ANKRD26 and Its Interacting Partners TRIO, GPS2, HMMR and DIPA Regulate Adipogenesis in 3T3-L1 Cells

Partial inactivation of the Ankyrin repeat domain 26 (Ankrd26) gene causes obesity and diabetes in mice and increases spontaneous and induced adipogenesis in mouse embryonic fibroblasts. However, it is not yet known how the Ankrd26 protein carries out its biological functions. We identified by yeast two-hybrid and immunoprecipitation assays the triple functional domain protein (TRIO), the G protein pathway suppressor 2 (GPS2), the delta-interacting protein A (DIPA) and the hyaluronan-mediated motility receptor (HMMR) as ANKRD26 interacting partners. Adipogenesis of 3T3-L1 cells was increased by selective down-regulation of Ankrd26, Trio, Gps2, Hmmr and Dipa. Furthermore, GPS2 and DIPA, which are normally located in the nucleus, were translocated to the cytoplasm, when the C-terminus of ANKRD26 was introduced into these cells. These findings provide biochemical evidence that ANKRD26, TRIO, GPS2 and HMMR are novel and important regulators of adipogenisis and identify new targets for the modulation of adipogenesis

Citrus aurantium L. Dry Extracts Ameliorate Adipocyte Differentiation of 3T3-L1 Cells Exposed to TNFα by Down-Regulating miR-155 Expression

Citrus aurantium L. dry extracts (CAde) improve adipogenesis in vitro. These effects are dependent from an early modulation of CCAAT/enhancer-binding protein beta (C/Ebpβ) expression and cyclic Adenosine Monophosphate (cAMP) response element-binding protein (CREB) activation. C/Ebpβ and Creb are also targets of miR-155. This study investigated whether CAde regulates miR-155 expression in the early stages of adipogenesis and whether it ameliorates adipocyte differentiation of cells exposed to tumor necrosis factor-alpha (TNFα). Adipogenic stimuli (AS) were performed in 3T3-L1 pre-adipocytes treated with CAde, TNFα, or both. Gene and miRNA expression were determined by quantitative real-time PCR. Adipogenesis was evaluated by Oil-Red O staining. CAde treatment enhanced AS effects during the early adipogenesis phases by further down-regulating miR-155 expression and increasing both C/Ebpβ and Creb mRNA and protein levels. At variance, TNFα inhibited 3T3-L1 adipogenesis and abolished AS effects on miR-155, C/Ebpβ, and Creb expression. However, in cells exposed to TNFα, CAde improved adipocyte differentiation and restored the AS effects on miRNA and gene expression at early time points. In conclusion, this study identified miR-155 down-regulation as part of the mechanism through which CAde enhances adipogenesis of pre-adipocytes in vitro. Furthermore, it provides evidence of CAde efficacy against TNFα negative effects on adipogenesis

shRNA knockdown of <i>Ankrd26</i>, <i>Trio</i>, <i>Gps2, HMMR</i> and <i>Dipa</i> induces adipocyte differentiation in 3T3-L1 cells.

<p>To down-regulate <i>Ankrd26</i>, <i>Trio</i>, <i>Gps2, HMMR</i> and <i>Dipa</i> expression cells were transfected with specific shRNA for <i>Ankrd26</i> (Ankrd-sh), <i>Trio</i> (Trio-sh), <i>Gps2</i> (Gps2-sh), <i>HMMR</i> (HMMR-sh), <i>Dipa</i> (Dipa-sh) or with non-targeting control shRNA (Co-sh). <b>A</b>) <i>Ankrd26</i> mRNA expression determined by Real-Time Quantitative PCR analysis of total RNA isolated from 3T3-L1 cells transfected with Ankrd-sh or Co-sh. mRNA levels in Ankrd-sh treated cells are relative expression units to those in control (Co-sh; mean ± SD; n = 3). **<i>p</i><0.01. Macroscopic images (<b>B</b>) and lipid quantification (<b>C</b>) in Ankrd-sh and Co-sh transfected cells stained with oil-red-O upon differentiation. Bars are expressed as means ± SEM of oil-red-O values measured at 490 nm. Ankrd-sh vs. Co-sh, **<i>p</i><0.01. mRNA expression (<b>D, G, J</b> and <b>M</b>), macroscopic images (<b>E, H, K</b> and <b>N</b>) and lipid quantification (<b>F, I, L</b> and <b>O</b>) of 3T3-L1 cells transduced with Trio-sh (<b>D–F</b>), Gps2-sh (<b>G–I</b>), HMMR-sh (<b>J–L</b>) and Dipa-sh (<b>M–O</b>) were evaluated as <b>A</b>, <b>B</b> and <b>C</b>, respectively.</p

Real Time Quantitative PCR primer sequences and primer efficiencies.

<p>Real Time Quantitative PCR primer sequences and primer efficiencies.</p

ANKRD26 yeast two-hybrid strategy.

<p>The position of ANKRD26's ankyrin and spectrin coiled-coil repeats are depicted on a schematic diagram of the full-length, 1710 amino acid, protein. The line below indicates the relative locations of ANKRD26 bait clones used in the <i>GAL4-</i>based yeast two-hybrid cDNA library screens. DIPA interacts with baits 7, 8, 9 and 17; GPS2 interacts with bait 17; HMMR interacts with baits 9 and 17; and TRIO interacts with bait 10.</p

Analysis of <i>Ankrd26</i>, <i>Trio</i>, <i>Gps2, HMMR</i> and <i>Dipa</i> RNA by rq-pcr during adipogenesis induction.

