposure of 3T3-L1 cells to the anti-adipogenic factors tumor necrosis factor α (TNF-α) or rapamycin during differentiation suppressed TrxR1 and Trx2 upregulation, concomitantly with inhibition of adipogenesis and lipogenesis. In contrast, TNF-α or rapamycin did not affect expression of TrxRs and their Trx substrates in mature adipocytes. These results indicate that upregulation of the thioredoxin-dependent redox system is linked to the development of an adipocyte phenotype.

Effects of uremic toxin p-cresol on proliferation, apoptosis, differentiation, and glucose uptake in 3T3-L1 cells.

Malnutrition is a common feature seen in chronic dialysis patients, and the survival rate of obese patients receiving such treatment is higher than that of lean patients. Irrespective of obesity or diabetes, dialysis patients commonly have insulin resistance, and the leading cause of death is cardiovascular (CV) disease. It has been reported that the concentration of p-cresol, a uremic toxin, is highly associated with CV events. As uremic toxin levels are high in dialysis patients, they may be involved in the pathogenesis of insulin resistance and CV disease in this population. However, little is known so far. Thus, we focused on this uremic toxin to examine its effects on adipocytes and their precursors. 3T3-L1 cells, a mouse preadipocyte cell line, were cultured until 90% confluency. The cells were then differentiated with 500 µM 3-isobutyl-methylxanthine, 250 nM dexamethasone, and 10 µg/mL insulin. Cell proliferation was evaluated by cell counting and bromodeoxyuridine (Brd-U) incorporation assay. Glucose uptake was estimated using radiolabeled 2-deoxyglucose. The range of concentrations of p-cresol used in the experiments was from 2 to 200 µM. The investigation of cell proliferation by cell counting revealed that, compared with control, 3T3-L1 cells treated with 100 and 200 µM p-cresol were significantly decreased in number at day 3 and day 7 of culture. The Brd-U incorporation assay also demonstrated similar inhibitory effects on cell proliferation, suggesting that p-cresol affected the normal cell cycle. Oil Red O staining at day 7 showed that the number of mature adipocytes was decreased by treatment with 200 µM p-cresol. Consistent with that finding, the number of apoptotic cells at day 7 was increased by treatment with 100 and 200 µM p-cresol. Peroxisome proliferator-activated receptor γ (PPARγ) mRNA expression increased time-dependently during the differentiation process of 3T3-L1 cells. p-Cresol dose-dependently decreased differentiation-induced mRNA expression of PPARγ. Uptake of 3H-labeled 2-deoxyglucose was markedly decreased by 200 µM p-cresol in the presence or in the absence of insulin, mainly because of the decreased number of mature adipocytes. High concentrations of p-cresol disturbed the cell cycle, induced apoptosis, inhibited the differentiation of preadipocytes into mature adipocytes, and decreased glucose uptake at baseline and after insulin stimulation. These findings indicate that accumulated p-cresol may induce reduction in adipose tissue, insulin resistance, and malnutrition, eventually leading to poor outcomes in chronic dialysis patients.

Adipocyte differentiation is affected by media height above the cell layer.

BACKGROUND: 3T3-L1 cells have been widely used as a model for adipogenesis. However, despite its popularity, differentiation of this cell line has been reported to be inconsistent with low efficiency. OBJECTIVE: To investigate the effect of media height during adipocyte differentiation on lipid accumulation and adipokine secretion in mature adipocytes. METHODS: Three cell lines (3T3-L1, OP9 and ChubS7) were used to test the influence of media volume on adipogenesis. Total lipid content and lipid droplet size and number were quantified. Adipocyte related gene expressions were quantified during the course of differentiation. Secretion of leptin and adiponectin from mature adipocytes were measured using enzyme-linked immunosorbent assays. The influence of oxygen partial pressure on adipogenesis was investigated using three oxygen percentages: 5, 21 and 30%. Insulin sensitivity was measured by insulin inhibition of isoproterenol-induced lipolysis and phosphorylation of insulin receptor substrate-1. RESULTS: A lower media height during adipogenesis increased total lipid accumulation, NEFA release and leptin and adiponectin secretion in mature adipocytes. Insulin sensitivity was not affected by media height during differentiation. CONCLUSION: Media height during adipogenesis was inversely correlated with lipid content in mature adipocytes. To achieve a high lipid content and greater adipokine secretion, it is best to use a low media volume during differentiation.

The use of Transwells™ improves the rates of differentiation and growth of cultured 3T3L1 cells.

