To understand the roles of thyroid hormone receptors (TRs) in adipogenesis, we adopted a loss-of-function approach. We generated 3T3-L1 cells stably expressing either TRα1 mutant (TRα1PV) or TRβ1 mutant (TRβ1PV). TRα1PV and TRβ1PV are dominant negative mutations with a frameshift in the C-terminal amino acids. In control cells, the thyroid hormone, tri-iodothyronine (T3), induced a 2.5-fold increase in adipogenesis in 3T3-L1 cells, as demonstrated by increased lipid droplets. This increase was mediated by T3-induced expression of the peroxisome proliferator-activated receptor γ (PPARγ) and CCAAT/enhancer-binding protein α (C/EBPα), which are master regulators of adipogenesis at both the mRNA and protein levels. In 3T3-L1 cells stably expressing TRα1PV (L1-α1PV cells) or TRβ1PV (L1-β1PV cells), adipogenesis was reduced 94 or 54% respectively, indicative of differential inhibitory activity of mutant TR isoforms. Concordantly, the expression of PPARγ and C/EBPα at the mRNA and protein levels was more repressed in L1-α1PV cells than in L1-β1PV cells. In addition, the expression of PPARγ downstream target genes involved in fatty acid synthesis – the lipoprotein lipase (Lpl) and aP2 involved in adipogenesis – was more inhibited by TRα1PV than by TRβ1PV. Chromatin immunoprecipitation assays showed that TRα1PV was more avidly recruited than TRβ1PV to the promoter to preferentially block the expression of the C/ebpα gene. Taken together, these data indicate that impaired adipogenesis by mutant TR is isoform dependent. The finding that induction of adipogenesis is differentially regulated by TR isoforms suggests that TR isoform-specific ligands could be designed for therapeutic intervention for lipid abnormalities.
Alok Mishra, Xu-guang Zhu, Kai Ge and Sheue-Yann Cheng
Jason F Ohlstein, Amy L Strong, John A McLachlan, Jeffrey M Gimble, Matthew E Burow and Bruce A Bunnell
Exposure of humans to the endocrine disrupter bisphenol A (BPA) has been associated with increased weight and obesity. However, the mechanism(s) by which BPA increases adipose tissue in humans remains to be determined. The goal of this study was to determine the effects of BPA on adipogenesis of cultured human adipose stromal/stem cells (ASCs), precursors to mature adipocytes. ASCs from three donors were cultured for either 14 or 21 days in adipogenic differentiation media containing increasing concentrations of BPA (100 pM–10 μM). The extent of adipogenic differentiation in the ASCs was assessed by staining with Oil Red O to visualize adipogenic differentiation and then quantified by extraction and optical density measurement of the retained dye. BPA significantly enhanced adipogenesis at a concentration of 1 μM after 21 days of culture. Additionally, we found that BPA increased transcription of the estrogen receptor (ER (ESR1)) and that treatment with the ER antagonist ICI 182 780, blocked the effects of BPA, indicating that BPA may act via an ER-mediated pathway. The results of molecular analyses indicated that the expression of the adipogenesis-associated genes dual leucine zipper-bearing kinase (DLK (MAP3K12)), IGF1, CCAAT/enhancer-binding protein alpha (C/EBP α (CEBPA)), peroxisome proliferator-activated receptor gamma (PPAR γ (PPARG)), and lipoprotein lipase (LPL) was temporally accelerated and increased by BPA. In summary, these results indicate that BPA significantly enhances adipogenesis in ASCs through an ER-mediated pathway at physiologically relevant concentrations.
