8 research outputs found

    The Demethylase Activity of FTO (Fat Mass and Obesity Associated Protein) Is Required for Preadipocyte Differentiation

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    <div><p>FTO (fat mass and obesity associated gene) was genetically identified to be associated with body mass index (BMI), presumably through functional regulation of energy homeostasis. However, the cellular and molecular mechanisms by which FTO functions remain largely unknown. Using 3T3-L1 preadipocyte as a model to study the role of FTO in adipogenesis, we demonstrated that FTO is functionally required for 3T3-L1 differentiation. FTO knock-down with siRNA inhibited preadipocyte differentiation, whereas ectopic over-expression of FTO enhanced the process. The demethylase activity of FTO is required for differentiation. Level of N<sup>6</sup>-methyladenosine (m<sup>6</sup>A) is decreased in cells over-expressing FTO. In contrast, overexpression of R96Q, a FTO missense mutant lack of demethylase activity, had no effect on cellular m<sup>6</sup>A level and impeded differentiation. Treatment with Rosiglitazone, a PPARγ agonist, could overcome the differentiation inhibition imposed by R96Q mutant, suggesting the effect of FTO is mediated through PPARγ.</p></div

    Confirmation of microarray findings with RT-qPCR.

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    <p>(A) Three genes from the focal adhesion, cytoskeleton and ECM gene set (Lamc1, Dock1 and Thbs2) were selected and confirmed to be decreased 6hr following adipogenic induction in FTO knock-down cells. (B) Three PPARγ target genes (CD36, Fabp5 and Adiponectin) were selected and verified to be down-regulated 3 days following adipogenic induction in siFTO transfected cells. All gene expression data are represented as mean ± SD of triplicates. * stands for p<0.05, ** stands for p<0.01 and *** stands for p<0.001in Student’s t-test (siFTO versus siCtrl).</p

    The demethylase activity of FTO is required during 3T3-L1 differentiation.

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    <p>Two lines of 3T3-L1 stably expressing wt FTO (hFTO-3 and hFTO-4) or R96Q (R96Q-2 and R96Q-4) were generated. (A) The protein expression level was confirmed by Western-blot using FTO and Myc antibodies, with β-actin as a loading control. (B) The sub-cellular localization of hFTO-4 or R96Q-4 was determined with confocal fluorescence imaging. Scale bars of 10μm are shown in the bottom row of images. (C) 3T3-L1differentiation was enhanced by over-expression of wt FTO and inhibited by R96Q. Level of differentiation was measured by extent of Oil Red-O staining at day 6 post differentiation induction (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0133788#sec002" target="_blank">materials and methods</a>). Demethylase activities of wt hFTO-4 and R96Q-4 in 3T3-L1 cells were determined by dot blotting (D) and quantified by Grayscale analysis with ImageJ software (E). Data are represented as means± SD of four replicates. * stands for p<0.05 and** stands for p<0.01in Student’s t-test.</p

    Rosiglitazone rescued adipogenesis inhibition in 3T3-L1 cells expressing R96Q.

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    <p>(A) Oil Red-O staining of 3T3-L1 cell lines treated with MDI (top and middle row), or MDI plus Rosiglitazone (bottom row). Scale bars are 500μm in 40x images and 50μm in 400x images respectively (left column). (B) Level of differentiation in 3T3-L1 lines expressing wt hFTO-4 or R96Q-4, measured by the extent of Oil Red-O staining. Expression levels of five PPARγ target genes (aP-2, Fabp5, Adiponectin, Perilipin and CD36) were evaluated in 3T3-L1 over expressing wt hFTO-4 (C) or R96Q-4(D) at day 3 post differentiation induction. All gene expression data are represented as mean ± SD of triplicates.* stands for p<0.05, ** stands for p<0.01 and *** stands for p<0.001 in Student’s t-test (Rosig versus DMSO)</p

    FTO knockdown inhibits differentiation of 3T3-L1 preadipocyte.