<p>3T3-L1 cells plated 6-well plates (1.8×10⁵/well) and harvested at various time points as: day 1 (pre-confluence), day 2 (confluence), day 4 (2 days after confluence), day 5 (IDM induction day 1), day 6 (IDM induction day 2), day 8 (IDM induction day 4), day12 (IDM induction day 8). RNA made by Trizol regent and subjected to Real Time Quantitative PCR using SYBR method. Bar represents triple for each sample.</p

Adipocyte precursor cells from first degree relatives of type 2 diabetic patients feature changes in hsa-mir-23a-5p, -193a-5p, and -193b-5p and insulin-like growth factor 2 expression

First-degree relatives (FDRs) of type 2 diabetics (T2D) feature dysfunction of subcutaneous adipose tissue (SAT) long before T2D onset. miRNAs have a role in adipocyte precursor cells (APC) differentiation and in adipocyte identity. Thus, impaired miRNA expression may contribute to SAT dysfunction in FDRs. In the present work, we have explored changes in miRNA expression associated with T2D family history which may affect gene expression in SAT APCs from FDRs. Small RNA-seq was performed in APCs from healthy FDRs and matched controls and omics data were validated by qPCR. Integrative analyses of APC miRNome and transcriptome from FDRs revealed down-regulated hsa-miR-23a-5p, -193a-5p and -193b-5p accompanied by up-regulated Insulin-like Growth Factor 2 (IGF2) gene which proved to be their direct target. The expression changes in these marks were associated with SAT adipocyte hypertrophy in FDRs. APCs from FDRs further demonstrated reduced capability to differentiate into adipocytes. Treatment with IGF2 protein decreased APC adipogenesis, while over-expression of hsa-miR-23a-5p, -193a-5p and -193b-5p enhanced adipogenesis by IGF2 targeting. Indeed, IGF2 increased the Wnt Family Member 10B gene expression in APCs. Down-regulation of the three miRNAs and IGF2 up-regulation was also observed in Peripheral Blood Leukocytes (PBLs) from FDRs. In conclusion, APCs from FDRs feature a specific miRNA/gene profile, which associates with SAT adipocyte hypertrophy and appears to contribute to impaired adipogenesis. PBL detection of this profile may help in identifying adipocyte hypertrophy in individuals at high risk of T2D

Altered H3K4me3 profile at the TFAM promoter causes mitochondrial alterations in preadipocytes from first-degree relatives of type 2 diabetics

Abstract Background First-degree relatives of type 2 diabetics (FDR) exhibit a high risk of developing type 2 diabetes (T2D) and feature subcutaneous adipocyte hypertrophy, independent of obesity. In FDR, adipose cell abnormalities contribute to early insulin-resistance and are determined by adipocyte precursor cells (APCs) early senescence and impaired recruitment into the adipogenic pathway. Epigenetic mechanisms signal adipocyte differentiation, leading us to hypothesize that abnormal epigenetic modifications cause adipocyte dysfunction and enhance T2D risk. To test this hypothesis, we examined the genome-wide histone profile in APCs from the subcutaneous adipose tissue of healthy FDR. Results Sequencing-data analysis revealed 2644 regions differentially enriched in lysine 4 tri-methylated H3-histone (H3K4me3) in FDR compared to controls (CTRL) with significant enrichment in mitochondrial-related genes. These included TFAM, which regulates mitochondrial DNA (mtDNA) content and stability. In FDR APCs, a significant reduction in H3K4me3 abundance at the TFAM promoter was accompanied by a reduction in TFAM mRNA and protein levels. FDR APCs also exhibited reduced mtDNA content and mitochondrial-genome transcription. In parallel, FDR APCs exhibited impaired differentiation and TFAM induction during adipogenesis. In CTRL APCs, TFAM-siRNA reduced mtDNA content, mitochondrial transcription and adipocyte differentiation in parallel with upregulation of the CDKN1A and ZMAT3 senescence genes. Furthermore, TFAM-siRNA significantly expanded hydrogen peroxide (H2O2)-induced senescence, while H2O2 did not affect TFAM expression. Conclusions Histone modifications regulate APCs ability to differentiate in mature cells, at least in part by modulating TFAM expression and affecting mitochondrial function. Reduced H3K4me3 enrichment at the TFAM promoter renders human APCs senescent and dysfunctional, increasing T2D risk. Graphical abstrac

Specific CpG hyper-methylation leads to Ankrd26 gene down-regulation in white adipose tissue of a mouse model of diet-induced obesity

Epigenetic modifications alter transcriptional activity and contribute to the effects of environment on the individual risk of obesity and Type 2 Diabetes (T2D). Here, we have estimated the in vivo effect of a fat-enriched diet (HFD) on the expression and the epigenetic regulation of the Ankyrin repeat domain 26 (Ankrd26) gene, which is associated with the onset of these disorders. In visceral adipose tissue (VAT), HFD exposure determined a specific hyper-methylation of Ankrd26 promoter at the â '436 and â '431 bp CpG sites (CpGs) and impaired its expression. Methylation of these 2 CpGs impaired binding of the histone acetyltransferase/transcriptional coactivator p300 to this same region, causing hypo-acetylation of histone H4 at the Ankrd26 promoter and loss of binding of RNA Pol II at the Ankrd26 Transcription Start Site (TSS). In addition, HFD increased binding of DNA methyl-transferases (DNMTs) 3a and 3b and methyl-CpG-binding domain protein 2 (MBD2) to the Ankrd26 promoter. More importantly, Ankrd26 down-regulation enhanced secretion of pro-inflammatory mediators by 3T3-L1 adipocytes as well as in human sera. Thus, in mice, the exposure to HFD induces epigenetic silencing of the Ankrd26 gene, which contributes to the adipose tissue inflammatory secretion profile induced by high-fat regimens