Silvia Senesi, Paola Marcolongo, Ivana Manini, Rosella Fulceri, Vincenzo Sorrentino, Miklós Csala, Gábor Bánhegyi and Angelo Benedetti
The reductase activity of 11β-hydroxysteroid dehydrogenase type 1 (HSD11B1) plays an important role in the growth and differentiation of adipose tissue via the prereceptorial activation of glucocorticoids. This enzyme colocalizes with hexose-6-phosphate dehydrogenase (H6PD) at the luminal surface of the endoplasmic reticulum membrane, and the latter enzyme provides NADPH to the former, which can thus act as an 11β-reductase. It was suggested that, during adipogenesis, the increased expression of H6PD causes a dehydrogenase-to-reductase switch in the activity of HSD11B1. However, only the expression of the HSD11B1 has been extensively studied, and little is known about the expression of H6PD. Here, we investigated the expression and the activity of H6PD in the course of the differentiation of human adipose-derived mesenchymal stem cells (ADMSCs) and murine 3T3-L1 cells. It was found that H6PD is already present in adipose-derived stem cells and in 3T3-L1 fibroblasts even before the induction of adipogenesis. Moreover, mRNA and protein levels, as well as the microsomal H6PD activities remained unchanged during the differentiation. At the same time a great induction of HSD11B1 was observed in both cell types. The observed constant expression of H6PD suggests that HSD11B1 acts as a reductase throughout the adipogenesis process in human ADMSCs and murine 3T3-L1 cells.
The development of obesity is characterised not only by increased storage of lipids in existing fat cells but also by the generation of new adipocytes from progenitor cells. This process, called adipogenesis, can be divided into two related steps. First, during determination, multipotent mesenchymal stem cells commit to preadipocytes. These cells exhibit similar morphology compared with stem cells; however, they are committed to the adipogenic lineage and are not longer able to transform into osteoblasts, myocytes or chondrocytes. Secondly, during differentiation, preadipocytes become mature fat cells. As in other developmental processes, adipogenesis is tightly regulated at a molecular level by several transcription factors. Within the last decade, it has also become clear how the activity of these transcription factors is coordinated by extracellular signals. In this respect, secreted WNT signalling molecules are particularly important. Several members of the WNT family have been shown to inhibit early steps of adipogenesis. Conversely, endogenous inhibitors of WNT signalling were found to promote generation of adipocytes, indicating a fundamental role of these bioactive peptides in adipogenesis. From a pathophysiological point of view, it is of interest that polymorphisms in genes of the WNT signalling system have been associated with the development of obesity and type 2 diabetes in humans. Moreover, recent findings indicate that certain WNT molecules are involved in the so-called low-grade inflammation of adipose tissue, which is crucial in the development of obesity-associated insulin resistance. These important findings in nutritional and metabolic medicine will be summarised in the present review.
Rihua Zhang, Dongming Su, Weidong Zhu, Qiong Huang, Menglan Liu, Yi Xue, Yuanyuan Zhang, Dong li, Allan Zhao and Yun Liu
The aim of this study is to determine the effects of E2 on metabolic syndrome and the molecular mechanisms involving S100A16. Ovariectomized (OVX) rat models and mouse embryonic fibroblasts cell models were used. E2 loss in OVX rats induced body weight gain and central abdominal fat accumulation, which were ameliorated by E2 treatment under chow and high-fat diet (HFD) conditions. E2 decreased the expression of the adipocyte marker genes PPAR γ, aP2, C/EBP α, and S100A16. E2 inhibited adipogenesis. Overexpression of S100A16 reversed the E2-induced adipogenesis effect. A luciferase assay showed that E2 inhibited the expression of S100A16. E2 treatment decreased body weight gain and central abdominal fat accumulation under both chow and HFD conditions. Also, E2 suppressed adipogenesis by inhibiting S100A16 expression.