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    <p>(A) The mRNA levels of FTO, PPAR-γ, and β-actin at different time point during3T3-L1 differentiation was analyzed with RT-qPCR. The relative mRNA levels were determined by the ratio to that of PPAR-γ at time 0.The level of FTO mRNA (B) and protein (C) in cells treated with siRNAs and controls were assessed with RT-qPCR and Western Blot. (D) 3T3-L1 preadipocyte differentiation was inhibited by FTO knock-down. The extent of differentiation was assessed by Oil Red-O staining of intracellular triglyceride and measuring the extract’s absorbance at 540 nm. (E) Expression levels of PPAR-γ in 3T3-L1 cells and those transfected with control or FTO siRNAs at different time point during differentiation. (F) Expression levels of C/EBP-α in 3T3-L1 cells and those transfected with control or FTO siRNAs at different time point during differentiation. All gene expression data are represented as mean ± SD of triplicates.* stands for p<0.05 and ** stands for p<0.01 in Student’s t-test (siFTO vs. siCtrl).</p

    Actin Microfilament Mediates Osteoblast Cbfa1 Responsiveness to BMP2 under Simulated Microgravity

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    <div><p>Microgravity decreases osteoblastic activity, induces actin microfilament disruption and inhibits the responsiveness of osteoblast to cytokines, but the mechanisms remains enigmatic. The F-actin cytoskeleton has previously been implicated in manifold changes of cell shape, function and signaling observed under microgravity. Here we investigate the involvement of microfilament in mediating the effects of microgravity and BMP2 induction on Cbfa1 activity. For this purpose we constructed a fluorescent reporter cell line (OSE-MG63) of Cbfa1 activity by stably transfecting MG63 cells with a reporter consisting of six tandem copies of OSE2 and a minimal mOG2 promoter upstream of enhanced green fluorescent protein (EGFP). The fluorescence intensity of OSE-MG63 showed responsiveness to bone-related cytokines (IGF-I, vitamin D3 and BMP2) and presented an accordant tendency with alkaline phosphatase (ALP) activity. Using OSE-MG63 reporter fluorescence, we performed a semi-quantitative analysis of Cbfa1 activity after treatment with simulated microgravity, microfilament-disrupting agent (cytochalasin B, CB), microfilament-stabilizing agent (Jasplakinolide, JAS) or any combination thereof. In parallel, ALP activity, DNA binding activity of Cbfa1 to OSE2 (ChIP), F-actin structure (immunofluorescence) and EGFP mRNA expression (RT-qPCR) were analyzed. Simulated microgravity inhibited Cbfa1 activity, affected the responsiveness of Cbfa1 to cytokine BMP2, and caused a thinning and dispersed distribution of microfilament. Under normal gravity, CB significantly attenuated BMP2 induction to Cbfa1 activity as well as DNA binding activity of Cbfa1 to OSE2. The addition of JAS reversed the inhibitory effects of microgravity on the responsiveness of Cbfa1 to BMP2. Our study demonstrates that disrupting the microfilament organization by CB or simulated microgravity attenuates the responsiveness of Cbfa1 to BMP2. A stabilization of the microfilament organization by JAS reverses this inhibition. Taken together, these results suggest that actin microfilament participates in BMP2’s induction to Cbfa1 activity and that their disruption might be an important contributor to microgravity’s inhibition on BMP2’s osteogenic induction.</p> </div

    Identification of A Novel Small-Molecule Binding Site of the Fat Mass and Obesity Associated Protein (FTO)

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    <i>N</i>-(5-Chloro-2,4-dihydroxyphenyl)-1-phenylcyclobutanecarboxamide (<i>N</i>-CDPCB, <b>1a</b>) is found to be an inhibitor of the fat mass and obesity associated protein (FTO). The crystal structure of human FTO with <b>1a</b> reveals a novel binding site for the FTO inhibitor and defines the molecular basis for recognition by FTO of the inhibitor. The identification of the new binding site offers new opportunities for further development of selective and potent inhibitors of FTO, which is expected to provide information concerning novel therapeutic targets for treatment of obesity or obesity-associated diseases

    Identification of A Novel Small-Molecule Binding Site of the Fat Mass and Obesity Associated Protein (FTO)

    No full text
    <i>N</i>-(5-Chloro-2,4-dihydroxyphenyl)-1-phenylcyclobutanecarboxamide (<i>N</i>-CDPCB, <b>1a</b>) is found to be an inhibitor of the fat mass and obesity associated protein (FTO). The crystal structure of human FTO with <b>1a</b> reveals a novel binding site for the FTO inhibitor and defines the molecular basis for recognition by FTO of the inhibitor. The identification of the new binding site offers new opportunities for further development of selective and potent inhibitors of FTO, which is expected to provide information concerning novel therapeutic targets for treatment of obesity or obesity-associated diseases
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