The use of Transwells™ for routine cultures of 3T3L1 cells results in a much improved rate of differentiation of fibroblasts to adipocytes (100% in 9 of 10 tests) compared with bottom-well layer cultures. Mean size of cells was not different, but the cell number and overall cell mass was 3× larger in transwell in spite of a smaller surface area. The difference between both models was the accessibility in transwells of both sides of the cells to the medium (and oxygen). Cells were counted, and their size estimated using a handheld cell counter, Scepter™, designed for blood cells, but adjusted to the larger size of adipocytes. Finally, the effect of nitric oxide was tested using spermineNONOate, a nitric oxide (NO·) donor. The product was released to cultures at a constant 1 µl/h rate for up to 3 days using osmotic Alzet™ minipumps held in wells with water and discharging their contents to the cultured cell-laden wells through a short capillary tube. A rate of 0.3 pmol/min/ml of medium did not affect the cells' size, but 0.4 pmol/min/ml significantly increased cell mass. The methodological improvements presented here allow for more uniform cultured cell yields and a more flexible environment for control of cell size and administration of signaling agents.

Increased cGMP promotes healthy expansion and browning of white adipose tissue.

With more than half a billion individuals affected worldwide, obesity has reached pandemic proportions. Development of "brown-like" or "brite" adipocytes within white adipose tissue (WAT) has potential antiobesity and insulin-sensitizing effects. We investigated the role of cyclic GMP (cGMP) signaling, focusing on cGMP-dependent protein kinase I (PKGI) in WAT. PKGI is expressed in murine WAT, primary adipocytes, and 3T3-L1. Treatment of adipocytes with cGMP resulted in increased adipogenesis, with a 54% increase in expression of peroxisome proliferator-activated receptor-γ. Lentiviral overexpression of PKGI further increased adipogenesis, whereas loss of PKGI significantly reduced adipogenic differentiation. In addition to adipogenic effects, PKGI had an antihypertrophic and anti-inflammatory effect via RhoA phosphorylation and reduction of proinflammatory adipokine expression. Moreover, PKGI induced a 4.3-fold increase in abundance of UCP-1 and the development of a brown-like thermogenic program in primary adipocytes. Notably, treatment of C57BL/6 mice with phosphodiesterase inhibitor sildenafil (12 mg/kg/d) for 7 d caused 4.6-fold increase in uncoupling protein-1 expression and promoted establishment of a brown fat cell-like phenotype ("browning") of WAT in vivo. Taken together, PKGI is a key regulator of cell size, adipokine secretion and browning of white fat depots and thus could be a valuable target in developing novel treatments for obesity.

Clk/STY (cdc2-like kinase 1) and Akt regulate alternative splicing and adipogenesis in 3T3-L1 pre-adipocytes.

The development of adipocytes from their progenitor cells requires the action of growth factors signaling to transcription factors to induce the expression of adipogenic proteins leading to the accumulation of lipid droplets, induction of glucose transport, and secretion of adipokines signaling metabolic events throughout the body. Murine 3T3-L1 pre-adipocytes sequentially express all the proteins necessary to become mature adipocytes throughout an 8-10 day process initiated by a cocktail of hormones. We examined the role of Clk/STY or Clk1, a cdc2-like kinase, in adipogenesis since it is known to be regulated by Akt, a pivotal kinase in development. Inhibition of Clk1 by a specific inhibitor, TG003, blocked alternative splicing of PKCßII and expression of PPARγ1 and PPARγ2. SiRNA depletion of Clk1 resulted in early expression of PKCßII and sustained PKCßI expression. Since Clk1 is a preferred Akt substrate, required for phosphorylation of splicing factors, mutation of Clk1 Akt phosphorylation sites was undertaken. Akt sites on Clk1 are in the serine/arginine-rich domain and not the kinase domain. Mutation of single and multiple sites resulted in dysregulation of PKCßII, PKCßI, and PPARγ1&2 expression. Additionally, adipogenesis was blocked as assessed by Oil Red O staining, adiponectin, and Glut1 and 4 expression. Immunofluorescence microscopy revealed that Clk1 triple mutant cDNA, transfected into pre-adipocytes, resulted in excluding SRp40 (SFSR6) from co-localizing to the nucleus with PFS, a perispeckle specific protein. This study demonstrates the role of Akt and Clk1 kinases in the early differentiation of 3T3-L1 cells to adipocytes.

Protocol for effective differentiation of 3T3-L1 cells to adipocytes.

In this note, we present a detailed procedure for highly effective and reproducible 3T3-L1 cell differentiation. Due to their potential to differentiate from fibroblasts to adipocytes, 3T3-L1 cells are widely used for studying adipogenesis and the biochemistry of adipocytes. However, using different kits and protocols published so far, we were not able to obtain full differentiation of the currently available American Type Culture Collection (ATCC) 3T3-L1 cell lots. Using rosiglitazone (2 µM) as an additional prodifferentiative agent, we achieved apparently complete differentiation of 3T3-L1 cells within 10 to 12 days that persisted for at least up to cell culture passage 10.

The ubiquitin ligase Siah2 regulates PPARγ activity in adipocytes.