Gabriela Silva Monteiro de Paula, Marianna Wilieman, Karina Ribeiro Silva, Leandra Santos Baptista, Sihem Boudina, Luana Lopes de Souza, Thais Bento-Bernardes, Karina Dutra Asensi, Regina Coeli dos Santos Goldenberg and Carmen Cabanelas Pazos-Moura
Neuromedin B, a bombesin-like peptide, and its receptor, are expressed in white adipose tissue with undefined roles. Female mice with disruption of neuromedin B receptor (NB-R) exhibited partial resistance to diet-induced obesity leading to our hypothesis that NB-R is involved in adipogenesis. Here, we showed that adipose stem/stromal cells (ASC) from perigonadal fat of female NB-R-knockout mice, exposed to a differentiation protocol in vitro, accumulated less lipid (45%) than wild type, suggesting reduced capacity to differentiate under adipogenic input. To further explore mechanisms, preadipocytes 3T3-L1 cells were incubated in the presence of NB-R antagonist (PD168368) during the first 3 days in culture. Cells were analyzed in the end of the treatment (Day 3) and later when fully differentiated (Day 21). NB-R antagonist induced lower number of cells at day 3 and 21 (33–39%), reduced cell proliferation at day 3 (−53%) and reduced lipid accumulation at day 21 (−86%). The mRNA expressions of several adipocyte differentiation markers were importantly reduced at both days: Cebpb and Pparg and Fabp4, Plin-1 and Adipoq, and additionally Lep mRNA at day 21. The antagonist had no effect when incubated with mature 3T3-L1 adipocytes. Therefore, genetically disruption of NB-R in mice ASC or pharmacological antagonism of NB-R in 3T3-L1 cells impairs adipogenesis. The mechanisms suggested by results in 3T3-L1 cells involve reduction of cell proliferation and of early gene expressions, leading to decreased number of mature adipocytes. We speculate that NB-R antagonism may be useful to limit the increase in adiposity due to pre-adipocyte differentiation.
Rui Wang, Jie Hong, Ruixin Liu, Maopei Chen, Min Xu, Wiqiong Gu, Yifei Zhang, Qinyun Ma, Feng Wang, Juan Shi, Jiqiu Wang, Weiqing Wang and Guang Ning
WNT/β-catenin signalling is involved in regulating adipogenesis, and its dysregulation occurs in obesity. Secreted frizzled-related protein 5 (SFRP5) is a WNT protein inhibitor; however, its role in adipogenesis and obesity is controversial. In this study, we observed that SFRP5 mRNA levels were increased in the fat tissues of obese humans and mice. Sfrp5 expression was gradually induced during differentiation of white and brown adipocytes and was highly increased in mature adipocytes rather than preadipocytes. However, the effects of the exogenous overexpression of Sfrp5 indicated that Sfrp5 may not directly regulate adipogenesis in vitro under the conditions studied. Moreover, SFRP5 did not inhibit the canonical WNT/β-catenin signalling pathway in preadipocytes. Subsequently, we measured the levels of circulating SFRP5 in obese patients and non-obese subjects using ELISA and did not find any significant difference. Collectively, these findings indicate that Sfrp5 represents a candidate for a mature adipocyte marker gene. Our data provide new evidence concerning the role of SFRP5 in adipogenesis of white and brown adipocytes and obesity.
Stephen Fitter, Kate Vandyke, Stan Gronthos and Andrew C W Zannettino
Improved glucose and lipid metabolism is a unique side effect of imatinib therapy in some chronic myeloid leukaemia (CML) patients. We recently reported that plasma levels of adiponectin, an important regulator of insulin sensitivity, are elevated following imatinib therapy in CML patients, which could account for these improved metabolic outcomes. Adiponectin is secreted exclusively from adipocytes, suggesting that imatinib modulates adiponectin levels directly, by transcriptional upregulation of adiponectin in pre-existing adipocytes, and/or indirectly, by stimulating adipogenesis. In this report, we have demonstrated that imatinib promotes adipogenic differentiation of human mesenchymal stromal cells (MSCs), which in turn secrete high-molecular-weight adiponectin. Conversely, imatinib does not stimulate adiponectin secretion from mature adipocytes. We hypothesise that inhibition of PDGFRα (PDGFRA) and PDGFRβ (PDGFRB) is the mechanism by which imatinib promotes adipogenesis. Supporting this, functional blocking antibodies to PDGFR promote adipogenesis and adiponectin secretion in MSC cultures. We have shown that imatinib is a potent inhibitor of PDGF-induced PI3 kinase activation and, using a PI3 kinase p110α-specific inhibitor (PIK-75), we have demonstrated that suppression of this pathway recapitulates the effects of imatinib on MSC differentiation. Furthermore, using mitogens that activate the PI3 kinase pathway, or MSCs expressing constitutively activated Akt, we have shown that activation of the PI3 kinase pathway negates the pro-adipogenic effects of imatinib. Taken together, our results suggest that imatinib increases plasma adiponectin levels by promoting adipogenesis through the suppression of PI3 kinase signalling downstream of PDGFR.
Chen Chen, Yongdong Peng, Yinglin Peng, Jian Peng and Siwen Jiang
MicroRNAs are endogenous, conserved, and non-coding small RNAs that function as post-transcriptional regulators of fat development and adipogenesis. Adipogenic marker genes, such as CCAAT/enhancer binding protein α (Cebpa), peroxisome proliferator-activated receptor γ (Pparg), adipocyte fatty acid binding protein (Ap2), and fatty acid synthase (Fas), are regarded as the essential transcriptional regulators of preadipocyte differentiation and lipid storage in mature adipocytes. Canonical Wnt/β-catenin signaling is recognized as a negative molecular switch during adipogenesis. In the present work we found that miR-135a-5p is markedly downregulated during the process of 3T3-L1 preadipocyte differentiation. Overexpression of miR-135a-5p impairs the expressions of adipogenic marker genes as well as lipid droplet accumulation and triglyceride content, indicating the importance of miR-135a-5p for adipogenic differentiation and adipogenesis. Further studies show that miR-135a-5p directly targets adenomatous polyposis coli (Apc), contributes to the translocation of β-catenin from cytoplasm to nucleus, and then activates the expressions of cyclin D1 (Ccnd1) and Cmyc, indicating the induction of canonical Wnt/β-catenin signaling. In addition, inhibition of APC with siRNA exhibits the same effects as overexpression of miR-135a-5p. Our findings demonstrate that miR-135a-5p suppresses 3T3-L1 preadipocyte differentiation and adipogenesis through the activation of canonical Wnt/β-catenin signaling by directly targeting Apc. Taken together, these results offer profound insights into the adipogenesis mechanism and the development of adipose tissue.
Shulin Zhang, Melinda Chan and Jane E Aubin
The steroid hormone 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) inhibits osteogenesis while stimulating adipogenesis in vitro. We hypothesized that 1,25(OH)2D3 redirects the fate of osteoblast/adipocyte bipotential progenitors and other potential progenitors towards adipogenesis, a process possibly underlying the pathogenesis of osteopenic diseases such as osteoporosis. We therefore tested the global effects of 1,25(OH)2D3 on the recruitment of mesenchymal progenitors including osteogenic, chondrogenic, adipogenic and myogenic lineages (colony forming cell (CFC)-osteoblast (CFC-O), CFC-chondrocyte (CFC-C), CFC-adipocyte (CFC-A), and CFC-myoblast (CFC-M) respectively) in rat calvaria (RC) cell populations using gene expression profiling of single cell-derived colonies. Based on expression of lineage specific transcripts, 86% of single cell-derived colonies in untreated cultures simultaneously co-expressed transcripts of two, three, or four of the mesenchymal lineages tested. The distribution of mesenchymal progenitors in 1,25(OH)2D3-treated cultures was significantly changed compared with the control group, i.e. CFC-O were reduced (from 6 to 0%) and CFC-O/A bipotential (0 to 8.2%), CFC-C (4 to 10.2%) and CFC-Fibroblast (CFC-F) (4 to 16%) were increased. 1,25(OH)2D3 did not affect the frequency of tri- or tetra-lineage colonies. Single lineage CFC-A colonies were not detected in either the control or 1,25(OH)2D3 treatment group under the conditions tested.Since the parietal bones used for cell isolation derive from neuroectoderm, we also analyzed for expression of the neural markers nestin and β3 tubulin in these colonies. Surprisingly, 90% (45 of 50) of the colonies in the control group expressed neural markers, a frequency not changed by 1,25(OH)2D3 treatment. The current studies demonstrate the global and developmental stage-specific effects of 1,25(OH)2D3 on mesenchymal lineage progenitors, and suggest that the effects of 1,25(OH)2D3 on osteogenesis and adipogenesis in RC populations are mediated, at least in part, by increased recruitment of CFC-O/A, but not CFC-A type precursors.