Moderate reductions in peroxisome proliferator-activated receptor (PPAR)γ levels control insulin sensitivity as effectively as activation of PPARγ in adipocytes by the thiazolidinediones. That observation suggests that PPARγ activity can be regulated by modulating the amount of PPARγ protein in adipocytes. Activation of PPARγ in adipocytes is linked to changes in PPARγ protein levels via increased degradation of PPARγ proteins by the ubiquitin proteasome system. Identification of the ubiquitin ligase or ligases that recognize ligand bound PPARγ is an essential step in determining the physiological significance of the relationship between activation and ubiquitin-dependent degradation of PPARγ. Using an RNA interference-based screen, we identified five RING (really interesting new gene)-type ubiquitin ligases that alter PPARγ protein levels in adipocytes. Here, we demonstrate that Drosophila seven-in-absentia homolog 2 (Siah2), a mammalian homolog of Drosophila seven-in-absentia, regulates PPARγ ubiquitylation and ligand-dependent activation of PPARγ in adipocytes. We also demonstrate that Siah2 expression is up-regulated during adipogenesis and that PPARγ interacts with Siah2 during adipogenesis. In addition, Siah2 is required for adipogenesis. These data suggest that modulation of PPARγ protein levels by the ubiquitin ligase Siah2 is essential in determining the physiological effects of PPARγ activation in adipocytes.

Static mechanical stretching accelerates lipid production in 3T3-L1 adipocytes by activating the MEK signaling pathway.

Understanding mechanotransduction in adipocytes is important for research of obesity and related diseases. We cultured 3T3-L1 preadipocytes on elastic substrata and applied static tensile strains of 12% to the substrata while inducing differentiation. Using an image processing method, we monitored lipid production for a period of 3-4 wk. The ratio of %-lipid area per field of view (FOV) in the stretched over nonstretched cultures was significantly greater than unity (P < 0.05), reaching ∼1.8 on average starting from experimental day ∼10. The superior coverage of the FOV by lipids in the stretched cultures was due to significantly greater sizes of lipid droplets (LDs) with respect to nonstretched cultures, starting from experimental day ∼10 (P < 0.05), and due to significantly more LDs per cell between days ∼10 and ∼17 (P < 0.05). The statically stretched cells also differentiated significantly faster than the nonstretched cells within the first ∼10 days (P < 0.05). Adding peroxisome proliferator-activated receptor-γ (PPARγ) antagonist did not change these trends, as the %-lipid area per FOV in the stretched cultures that received this treatment was still significantly greater than in the nonstretched cultures without the PPARγ antagonist (14.44 ± 1.96% vs. 10.21 ± 3%; P < 0.05). Hence, the accelerated adipogenesis in the stretched cultures was not mediated through PPARγ. Nonetheless, inhibiting the MEK/MAPK signaling pathway reduced the extent of adipogenesis in the stretched cultures (13.53 ± 5.63%), bringing it to the baseline level of the nonstretched cultures without the MEK inhibitor (10.21 ± 3.07%). Our results hence demonstrate that differentiation of adipocytes can be enhanced by sustained stretching, which activates the MEK signaling pathway.

Functional ion channels and cell proliferation in 3T3-L1 preadipocytes.

Mouse 3T3-L1 preadipocytes are widely used for metabolic study of obesity; however, their cellular physiology is not fully understood. The present study investigates functional ion channels and their role in the regulation of cell proliferation using whole-cell patch voltage-clamp, RT-PCR, Western blot, and cell proliferation assay in undifferentiated 3T3-L1 preadipocytes. We found three types of ionic currents present in 3T3-L1 preadipocytes, including an inwardly-rectifying K(+) current (I(Kir), recorded in 15% of cells) inhibited by Ba(2+), a Ca(2+)-activated intermediate K(+) current (IK(Ca), recorded in 44% of cells) inhibited by clotrimazole (or TRAM-34) as well as a chloride current (I(Cl)) inhibited by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS) in 12% of cells, which can be activated in all cells with hypotonic (0.8 T) insult, implicating a volume-sensitive I(Cl) (I(Cl.vol)). RT-PCR and Western blot analysis revealed the expression of KCa3.1 (for IK(Ca)), Kir2.1 (for I(Kir)), and Clcn3 (for I(Cl.vol)). Blockade of IK(Ca) with TRAM-34 or I(Cl.vol) with DIDS inhibited cell proliferation in a concentration-dependent manner. Knockdown of KCa3.1 or Clcn3 with specific siRNAs also suppressed cell proliferation. Flow cytometry analysis showed that blockade or silencing of KCa3.1 or Clcn3 channels with corresponding blockers or siRNAs caused an accumulation of cells at the G0/G1 phase. These results demonstrate that three functional ion channel currents, I(KCa), I(Cl.vol), and I(Kir), are heterogeneously present in 3T3-L1 preadipocytes. I(KCa) and I(Cl.vol) participate in the regulation of cell proliferation.